29305972	 BACKGROUND: Late embryogenesis abundant (LEA) proteins were initially discovered  about 35years ago as accumulating late in embryogenesis of cotton seeds. Although abundant in seeds and pollens, these proteins have been found to protect cells against desiccation, cold, high temperature, and high salinity. OBJECTIVE: Here, we present the first comprehensive survey of LEA proteins and their encoding genes in Gastrodia elata, a well-known medicinal orchid in China.  Moreover, we researched on LEA family evolutionary relationships and functional characteristics. METHODS: The LEA gene family in G. elata (GeLEAs) was cloned based on RNA-Seq data. In addition, all of GeLEA genes were introduced into Escherichia coli to assess the function of GeLEAs under low temperature stress. RESULTS: Based on the phylogenetic analysis with Arabidopsis and Oryza LEA proteins, we identified 8 LEA protein encoding genes in G. elata that could be classified into 6 distinct subgroups. The motif composition of these proteins was an important feature specific to LEA groups. Compared with control, the expressions of 5 GeLEAs in E. coli exhibited enhanced cold resistance and viability, indicating that GeLEAs protein could play a protective role in cells under low temperature stress. CONCLUSION: Our results suggest that LEAs from G. elata play an important role in responses to abiotic stress. 
29240998	 Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network  being orthogonal to the host background. However, introduced circuits may interfere with the host's physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behavior of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guidance for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host. 
29467745	 As dormant phenotypic variants of bacteria, persisters account for many chronic infections affecting human health. Despite numerous studies, the role of small non-coding RNA (sRNA) in bacterial persistence has not been reported. To investigate the role of Hfq-interacting sRNA in persistence, we constructed the deletion mutants of 20 Hfq-interacting sRNAs (RyhB, GcvB, MgrR, RybB, MicF, SgrS, RprA, DicF, SsrS, FnrS, GadY, DsrA, OmrB, ArcZ, RyeB, RydC, OmrA, MicA, MicC, and ChiX) to assess their persistence capacity in uropathogenic Escherichia coli strain UTI89 and identified a new sRNA RyhB being involved in persister formation. The ryhB-knockout mutant had significant defect in persistence to a diverse range of antibiotics (levofloxacin, cefotaxime, gentamicin) and stresses  (hyperosmosis, acid, and heat) in both exponential phase and stationary phase. In addition, the effect of RyhB on persistence was synergistic with ppGpp and Fur protein. RNA-Seq analysis indicated that the ryhB-knockout mutant had a hyperactive metabolic state compared with the parent strain. Interestingly, increased adenosine triphosphate (ATP) levels and altered NAD+/NADH ratios were observed in the ryhB-knockout mutant. Our findings represent a new level of persistence regulation via sRNA and may provide novel therapeutic targets for interventions. 
29338696	 BACKGROUND: The protean chemical properties of mercury have long made it attractive for diverse applications, but its toxicity requires great care in its  use, disposal, and recycling. Mercury occurs in multiple chemical forms, and the  molecular basis for the distinct toxicity of its various forms is only partly understood. Global transcriptomics applied over time can reveal how a cell recognizes a toxicant and what cellular subsystems it marshals to repair and recover from the damage. The longitudinal effects on the transcriptome of exponential phase E. coli were compared during sub-acute exposure to mercuric chloride (HgCl2) or to phenylmercuric acetate (PMA) using RNA-Seq. RESULTS: Differential gene expression revealed common and distinct responses to the mercurials throughout recovery. Cultures exhibited growth stasis immediately  after each mercurial exposure but returned to normal growth more quickly after PMA exposure than after HgCl2 exposure. Correspondingly, PMA rapidly elicited up-regulation of a large number of genes which continued for 30 min, whereas fewer genes were up-regulated early after HgCl2 exposure only some of which overlapped with PMA up-regulated genes. By 60 min gene expression in PMA-exposed  cells was almost indistinguishable from unexposed cells, but HgCl2 exposed cells  still had many differentially expressed genes. Relative expression of energy production and most metabolite uptake pathways declined with both compounds, but  nearly all stress response systems were up-regulated by one or the other mercurial during recovery. CONCLUSIONS: Sub-acute exposure influenced expression of ~45% of all genes with many distinct responses for each compound, reflecting differential biochemical damage by each mercurial and the corresponding resources available for repair. This study is the first global, high-resolution view of the transcriptional responses to any common toxicant in a prokaryotic model system from exposure to recovery of active growth. The responses provoked by these two mercurials in this model bacterium also provide insights about how higher organisms may respond to these ubiquitous metal toxicants. 
28904250	 The model organism, Escherichia coli, contains a total of more than 4,500 genes,  but the total number of RNA polymerase (RNAP) core enzyme or the transcriptase is only about 2,000 molecules per genome. The regulatory targets of RNAP are, however, modulated by changing its promoter selectivity through two-steps of protein-protein interplay with 7 species of the sigma factor in the first step, and then 300 species of the transcription factor (TF) in the second step. Scientists working in the field of prokaryotic transcription in Japan have made considerable contributions to the elucidation of genetic frameworks and regulatory modes of the genome transcription in E. coli K-12. This review summarizes the findings by this group, first focusing on three sigma factors, the stationary-phase sigma RpoS, the heat-shock sigma RpoH, and the flagellar-chemotaxis sigma RpoF, as examples. It also presents an overview of the current state of the systematic research being carried out to identify the regulatory functions of all TFs from a single and the same bacterium E. coli K-12, using the genomic SELEX and PS-TF screening systems. All these studies have been undertaken with the aim of understanding the genome regulation in E. coli K-12 as a whole. 
29246112	 BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death.  Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions. RESULTS: Quantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments. We characterized hepP and its protein product HepP. hepP was cloned  from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14ΔhepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14. CONCLUSIONS: Changes within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing. 
29247194	 Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5' UTR of mRNA and the anti-SD (aSD) sequence at the free 3' end of the 16S rRNA (3' TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3' TAIL and 2) extent of the core aSD sequence within the 3' TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves  1) mapping the 3' boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3' TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3' TAIL in B. subtilis and recovered the established 3' TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein. 
29199958	 Cyclin D1 is a critical regulator of cell cycle progression and works at the G1 to S-phase transition. Here, we report the isolation and characterization of the  novel c-Myc-regulated lncRNA LAST (LncRNA-Assisted Stabilization of Transcripts), which acts as a CCND1 mRNA stabilizer. Mechanistically, LAST was shown to cooperate with CNBP to bind to the 5'UTR of CCND1 mRNA to protect against possible nuclease targeting. In addition, data from CNBP RIP-seq and LAST RNA-seq showed that CCND1 mRNA might not be the only target of LAST and CNBP; three additional mRNAs were shown to be post-transcriptional targets of LAST and CNBP.  In a xenograft model, depletion of LAST diminished and ectopic expression of LAST induced tumor formation, which are suggestive of its oncogenic function. We thus  report a previously unknown lncRNA involved in the fine-tuned regulation of CCND1 mRNA stability, without which CCND1 exhibits, at most, partial expression. 
29087459	 The genome of Escherichia coli K-12 contains ten cryptic phages, altogether constituting about 3.6% of the genome in sequence. Among more than 200 predicted  genes in these cryptic phages, 14 putative transcription factor (TF) genes exist, but their regulatory functions remain unidentified. As an initial attempt to make a breakthrough for understanding the regulatory roles of cryptic phage-encoded TFs, we tried to identify the regulatory function of CP4-6 cryptic prophage-encoded YagI with unknown function. After SELEX screening, YagI was found to bind mainly at a single site within the spacer of bidirectional transcription units, yagA (encoding another uncharacterized TF) and yagEF (encoding 2-keto-3-deoxy gluconate aldolase, and dehydratase, respectively) within this prophage region. YagEF enzymes are involved in the catabolism of xylose downstream from xylonate. We then designated YagI as XynR (regulator of xylonate catabolism), one of the rare single-target TFs. In agreement with this predicted regulatory function, the activity of XynR was suggested to be controlled by xylonate. Even though low-affinity binding sites of XynR were identified in the E. coli K-12 genome, they all were inside open reading frames,  implying that the regulation network of XynR is still fixed within the CR4-6 prophage without significant influence over the host E. coli K-12. 
29069500	 Although many tools have been developed to analyze small RNA sequencing (sRNA-Seq) data, it remains challenging to accurately analyze the small RNA population, mainly due to multiple sequence ID assignment caused by short read length. Additional issues in small RNA analysis include low consistency of microRNA (miRNA) measurement results across different platforms, miRNA mapping associated with miRNA sequence variation (isomiR) and RNA editing, and the origin of those unmapped reads after screening against all endogenous reference sequence databases. To address these issues, we built a comprehensive and customizable sRNA-Seq data analysis pipeline-sRNAnalyzer, which enables: (i) comprehensive miRNA profiling strategies to better handle isomiRs and summarization based on each nucleotide position to detect potential SNPs in miRNAs, (ii) different sequence mapping result assignment approaches to simulate results from microarray/qRT-PCR platforms and a local probabilistic model to assign mapping results to the most-likely IDs, (iii) comprehensive ribosomal RNA filtering for accurate mapping of exogenous RNAs and summarization based on taxonomy annotation. We evaluated our pipeline on both artificial samples (including synthetic miRNA and Escherichia coli cultures) and biological samples (human tissue and plasma). sRNAnalyzer is implemented in Perl and available at: http://srnanalyzer.systemsbiology.net/. 
28818557	 Escherichia coli (E. coli) O157:H7 is a foodborne pathogen that causes symptoms in humans. Its rapid identification should be considered to avoid toxic effects of the pathogen. In this study, systematic evolution of ligands by exponential enrichment using whole cells (Cell-SELEX) method was used for recognizing E. coli strain, O157 by single-stranded DNA library of aptamer. Nine rounds of cell-selex procedure were applied using O157, as a whole-cell target, with O42, K12, Top10,  DH5α E. coli cells, Shigella flexneri and Salmonella typhi as counterparts. The specific interaction between selected DNA aptamers and targeted cell was assessed. After applying six rounds of SELEX for selection of DNA aptamers, the candidate sequences were obtained. Finally, specific aptamer was selected as an ideal aptamer for detection and capturing of E. coli O157. Dissociation constant  of the selected aptamer were calculated (107.6 ± 67.8 pM). In addition, the secondary structure prediction and cross reactivity assays were performed. The isolated aptamer efficiency was confirmed and it was shown that the new DNA aptamer sequence has the ability to use for detection. This specific O157:H7 aptamer have the potential for application as a diagnostic ligand and could be used for detection of the related food borne diseases. 
29089595	 Phosphate control of the biosynthesis of secondary metabolites in Streptomyces is mediated by the two component system PhoR-PhoP. Linked to the phoR-phoP cluster,  and expressed in the opposite orientation, is a phoU-like encoding gene with low  identity to the phoU gene of Escherichia coli. Expression of this phoU-like gene  is strictly dependent on PhoP activation. We have isolated a PhoU-null mutant and used transcriptomic and RNA-sequencing (RNA-seq) procedures to identify its transcription start site and regulation. RNA-seq studies identified two transcription start sites, one upstream of phoU and the second upstream of the mptA gene. Whereas transcription of PhoU is entirely dependent on PhoP, expression of the downstream mtpA gene is only partially dependent on PhoP activation. The phoU mutant grows more slowly than the parental strain, sporulates poorly and the spores lack pigmentation. Production of actinorhodin and undecylprodigiosin decreased in the phoU mutant, indicating that PhoU has a positive modulating effect on production of these antibiotics. Indeed, transcriptional studies of expression of the actII-ORF4 and redD genes indicated  that the PhoU protein activates expression of these antibiotic regulators. Using  the glpQ1 promoter as in vivo reporter of the activity of the PHO regulon genes,  we observed that expression of glpQ1 is negatively modulated by PhoU. These results were confirmed by reverse transcription-PCR studies of three genes of the PHO regulon; that is, glpQ1, pstS and phoR. In conclusion, PhoU acts as a negative modulator of expression of the PHO regulon genes and as phoU expression  is strictly dependent on PhoP activation, this mechanism appears to work as a feed-back control mechanism (self-regulation).The Journal of Antibiotics advance  online publication, 1 November 2017; doi:10.1038/ja.2017.130. 
29031842	 Heat shock response is a classical stress-induced regulatory system in bacteria,  characterized by extensive transcriptional reprogramming. To compare the impact of heat stress on the transcriptome and translatome in Escherichia coli, we conducted ribosome profiling in parallel with RNA-Seq to investigate the alterations in transcription and translation efficiency when E. coli cells were exposed to a mild heat stress (from 30 °C to 45 °C). While general changes in ribosome footprints correlate with the changes of mRNA transcripts upon heat stress, a number of genes show differential changes at the transcription and translation levels. Translation efficiency of a few genes that are related to environment stimulus response is up-regulated, and in contrast, some genes functioning in mRNA translation and amino acid biosynthesis are down-regulated at the translation level in response to heat stress. Moreover, our ribosome occupancy data suggest that in general ribosomes accumulate remarkably in the starting regions of ORFs upon heat stress. This study provides additional insights into bacterial gene expression in response to heat stress, and suggests  the presence of stress-induced but yet-to-be characterized cellular regulatory mechanisms of gene expression at translation level. 
28870757	 The post-transcriptional regulator CsrA regulates multiple unrelated processes such as central carbon metabolism, motility, biofilm formation and bacterial virulence in different bacteria. However, regulation by CsrA in enterohemorrhagic Escherichia coli (EHEC) O157:H7 is still largely unknown. In this study, we performed a detailed analysis of gene expression differences between the EHEC O157:H7 wild-type strain and a corresponding csrA::kan mutant using RNA-seq technology. Genes whose expression was affected by CsrA were identified and grouped into different clusters of orthologous group categories. Genes located in the locus of enterocyte effacement (LEE) pathogenicity island were significantly  upregulated, whereas expression of flagella-related genes was significantly reduced in the csrA::kan mutant. Subsequent bacterial adherence and motility assays showed that inactivation of CsrA in EHEC O157:H7 resulted in a significant increase in bacterial adherence to host epithelial cells, with a concomitant loss of swimming motility on semi-solid agar plates. Furthermore, we also found that CsrA regulates genes not previously identified in other bacterial species, including genes encoding cytochrome oxidases and those required for nitrogen metabolism. Our results provide essential insight into the regulatory function of CsrA. 
28959742	 The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing  of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and  determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9  (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as  distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits  AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria. 
28924029	 CsrA of Escherichia coli is an RNA-binding protein that globally regulates a wide variety of cellular processes and behaviors including carbon metabolism, motility, biofilm formation, and the stringent response. CsrB and CsrC are sRNAs  that sequester CsrA, thereby preventing CsrA-mRNA interaction. RpoE (σE) is the extracytoplasmic stress response sigma factor of E. coli Previous RNA-seq studies identified rpoE mRNA as a CsrA target. Here we explored the regulation of rpoE by CsrA and found that CsrA represses rpoE translation. Gel mobility shift, footprint and toeprint studies identified three CsrA binding sites in the rpoE leader transcript, one of which overlaps the rpoE Shine-Dalgarno (SD) sequence, while another overlaps the rpoE translation initiation codon. Coupled in vitro transcription-translation experiments showed that CsrA represses rpoE translation by binding to these sites. We further demonstrate that σE indirectly activates transcription of csrB and csrC, leading to increased sequestration of CsrA such that repression of rpoE by CsrA is reduced. We propose that the Csr system fine-tunes the σE-dependent cell envelope stress response. We also identified a 51 amino acid coding sequence whose stop codon overlaps the rpoE start codon, and demonstrate that rpoE is translationally coupled with this upstream open reading  frame (ORF51). Loss of coupling reduces rpoE translation by more than 50%. Identification of a translationally coupled ORF upstream of rpoE suggests that this previously unannotated protein may participate in the cell envelope stress response. In keeping with existing nomenclature, we name ORF51 as rseD, resulting in an operon arrangement of rseD-rpoE-rseA-rseB-rseCIMPORTANCE CsrA posttranscriptionally represses genes required for bacterial stress responses, including the stringent response, catabolite repression, and the RpoS (σS)-mediated general stress response. We show that CsrA represses translation of rpoE, encoding the extracytoplasmic stress response sigma factor and that σE indirectly activates transcription of csrB and csrC, resulting in reciprocal regulation of these two global regulatory systems. These findings suggest that extracytoplasmic stress leads to derepression of rpoE translation by CsrA, and CsrA-mediated repression helps to reset RpoE abundance to pre-stress levels once  envelope damage is repaired. The discovery of an ORF, RseD, translationally coupled with rpoE adds further complexity to translational control of rpoE. 
28878825	 BACKGROUND: Measuring how gene expression changes in the course of an experiment  assesses how an organism responds on a molecular level. Sequencing of RNA molecules, and their subsequent quantification, aims to assess global gene expression changes on the RNA level (transcriptome). While advances in high-throughput RNA-sequencing (RNA-seq) technologies allow for inexpensive data  generation, accurate post-processing and normalization across samples is required to eliminate any systematic noise introduced by the biochemical and/or technical  processes. Existing methods thus either normalize on selected known reference genes that are invariant in expression across the experiment, assume that the majority of genes are invariant, or that the effects of up- and down-regulated genes cancel each other out during the normalization. RESULTS: Here, we present a novel method, moose2 , which predicts invariant genes in silico through a dynamic programming (DP) scheme and applies a quadratic normalization based on this subset. The method allows for specifying a set of known or experimentally validated invariant genes, which guides the DP. We experimentally verified the predictions of this method in the bacterium Escherichia coli, and show how moose2 is able to (i) estimate the expression value distances between RNA-seq samples, (ii) reduce the variation of expression  values across all samples, and (iii) to subsequently reveal new functional groups of genes during the late stages of DNA damage. We further applied the method to three eukaryotic data sets, on which its performance compares favourably to other methods. The software is implemented in C++ and is publicly available from http://grabherr.github.io/moose2/. CONCLUSIONS: The proposed RNA-seq normalization method, moose2 , is a valuable alternative to existing methods, with two major advantages: (i) in silico prediction of invariant genes provides a list of potential reference genes for downstream analyses, and (ii) non-linear artefacts in RNA-seq data are handled adequately to minimize variations between replicates. 
28919886	 Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. The purpose of this study was to determine the role played by the dam ortholog identified in the entomopathogenic bacterium Photorhabdus luminescens. Complementation assays of an Escherichia coli dam mutant showed the restoration of the DNA methylation state of the parental strain. Overexpression of dam in P.  luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. A transcriptome analysis revealed the  differential expression of 208 genes between the two strains. In particular, the  downregulation of flagellar genes was observed in the dam-overexpressing strain.  In the closely related bacterium Xenorhabdus nematophila, dam overexpression also impaired motility. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection. 
28841711	 Translation-independent mRNA localization represents an emerging concept in cell  biology. In Escherichia coli, mRNAs encoding integral membrane proteins (MPRs) are targeted to the membrane where they are translated by membrane associated ribosomes and the produced proteins are inserted into the membrane co-translationally. In order to better understand aspects of the biogenesis and localization of MPRs, we investigated their subcellular distribution using cell fractionation, RNA-seq and qPCR. The results show that MPRs are overrepresented in the membrane fraction, as expected, and depletion of the signal recognition particle-receptor, FtsY reduced the amounts of all mRNAs on the membrane. Surprisingly, however, MPRs were also found relatively abundant in the soluble ribosome-free fraction and their amount in this fraction is increased upon overexpression of CspE, which was recently shown to interact with MPRs. CspE also conferred a positive effect on the membrane-expression of integral membrane proteins. We discuss the possibility that the effects of CspE overexpression may  link the intriguing subcellular localization of MPRs to the cytosolic ribosome-free fraction with their translation into membrane proteins and that the ribosome-free pool of MPRs may represent a stage during their targeting to the membrane, which precedes translation. 
28652307	 During infection, Neisseria gonorrhoeae senses and responds to stress; such responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system that is a homolog of CpxRA. In Escherichia coli, CpxRA senses and responds to envelope stress; CpxA is a sensor kinase/phosphatase for CpxR, a response regulator. When a cpxA mutant is grown in medium containing glucose, CpxR is phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J. L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszyński, R. W. Carlson, and W. M. Shafer, Antimicrob Agents Chemother 60:4690-4700, 2016, https://doi.org/10.1128/AAC.00823-16) showed that MisR (CpxR) is required for the maintenance of membrane integrity and resistance to antimicrobial peptides, suggesting a role in gonococcal survival in vivo Here, we evaluated the contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model of cervicovaginal colonization and identified MisR-regulated genes using RNA sequencing (RNA-Seq). The deletion of misR or misS severely reduced the capacity  of N. gonorrhoeae to colonize mice or maintain infection over a 7-day period and  reduced microbial fitness after exposure to heat shock. Compared to the wild type (WT), the inactivation of misR identified 157 differentially regulated genes, most of which encoded putative envelope proteins. The inactivation of misS identified 17 differentially regulated genes compared to the WT and 139 differentially regulated genes compared to the misR mutant, 111 of which overlapped those differentially expressed in the comparison of the WT versus the  misR mutant. These data indicate that an intact MisRS system is required for gonococcal infection of mice. Provided the MisR is constitutively phosphorylated  in the misS mutant, the data suggest that controlled but not constitutive activation is required for gonococcal infection in mice. 
28800583	 Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with  DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can  be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis  from some genes via competition with RNA polymerase or, vice versa, competing with inhibitors to activate transcription. 
28794460	 The host mechanisms involved in Escherichia coli O157 super-shedding in cattle is largely unknown. In this study, the comparison of transcriptomes of intestinal tissues between super-shedders (SS) and cattle negative for E. coli O157 (NS) was performed, aiming to identify genes that are potentially associated with super-shedding. In total, 16,846 ± 639 (cecum) to 18,137 ± 696 (distal jejunum) were expressed throughout the intestine, with the expressed genes associated with immune functions more pronounced in the small intestine. In total, 351 differentially expressed (DE) genes were identified throughout the intestine between SS and NS, with 101 being up-regulated and 250 down-regulated in SS. Functional analysis revealed DE genes were involved in increased T-cell responses and cholesterol absorption in the distal jejunum and descending colon, and decreased B-cell maturation in the distal jejunum of SS. RNA-Seq based SNP discovery revealed that the mutations in seven DE genes involved in leukocyte activation and cholesterol transportation were associated with E. coli O157 shedding. Our findings suggest that T-cell responses and cholesterol metabolism in the intestinal tract may be associated with super-shedding phenomenon, and the SNPs in the DE genes are possibly associated with the observed gene expression difference between SS and NS. 
28224267	 BACKGROUND: Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas. METHODS: We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate  was also verified with the tissue samples of breast cancer patients by immunohistochemistry process. RESULTS: Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K d = 6.33 ± 0.86 nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at  490 nm wavelength) in the dynamic range of 100-700 nM ECD_Apt1 with a detection limit of 12.5 ± 2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche  of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes. CONCLUSIONS: Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1  could be an effective low-cost alternative to conventional anti-Her2 antibody in  solid phase immunoassays for cancer diagnosis and related applications. 
28585781	 Pseudomonas putida and Escherichia coli are ubiquitous microorganisms that can be isolated from soil rhizosphere, the surface of vegetables, fresh waters and wastewaters - environments in which they likely co-exist. Despite this, the potential interactions between these microbes have not been studied in detail. To analyse these interactions, we carried out RNA-seq transcriptomic analysis of these microbes as monocultures and as co-cultures. Our results show that co-culture of these microbes significantly alters transcriptional profiles. The most dramatic transcriptional changes in both microorganisms were involved in central carbon metabolism, as well as adhesion to surfaces and the activation of  drug efflux pumps. We also found that acetate production was one of the mechanisms used by E. coli K-12 MG1655 in response to the presence of P. putida DOT-T1E. 
28499021	 The RNase P family comprises structurally diverse endoribonucleases ranging from  complex ribonucleoproteins to single polypeptides. We show that the organellar (AtPRORP1) and the two nuclear (AtPRORP2,3) single-polypeptide RNase P isoenzymes from Arabidopsis thaliana confer viability to Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. RNA-Seq revealed that AtPRORP1, compared with bacterial RNase P or AtPRORP3, cleaves several precursor  tRNAs (pre-tRNAs) aberrantly in E. coli. Aberrant cleavage by AtPRORP1 was mainly observed for pre-tRNAs that can form short acceptor-stem extensions involving G:C base pairs, including tRNAAsp(GUC), tRNASer(CGA) and tRNAHis. However, both AtPRORP1 and 3 were defective in processing of E. coli pre-tRNASec carrying an acceptor stem expanded by three G:C base pairs. Instead, pre-tRNASec was degraded, suggesting that tRNASec is dispensable for E. coli under laboratory conditions. AtPRORP1, 2 and 3 are also essentially unable to process the primary  transcript of 4.5S RNA, a hairpin-like non-tRNA substrate processed by E. coli RNase P, indicating that PRORP enzymes have a narrower, more tRNA-centric substrate spectrum than bacterial RNA-based RNase P enzymes. The cells' viability also suggests that the essential function of the signal recognition particle can  be maintained with a 5΄-extended 4.5S RNA. 
28648779	 In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for  RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription. 
28677629	 Toxin-antitoxin (TA) systems are key regulators of bacterial persistence, a multidrug-tolerant state found in bacterial species that is a major contributing  factor to the growing human health crisis of antibiotic resistance. Type II TA systems consist of two proteins, a toxin and an antitoxin; the toxin is neutralized when they form a complex. The ratio of antitoxin to toxin is significantly greater than 1.0 in the susceptible population (non-persister state), but this ratio is expected to become smaller during persistence. Analysis of multiple datasets (RNA-seq, ribosome profiling) and results from translation initiation rate calculators reveal multiple mechanisms that ensure a high antitoxin-to-toxin ratio in the non-persister state. The regulation mechanisms include both translational and transcriptional regulation. We classified E. coli  type II TA systems into four distinct classes based on the mechanism of differential protein production between toxin and antitoxin. We find that the most common regulation mechanism is translational regulation. This classification scheme further refines our understanding of one of the fundamental mechanisms underlying bacterial persistence, especially regarding maintenance of the antitoxin-to-toxin ratio. 
28854680	 The dissemination of extended-spectrum β-lactamases (ESBLs) genes among bacteria  is commonly achieved by plasmid conjugation. In the last decade, the CTX-M type enzyme was the most widespread and prevalent ESBLs in the world. In Hong Kong and mainland China, among the commonly found CTX-M-carrying plasmids were pHK01 and pHK01-like plasmids, which belong to incompatibility group FII (IncFII). In this  work, we studied the physiological effect caused by the pHK01 plasmid in bacterial host Escherichia coli J53. The plasmid did not affect cell growth of the host but reduced their motility. The reduction of host motility was attributed to downregulation of genes that encode the flagellar system. We also identified several plasmid-encoded sRNAs, and showed that the overexpression of one of them, AS-traI, in the presence of pHK01 plasmid shortened the lag phase of host growth. In addition to the study of pHK01 in bacteria, we also developed a fast and incompatibility group-specific curing method using countertranscribed RNA, which could be of general usage for studying plasmid-host interaction in clinical aspects. 
28666008	 The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined  by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. For identification of the "constitutive promoters" that are recognized by each RNAP holoenzyme alone in the absence of other supporting factors, we have performed the genomic SELEX screening in vitro for their binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of constitutive  promoters for each RNAP holoenzyme was then estimated based on the location of RNAP-binding sites. The first successful screening of the constitutive promoters  was achieved for RpoD (σ70), the principal sigma for transcription of growth-related genes. As an extension, we performed in this study the screening of constitutive promoters for four minor sigma subunits, stationary-phase specific RpoS (σ38), heat-shock specific RpoH (σ32), flagellar-chemotaxis specific RpoF (σ28) and extra-cytoplasmic stress-response RpoE (σ24). The total number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41  for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared with that of known promoters identified in vivo under various conditions and using varieties of E. coli strains, altogether allowing the estimation of "inducible promoters" in the presence of additional supporting factors. 
28607761	 The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic  studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem,  we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9. When targeting the promoter region, both strands showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome  RNA-seq technique was further employed to demonstrate the high specificity of ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity  in ddCpf1 was capable of processing a precursor CRISPR array to simply generate multiple mature crRNAs in vivo, facilitating multiplex gene regulation. With the  employment of this multiplex gene regulation strategy, we also showed how to quickly screen a library of candidate targets, that is, the two-component systems in E. coli. Therefore, based on our findings here, the CRISPR-ddCpf1 system may be further developed and widely applied in both biological research and clinical  studies. 
28389540	 Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains  revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help  neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3  had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads  to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain. 
28542466	 BACKGROUND: Biochemical methods are available for enriching 5' ends of RNAs in prokaryotes, which are employed in the differential RNA-seq (dRNA-seq) and the more recent Cappable-seq protocols. Computational methods are needed to locate RNA 5' ends from these data by statistical analysis of the enrichment. Although statistical-based analysis methods have been developed for dRNA-seq, they may not be suitable for Cappable-seq data. The more efficient enrichment method employed  in Cappable-seq compared with dRNA-seq could affect data distribution and thus algorithm performance. RESULTS: We present Transformation of Nucleotide Enrichment Ratios (ToNER), a tool for statistical modeling of enrichment from RNA-seq data obtained from enriched and unenriched libraries. The tool calculates nucleotide enrichment scores and determines the global transformation for fitting to the normal distribution using the Box-Cox procedure. From the transformed distribution, sites of significant enrichment are identified. To increase power of detection, meta-analysis across experimental replicates is offered. We tested the tool on Cappable-seq and dRNA-seq data for identifying Escherichia coli transcript 5' ends and compared the results with those from the TSSAR tool, which is designed for analyzing dRNA-seq data. When combining results across Cappable-seq replicates, ToNER detects more known transcript 5' ends than TSSAR. In general, the transcript 5' ends detected by ToNER but not TSSAR occur in regions which cannot be locally modeled by TSSAR. CONCLUSION: ToNER uses a simple yet robust statistical modeling approach, which can be used for detecting RNA 5'ends from Cappable-seq data, in particular when combining information from experimental replicates. The ToNER tool could potentially be applied for analyzing other RNA-seq datasets in which enrichment for other structural features of RNA is employed. The program is freely available for download at ToNER webpage (http://www4a.biotec.or.th/GI/tools/toner) and GitHub repository (https://github.com/PavitaKae/ToNER). 
28536480	 Microorganisms are key components for plant biomass breakdown within rumen environments. Fibrobacter succinogenes have been identified as being active and dominant cellulolytic members of the rumen. In this study, F. succinogenes type strain S85 was adapted for steady state growth in continuous culture at pH 5.75 and confirmed to grow in the range of pH 5.60-5.65, which is lower than has been  reported previously. Wild type and acid tolerant strains digested corn stover with equal efficiency in batch culture at low pH. RNA-seq analysis revealed 268 and 829 genes were differentially expressed at pH 6.10 and 5.65 compared to pH 6.70, respectively. Resequencing analysis identified seven single nucleotide polymorphisms (SNPs) in the sufD, yidE, xylE, rlmM, mscL and dosC genes of acid tolerant strains. Due to the absence of a F. succinogenes genetic system, homologues in Escherichia coli were mutated and complemented and the resulting strains were assayed for acid survival. Complementation with wild-type or acid tolerant F. succinogenes sufD restored E. coli wild-type levels of acid tolerance, suggesting a possible role in acid homeostasis. Recent genetic engineering developments need to be adapted and applied in F. succinogenes to further our understanding of this bacterium. 
28545122	 Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA  analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome. 
28437463	 The CreBC two-component system (TCS) is a conserved regulatory system found in Escherichia coli, Aeromonas spp., Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. In this study, we determined how CreBC TCS regulates secreted protease activities and swimming motility using creB, creC, and creBC in-frame deletion mutants (KJΔCreB, KJΔCreC, and KJΔBC) of S. maltophilia KJ. Compared to  wild-type KJ, KJΔCreB had a comparable secreted protease activity; however, the secreted protease activities were obviously reduced in KJΔCreC and KJΔBC, suggesting that CreC works together with another unidentified response regulator  (not CreB) to regulate secreted protease activity. Single gene inactivation of creB or creC resulted in mutants with an enhanced swimming motility, and this phenotype was exacerbated in a double mutant KJΔBC. To elucidate the underlying mechanism responsible for the ΔcreBC-mediated swimming enhancement, flagella morphology observation, RNA-seq based transcriptome assay, qRT-PCR, and membrane  integrity and potential assessment were performed. Flagella morphological observation ruled out the possibility that swimming enhancement was due to altered flagella morphology. CreBC inactivation upregulated the expression of creD and flagella-associated genes encoding the basal body- and motor-associated  proteins. Furthermore, KJΔBC had an increased membrane susceptibility to Triton X-100 and CreD upregulation in KJΔBC partially alleviated the compromise of membrane integrity. The impact of creBC TCS on bacterial membrane potential was assessed by carbonyl cyanide m-chlorophenyl hydrazine (CCCP50) concentration at which 50% of bacterial swimming is inhibited. CCCP50 of wild-type KJ increased when creBC was deleted, indicating an association between the higher membrane potential of KJΔBC cells and enhanced motility. Upregulation of the basal body- and motor-associated genes of flagella in KJΔBC cells may explain the increased membrane potential. Collectively, inactivation of creBC increased swimming motility through membrane potential increase and creD upregulation in S. maltophilia. The increased membrane potential may supply more energy for flagella propelling and CreD upregulation supports membrane stability, providing a strong  membrane for flagellum function. 
28189052	 The annual Zea mays ssp. mexicana L., a member of the teosinte group, is a close  wild relative of maize and thus can be effectively used in maize improvement. In  this study, an ICE-like gene, ZmmICE1, was isolated from a cDNA library of RNA-Seq from cold-treated seedling tissues of Zea mays ssp. mexicana L. The deduced protein of ZmmICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE-like proteins. The ZmmICE1 protein localizes to the nucleus and shows sumoylation when expressed in an Escherichia coli reconstitution system. In addition, yeast one hybrid assays indicated that ZmmICE1 has transactivation activities. Moreover, ectopic expression of ZmmICE1 in the Arabidopsis ice1-2 mutant increased freezing tolerance. The ZmmICE1 overexpressed plants showed lower electrolyte leakage (EL), reduced contents of malondialdehyde (MDA). The expression of downstream cold related genes of Arabidopsis C-repeat-binding factors (AtCBF1, AtCBF2 and AtCBF3), cold-responsive genes (AtCOR15A and AtCOR47), kinesin-1 member gene (AtKIN1) and responsive to desiccation gene (AtRD29A) was significantly induced when compared with wild type under low temperature treatment. Taken together, these results indicated that ZmmICE1 is the homolog of Arabidopsis inducer of CBF expression genes (AtICE1/2)  and plays an important role in the regulation of freezing stress response. 
28382047	 Peach (Prunus persica L. Batsch) is a commercial grown fruit trees, important because of its essential nutrients and flavor promoting secondary metabolites. The glycosylation processes mediated by UDP-glycosyltransferases (UGTs) play an important role in regulating secondary metabolites availability. Identification and characterization of peach UGTs is therefore a research priority. A total of 168 peach UGT genes that distributed unevenly across chromosomes were identified  based on their conserved PSPG motifs. Phylogenetic analysis of these genes with plant UGTs clustered them into 16 groups (A-P). Comparison of the patterns of intron-extron and their positions within genes revealed one highly conserved intron insertion event in peach UGTs. Tissue specificity, temporal expression patterns in peach fruit during development and ripening, and in response to abiotic stress UV-B irradiation was investigated using RNA-seq strategy. The relationship between UGTs transcript levels and concentrations of glycosylated volatiles was examined to select candidates for functional analysis. Heterologous expressing these candidate genes in Escherichia coli identified UGTs that were involved in the in vitro volatile glycosylation. Our results provide an important source for the identification of functional UGT genes to potential manipulate secondary biosynthesis in peach. 
28426097	 Polyadenylation is thought to be involved in the degradation and quality control  of bacterial RNAs but relatively few examples have been investigated. We used a combination of 5΄-tagRACE and RNA-seq to analyze the total RNA content from a wild-type strain and from a poly(A)polymerase deleted mutant. A total of 178 transcripts were either up- or down-regulated in the mutant when compared to the  wild-type strain. Poly(A)polymerase up-regulates the expression of all genes related to the FliA regulon and several previously unknown transcripts, including numerous transporters. Notable down-regulation of genes in the expression of antigen 43 and components of the type 1 fimbriae was detected. The major consequence of the absence of poly(A)polymerase was the accumulation of numerous  sRNAs, antisense transcripts, REP sequences and RNA fragments resulting from the  processing of entire transcripts. A new algorithm to analyze the position and composition of post-transcriptional modifications based on the sequence of unencoded 3΄-ends, was developed to identify polyadenylated molecules. Overall our results shed new light on the broad spectrum of action of polyadenylation on  gene expression and demonstrate the importance of poly(A) dependent degradation to remove structured RNA fragments. 
28115545	 The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the  production of only a little RpoS ("sensitive" genes), and genes whose expression  changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute  to the diversity of stress responses directed by RpoS.IMPORTANCE The sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses. 
28281578	 Cold shock proteins (CSPs) enhance acclimatization of bacteria to adverse environmental circumstances. The Escherichia coli CSP genes CspA and CspB were modified to plant-preferred codon sequences and named as SeCspA and SeCspB. Overexpression of exogenous SeCspA and SeCspB in transgenic Arabidopsis lines increased germination rates, survival rates, and increased primary root length compared to control plants under drought and salt stress. Investigation of several stress-related parameters in SeCspA and SeCspB transgenic wheat lines indicated that these lines possessed stress tolerance characteristics, including  lower malondialdehyde (MDA) content, lower water loss rates, lower relative Na+ content, and higher chlorophyll content and proline content than the control wheat plants under drought and salt stresses. RNA-seq and qRT-PCR expression analysis showed that overexpression of SeCsp could enhance the expression of stress-responsive genes. The field experiments showed that the SeCspA transgenic  wheat lines had great increases in the 1000-grain weight and grain yield compared to the control genotype under drought stress conditions. Significant differences  in the stress indices revealed that the SeCspA transgenic wheat lines possessed significant and stable improvements in drought tolerance over the control plants. No such improvement was observed for the SeCspB transgenic lines under field conditions. Our results indicated that SeCspA conferred drought tolerance and improved physiological traits in wheat plants. 
28274217	 BACKGROUND: Escherichia coli O157:H7 (O157) strain 86-24, linked to a 1986 disease outbreak, displays curli- and biofilm-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR-) on a CR-containing medium. However, on a CR medium this strain produces red isolates (CR+) capable of producing curli fimbriae and biofilms. RESULTS: To identify genes controlling differential expression of curli fimbriae  and biofilm formation, the RNA-Seq profile of a CR+ isolate was compared to the CR- parental isolate. Of the 242 genes expressed differentially in the CR+ isolate, 201 genes encoded proteins of known functions while the remaining 41 encoded hypothetical proteins. Among the genes with known functions, 149 were down- and 52 were up-regulated. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes  encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH,  htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR+ isolate. Many genes mediating acid resistance and colanic acid biosynthesis,  which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR+ and CR- isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR+ isolate. The  alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR+ isolate at the insertion site of the duplicated sequence. Complementation of CR+ isolate with rcsB of the CR- parent restored parental phenotypes to the CR+ isolate. CONCLUSIONS: The results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. Understanding whether rcsB inactivation enhances persistence and survival of O157 in carrier animals and the environment would be important in developing strategies for controlling this bacterial pathogen in these niches. 
28272554	 Conventional cell-SELEX aims to isolate aptamers to a single unique target bacteria species. We propose a method to isolate single-stranded DNA aptamers that have broad reactivity to multiple bacterial targets belonging to different genera. The key of the proposed method is that targets of interest are changed sequentially at each SELEX round. The general scheme was examined using six bacteria from different genera, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Citrobacter freundii, Bacillus subtilis, and Staphylococcus epidermidis (four gram-negative and two gram-positive bacteria). In the first round of SELEX, the DNA library was incubated with E. coli and amplicons bound to E. coli were separated. The amplicons were sequentially separated by incubation with E. aerogenes, K. pneumoniae, C. freundii, B. subtilis, and S. epidermidis at each SELEX. The amplicons obtained using the last bacterial species were incubated again with the first bacterial species and this  loop was repeated two more times. We refer to this method as sequential toggle cell-SELEX (STC-SELEX). The isolated aptamers had dissociation constants of 9.22-38.5 nM and had no affinity to other bacteria that were not included in STC-SELEX. These results demonstrate the potential to isolate aptamers with broad affinity to bacterial taxa in different genera. 
27655246	 Enterohemorrhagic E. coli O157:H7 (EHEC) shorten the lifespan of Caenorhabditis elegans compared to avirulent bacteria. Co-feeding EHEC with Enterococcus faecalis Symbioflor® significantly increased the worms' lifespan. The transcriptome of EHEC grown in vitro with or without Symbioflor® was analyzed using RNA-seq. The analysis revealed downregulation of several virulence-associated genes in the presence of Symbioflor®, including virulence key genes (e.g., LEE, flagellum, quorum-sensing). The downregulation of the LEE genes was corroborated by lux-transposon mutants. Upregulated genes included acid response genes, due to a decrease in pH exerted by Symbioflor®. Further genes indicate cellular stress in EHEC (e.g. prophage/mobile elements involved in excision, cell lysis, and cell division inhibition). Thus, the observed protection of C. elegans during an EHEC infection by the probiotic Symbioflor® is suggested to be caused by triggering concomitant transcriptomic changes. To verify the biological relevance of this modulation, exemplary genes found to be influenced by Symbioflor® were knocked out (fliD, espB, Z3136, Z3917, and L7052). The lifespan of nematodes changed when using knock-outs as food source and the effect could be complemented in trans. In summary, Symbioflor® appears to be a protective probiotic in the nematode model. 
28009914	 In an effort to expand the binding and recognition capabilities of aptamers, a nucleoside triphosphate modified with a phenol that mimics the side chain of tyrosine was used in the selection of DNA aptamers against live bacteria. Of multiple modified aptamers that were isolated against Escherichia coli DH5α cells, one aptamer displays high selectivity and affinity for the target cells and is greatly enriched for phenol-modified dU nucleotides (dUy, 47.5%). When the same sequences are synthesized with TTP, no binding is observed. Taken together,  these findings highlight the value of using modified nucleotide triphosphates in  aptamer selections and portends success in SELEX against an array of whole cells  as targets. 
28280778	 Author information:  (1)CIBIO, University of Trento , via Sommarive 9, 38123 Povo, Italy. (2)Institute for Bioscience and Biotechnology Research, University of Maryland ,  College Park, Maryland 20742, United States. 
28193207	 BACKGROUND: Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory. RESULTS: Here, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a "pre-defense" mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the  presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype. CONCLUSIONS: The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential "pre-defense" mechanism under non-stressed conditions. The findings of  this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production. 
28479747	 Functional genomics and gene regulation inference has readily expanded our knowledge and understanding of gene interactions with regards to expression regulation. With the advancement of transcriptome sequencing in time-series comes the ability to study the sequential changes of the transcriptome. Here, we present a new method to augment regulation networks accumulated in literature with transcriptome data gathered from time-series experiments to construct a sequential representation of transcription factor activity. We apply our method on a time-series RNA-Seq data set of Escherichia coli as it transitions from growth to stationary phase over five hours and investigate the various activity in gene regulation process by taking advantage of the correlation between regulatory gene pairs to examine their activity on a dynamic network. We analyse  the changes in metabolic activity of the pagP gene and associated transcription factors during phase transition, and visualize the sequential transcriptional activity to describe the change in metabolic pathway activity originating from the pagP transcription factor, phoP. We observe a shift from amino acid and nucleic acid metabolism, to energy metabolism during the transition to stationary phase in E. coli. 
28005933	 Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX  screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing  RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of  all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed. 
27901120	 A novel marine strain representing efficient degradation ability toward brown algae was isolated, identified, and assigned to Bacillus weihaiensis Alg07. The alga-associated marine bacteria promote the nutrient cycle and perform important  functions in the marine ecosystem. The de novo sequencing of the B. weihaiensis Alg07 genome was carried out. Results of gene annotation and carbohydrate-active  enzyme analysis showed that the strain harbored enzymes that can completely degrade alginate and laminarin, which are the specific polysaccharides of brown algae. We also found genes for the utilization of mannitol, the major storage monosaccharide in the cell of brown algae. To understand the process of brown algae decomposition by B. weihaiensis Alg07, RNA-seq transcriptome analysis and qRT-PCR were performed. The genes involved in alginate metabolism were all up-regulated in the initial stage of kelp degradation, suggesting that the strain Alg07 first degrades alginate to destruct the cell wall so that the laminarin and mannitol are released and subsequently decomposed. The key genes involved in alginate and laminarin degradation were expressed in Escherichia coli and characterized. Overall, the model of brown algae degradation by the marine strain Alg07 was established, and novel alginate lyases and laminarinase were discovered. 
27419274	 The aims of this study were to examine the removal of bacteria and viruses by household point-of-use (POU) treatments and to apply a previously developed large-volume virus concentration method (∼20 L). First, the removal of microbes by household POU treatment was investigated in the laboratory. Second, the prevalence of viruses in drinking water sources for households and the removal efficiency of microbes by POU treatments in two suburban communities in Hanoi, Vietnam, were investigated. Indigenous pepper mild mottle virus (PMMoV) was used  as the main target together with adenovirus, Aichi virus, enterovirus, F-specific bacteriophage genogroup 1, and Escherichia coli to investigate the removal efficiency of household treatments. The results from laboratory and field survey  were compared. From the laboratory study, ceramic membranes were not effective for removing viruses and bacteria from water; pathogen reduction was less than 1.5 log10. By contrast, reverse osmosis (RO) devices reduced microbes by 3 to > 5 log10. In a field study, PMMoV was found to be the most prevalent waterborne virus. Household sand filtration was ineffective for removing E. coli, total coliforms and PMMoV; the reduction was less than 1 order of magnitude. Boiling the water and then filtering it with a ceramic membrane reduced E. coli by 3 orders of magnitude, but this was not effective for removing PMMoV. RO filtration was one of the promising methods for removing E. coli, total coliforms and PMMoV  to below their detection limits in most of the samples studied. The removal of E. coli, total coliforms and PMMoV was >2.3, >4 and >3 log10, respectively. The laboratory results of virus removal efficiency by POU devices agreed with the field study. Due to the prevalence and characteristics of PMMoV, it is a strong candidate for an indigenous indicator to investigate the viral removal efficiency of household POU treatments. 
27459147	 There is a growing interest in applying tobacco agroinfiltration for recombinant  protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely  degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production. 
27038308	 Transcriptome profiling is a powerful tool for identifying gene networks from whole genome expression analysis in many living species. Here is described the first extensively characterized platform using Agilent microarray technology for  transcriptome analysis in the filamentous fungus Aspergillus (Emericella) nidulans. We developed and validated a reliable gene expression microarray in 8 × 15 K format, with predictive and experimental data establishing its specificity and sensitivity. Either one or two 60-mer oligonucleotide probes were selected for each of 10,550 nuclear as well as 20 mitochondrial coding sequences. More than 99 % of probes were predicted to hybridize with 100 % identity to their aimed specific A. nidulans target only. Probe sensitivity was supported by a highly narrow distribution of melting temperatures together with thermodynamic features, which strongly favored probe-target perfect match hybridization, in comparison with predicted secondary structures. Array quality was evaluated through transcriptome comparison of two A. nidulans strains, differing by the presence or not of Escherichia coli LacZ transgene. High signal-to-noise ratios were measured, and signal reproducibility was established at intra-probe and inter-probe levels. Reproducibility of microarray performances was assessed by high correlation between two-color dye signals and between technical replicates.  Results were confirmed by RT-qPCR analysis on five genes. Though it covers 100 %  of the A. nidulans targeted coding sequences, this low density array allows limited experimental costs and simplified data analysis process, making it suitable for studying gene expression in this model organism through large numbers of experimental conditions, in basic, biomedical or industrial microbiology research fields. 
27591771	 KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS  were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription  polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli  individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+ binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants. 
27683109	 Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have  screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T>G in intron 6, c.1408+729A>G in intron 11, and c.1408+731C>T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation  sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families. 
26936422	 Enterotoxigenic Escherichia coli (ETEC) is a type of pathogenic bacteria that cause diarrhea in piglets through colonizing pig small intestine epithelial cells by their surface fimbriae. Different fimbriae type of ETEC including F4, F18, K99 and F41 have been isolated from diarrheal pigs. In this study, we performed a genome-wide association study to map the loci associated with the susceptibility  of pigs to ETEC F41 using 39454 single nucleotide polymorphisms (SNPs) in 667 F2  pigs from a White Duroc×Erhualian F2 cross. The most significant SNP (ALGA0022658, P=5.59×10-13) located at 6.95 Mb on chromosome 4. ALGA0022658 was in high linkage disequilibrium (r 2>0.5) with surrounding SNPs that span a 1.21 Mb interval. Within this 1.21 Mb region, we investigated ZFAT as a positional candidate gene. We re-sequenced cDNA of ZFAT in four pigs with different susceptibility phenotypes, and identified seven coding variants. We genotyped these seven variants in 287 unrelated pigs from 15 diverse breeds that were measured with ETEC F41 susceptibility phenotype. Five variants showed nominal significant association (P<0.05) with ETEC F41 susceptibility phenotype in International commercial pigs. This study provided refined region associated with susceptibility of pigs to ETEC F41 than that reported previously. Further works are needed to uncover the underlying causal mutation(s). 
27668277	 Ni is recognized as an element that is toxic to humans, acting as an allergen and a carcinogenic agent, and it is also toxic to plants. The toxicity of Ni has been understudied in microorganisms. The data presented here were obtained by submitting the model bacterium Escherichia coli K-12 to nickel stress. To identify expressed genes, RNA-Seq was performed. Bacteria were exposed to 50 µM NiCl2 during 10 min. Exposure to Ni lead to the deregulation of 57% of the E. coli transcripts. Further analysis using DAVID identified most affected biological pathways. The list of differentially expressed genes and physiological consequences of Ni stress are described in "Ni exposure impacts the pool of free  Fe and modifies DNA supercoiling via metal-induced oxidative stress in Escherichia coli K-12" (M. Gault, G. Effantin, A. Rodrigue, 2016) [1]. 
27435271	 YbaO is an uncharacterized AsnC-family transcription factor of Escherichia coli.  In both Salmonella enterica and Pantoea ananatis, YbaO homologues were identified to regulate the adjacent gene encoding cysteine desulfhydrase for detoxification  of cysteine. Using the genomic SELEX (systematic evolution of ligands by exponential enrichment) screening system, we identified the yhaOM operon, located far from the ybaO gene on the E. coli genome, as a single regulatory target of YbaO. In both gel shift assay in vitro and reporter and Northern blot assays in vivo, YbaO was found to regulate the yhaOM promoter. The growth of mutants lacking either ybaO or its targets yhaOM was delayed in the presence of cysteine, indicating involvement of these genes in cysteine detoxification. In the major pathway of cysteine degradation, hydrogen sulfide is produced in wild-type E. coli, but its production was not observed in each of the ybaO, yhaO and yhaM mutants. The yhaOM promoter was activated in the presence of cysteine, implying the role of cysteine in activation of YbaO. Taken together, we propose that YbaO  is the cysteine-sensing transcriptional activator of the yhaOM operon, which is involved in the detoxification of cysteine. We then propose the naming of ybaO as decR (regulator of detoxification of cysteine). 
27578754	 The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were  mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state  formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state  formation.IMPORTANCE: Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by  HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment. 
27552843	 BACKGROUND: Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs  and to identify enzymes acting on hydrolysis of polysaccharides or glycans. RESULTS: This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database. Probes were designed using two softwares, and microarrays were directly synthesized using the in situ ink-jet technology. CAZyChip specificity and reproducibility was validated by hybridization of known  GHs RNA extracted from recombinant E. coli strains, which were previously identified by a functional metagenomic approach. The GHs arsenal was also studied in bioprocess conditions using rumen derived microbiota. CONCLUSIONS: The CAZyChip appears to be a user friendly tool for profiling the expression of a large variety of GHs. It can be used to study temporal variations of functional diversity, thereby facilitating the identification of new efficient candidates for enzymatic conversions from various ecosystems. 
26908653	 Flexible adaptation to environmental stress is vital for bacteria. An energy-efficient post-transcriptional stress response mechanism in Escherichia coli is governed by the toxin MazF. After stress-induced activation the endoribonuclease MazF processes a distinct subset of transcripts as well as the 16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes' are specific for the MazF-processed mRNAs, the translational program is changed.  To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation coupled with RNA-seq analysis) and analyzed alterations introduced into the transcriptome and translatome after mazF overexpression. Unexpectedly, our results reveal that the corresponding protein products are involved in all cellular processes and do not particularly contribute to the general stress response. Moreover, our findings suggest that translational reprogramming serves  as a fast-track reaction to harsh stress and highlight the so far underestimated  significance of selective translation as a global regulatory mechanism in gene expression. Considering the reported implication of toxin-antitoxin (TA) systems  in persistence, our results indicate that MazF acts as a prime effector during harsh stress that potentially introduces translational heterogeneity within a bacterial population thereby stimulating persister cell formation. 
27375130	 The biology of nickel has been widely studied in mammals because of its carcinogenic properties, whereas few studies have been performed in microorganisms. In the present work, changes accompanying stress caused by nickel were evaluated at the cellular level using RNA-Seq in Escherichia coli K-12. Interestingly, a very large number of genes were found to be deregulated by Ni stress. Iron and oxidative stress homeostasis maintenance were among the most highly enriched functional categories, and genes involved in periplasmic copper efflux were among the most highly upregulated. These results suggest that the deregulation of Fe and Cu homeostatic genes is caused by a release of free Cu and Fe ions in the cell which in turn activate the Cu and Fe homeostatic systems. The content of Cu was not significantly affected upon the addition of Ni to the growth medium, nor were the Cus and CopA Cu-efflux systems important for the survival of bacteria under Ni stress In contrast the addition of Ni slightly decreased the amount of cellular Fe and activated the transcription of Fur regulated genes in a Fur-dependent manner. Cu or Fe imbalance together with oxidative stress might affect the structure of DNA. Further experiments revealed  that Ni alters the state of DNA folding by causing a relaxed conformation, a phenomenon that is reversible by addition of the antioxidant Tiron or the Fe chelator Dip. The Tiron-reversible DNA relaxation was also observed for Fe and to a lesser extent with Cu but not with Co. DNA supercoiling is well recognized as an integral aspect of gene regulation. Moreover our results show that Ni modifies the expression of several nucleoid-associated proteins (NAPs), important agents of DNA topology and global gene regulation. This is the first report describing the impact of metal-induced oxidative on global regulatory networks. 
27246569	 The iprA gene (formerly known as yaiV or STM0374) is located in a two-gene operon in the Salmonella enterica serovar Typhimurium genome and is associated with altered expression during spaceflight and rotating-wall-vessel culture conditions that increase virulence. However, iprA is uncharacterized in the literature. In this report, we present the first targeted characterization of this gene, which revealed that iprA is highly conserved across Enterobacteriaceae We found that S  Typhimurium, Escherichia coli, and Enterobacter cloacae ΔiprA mutant strains display a multi-log-fold increase in oxidative stress resistance that is complemented using a plasmid-borne wild-type (WT) copy of the S Typhimurium iprA  gene. This observation was also associated with increased catalase activity, increased S Typhimurium survival in macrophages, and partial dependence on the katE gene and full dependence on the rpoS gene. Our results indicate that IprA protein activity is sensitive to deletion of the N- and C-terminal 10 amino acids, while a region that includes amino acids 56 to 80 is dispensable for activity. RNA sequencing (RNA-Seq) analysis revealed several genes altered in expression in the S Typhimurium ΔiprA mutant strain compared to the WT, including those involved in fimbria formation, spvABCD-mediated virulence, ethanolamine utilization, the phosphotransferase system (PTS) transport, and flagellin phase switching from FlgB to FliC (likely a stochastic event) and several genes of hypothetical or putative function.IMPORTANCE: Overall, this work reveals that the conserved iprA gene measurably influences bacterial biology and highlights the pool of currently uncharacterized genes that are conserved across bacterial genomes. These genes represent potentially useful targets for bacterial engineering, vaccine design, and other possible applications. 
27190161	 Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional control of gene expression. To improve their detection by conventional microarrays, we designed a custom microarray containing a group of probes targeting known and some putative Escherichia coli sRNAs. To assess its potential in detection of sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells exponentially grown in rich (Luria-Bertani) and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably strong and statistically significant signals corresponding to nearly all sRNAs annotated in the EcoCyc database. Besides differential expression of two sRNAs (GcvB and RydB), expression of other sRNAs was less affected by the composition of the growth media. Other examples of the differentially expressed sRNAs were revealed by comparing gene expression of the wild-type strain and its isogenic mutant lacking functional poly(A) polymerase I (pcnB). Further, northern blot analysis was employed to validate these data and to assess the existence of new putative sRNAs. Our results suggest that the use of custom microarrays with improved capacities for detection of sRNAs can offer an attractive opportunity for efficient gene expression profiling of sRNAs and their target mRNAs at the whole transcriptome level. 
27112147	 Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening was performed for identification of the binding site of YbiH, an as yet uncharacterized TetR-family transcription factor, on the Escherichia coli genome. YbiH was found to be a unique single-target regulator that binds in vitro within  the intergenic spacer located between the divergently transcribed ybiH-ybhGFSR and rhlE operons. YbhG is an inner membrane protein and YbhFSR forms a membrane-associated ATP-binding cassette (ABC) transporter while RhlE is a ribosome-associated RNA helicase. Gel shift assay and DNase footprinting analyses indicated one clear binding site of YbiH, including a complete palindromic sequence of AATTAGTT-AACTAATT. An in vivo reporter assay indicated repression of  the ybiH operon and activation of the rhlE operon by YbiH. After phenotype microarray screening, YbiH was indicated to confer resistance to chloramphenicol  and cefazoline (a first-generation cephalosporin). A systematic survey of the participation of each of the predicted YbiH-regulated genes in the antibiotic sensitivity indicated involvement of the YbhFSR ABC-type transporter in the sensitivity to cefoperazone (a third-generation cephalosporin) and of the membrane protein YbhG in the control of sensitivity to chloramphenicol. Taken together with the growth test in the presence of these two antibiotics and in vitro transcription assay, it was concluded that the hitherto uncharacterized YbiH regulates transcription of both the bidirectional transcription units, the ybiH-ybhGFSR operon and the rhlE gene, which altogether are involved in the control of sensitivity to cefoperazone and chloramphenicol. We thus propose to rename YbiH as CecR (regulator of cefoperazone and chloramphenicol sensitivity). 
27349222	 BACKGROUND: Escherichia coli K-12 strains contain DNA cytosine methyltransferase  (Dcm), which generates 5-methylcytosine at 5'CCWGG3' sites. Although the role of  5-methylcytosine in eukaryotic gene expression is relatively well described, the  role of 5-methylcytosine in bacterial gene expression is largely unknown. RESULTS: To identify genes that are controlled by 5-methylcytosine in E. coli, we compared the transcriptomes of cells grown in the absence and presence of the DNA methylation inhibitor 5-azacytidine. We observed expression changes for 63 genes. The majority of the gene expression changes occurred at early stationary phase and were up-regulations. To identify gene expression changes due to a loss of DNA methylation, we compared the expression of selected genes in a wild-type and dcm  knockout strain via reverse transcription quantitative PCR. CONCLUSIONS: Our data indicate that 5-azacytidine can influence gene expression by at least two distinct mechanisms: DNA methylation loss and a mechanism that is independent of DNA methylation loss. In addition, we have identified new targets  of 5-methylcytosine-mediated regulation of gene expression. In summary, our data  indicate that 5-azacytidine impacts the composition of the bacterial transcriptome, and the primary effect is increased gene expression at early stationary phase. 
27295952	 BACKGROUND: Microarray is an efficient apparatus to interrogate the whole transcriptome of species. Microarray can be designed according to annotated gene  sets, but the resulted microarrays cannot be used to identify novel transcripts and this design method is not applicable to unannotated species. Alternatively, a whole-genome tiling microarray can be designed using only genomic sequences without gene annotations, and it can be used to detect novel RNA transcripts as well as known genes. The difficulty with tiling microarray design lies in the tradeoff between probe-specificity and coverage of the genome. Sequence comparison methods based on BLAST or similar software are commonly employed in microarray design, but they cannot precisely determine the subtle thermodynamic competition between probe targets and partially matched probe nontargets during hybridizations. FINDINGS: Using the whole-genome thermodynamic analysis software PICKY to design  tiling microarrays, we can achieve maximum whole-genome coverage allowable under  the thermodynamic constraints of each target genome. The resulted tiling microarrays are thermodynamically optimal in the sense that all selected probes share the same melting temperature separation range between their targets and closest nontargets, and no additional probes can be added without violating the specificity of the microarray to the target genome. CONCLUSIONS: This new design method was used to create two whole-genome tiling microarrays for Escherichia coli MG1655 and Agrobacterium tumefaciens C58 and the experiment results validated the design. 
27195891	 Genome-wide measurements of transcriptional activity in bacteria indicate that the transcription of successive genes is strongly correlated beyond the scale of  operons. Here, we analyze hundreds of bacterial genomes to identify supra-operonic segments of genes that are proximal in a large number of genomes.  We show that these synteny segments correspond to genomic units of strong transcriptional co-expression. Structurally, the segments contain operons with specific relative orientations (co-directional or divergent) and nucleoid-associated proteins are found to bind at their boundaries. Functionally, operons inside a same segment are highly co-expressed even in the apparent absence of regulatory factors at their promoter regions. Remote operons along DNA can also be co-expressed if their corresponding segments share a transcriptional  or sigma factor, without requiring these factors to bind directly to the promoters of the operons. As evidence that these results apply across the bacterial kingdom, we demonstrate them both in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The underlying process that we propose involves only RNA-polymerases and DNA: it implies that the transcription of an operon mechanically enhances the transcription of adjacent operons. In support of a primary role of this regulation by facilitated co-transcription, we show that the transcription en bloc of successive operons as a result of transcriptional read-through is strongly and specifically enhanced in synteny segments. Finally, our analysis indicates that facilitated co-transcription may be evolutionary primitive and may apply beyond bacteria. 
26837900	 Citrobacter rodentium (Cr) is a mouse pathogen that mimics many aspects of enteropathogenic Escherichia coli infections including producing attaching and effacing (A/E) lesions. Host-adapted (HA) Cr cells that are shed at the peak of infection have been reported to be hyper-infective. The exact mechanism underlying this phenomenon has remained elusive since the pathogen loses its HA 'status' immediately upon subculturing in laboratory media. We sequenced the entire transcriptome of Cr directly from the feces of infected mice and analyzed  the gene expression pattern. We observed that the entire transcriptional machinery as well as several transcriptional regulators to be differentially expressed when compared with the transcriptome of cells grown on laboratory media. Major adhesion and effector genes, tir and eae, were highly expressed in HA along with many genes located on all five loci of enterocyte effacement regions (LEE 1-5). Notable absent among the HA expressed genes were 19 fimbrial operons and non-fimbrial adhesions and several non-LEE encoded effectors. These results demonstrate that host-adapted Cr has a unique transcriptome that is associated with increased host transmission. 
27104834	 Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers-called P12-31-is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies. 
26971973	 Bioconversion processes of organic acid or acid hydrolysis of raw material for microbial metabolism often suffer limitations as a result of microbial sensitivity in low-pH conditions. We adopted a three-step method called RAndom Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the components of global regulator Sigma D factor (RpoD) of Escherichia coli to improve its acid tolerance. The best strain Mutant VII was identified from random mutagenesis libraries based on the growth performance, which exhibited much higher growth rate than the control (0.22h(-1) vs. 0.15h(-1)) at pH as low as 3.17. Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks. Our analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results demonstrated that RpoD acts as global regulator in the growth phase of E. coli and consequently improves acid tolerances. 
27572646	 Bacteria pose unique challenges for genome-wide association studies because of strong structuring into distinct strains and substantial linkage disequilibrium across the genome(1,2). Although methods developed for human studies can correct  for strain structure(3,4), this risks considerable loss-of-power because genetic  differences between strains often contribute substantial phenotypic variability(5). Here, we propose a new method that captures lineage-level associations even when locus-specific associations cannot be fine-mapped. We demonstrate its ability to detect genes and genetic variants underlying resistance to 17 antimicrobials in 3,144 isolates from four taxonomically diverse clonal and recombining bacteria: Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. Strong selection, recombination and penetrance confer high power to recover known antimicrobial resistance mechanisms and reveal a candidate association between the outer membrane porin nmpC and cefazolin resistance in E. coli. Hence, our method pinpoints locus-specific effects where possible and boosts power by detecting lineage-level differences when fine-mapping is intractable. 
27006690	 BACKGROUND: The conversion of biomass-derived sugars via enzymatic hydrolysis for biofuel production is a challenge. Therefore, the search for microorganisms and key enzymes that increase the efficiency of the saccharification of cellulosic substrates remains an important and high-priority area of study. Trichoderma harzianum is an important fungus known for producing high levels of cellulolytic  enzymes that can be used for cellulosic ethanol production. In this context, β-glucosidases, which act synergistically with cellobiohydrolases and endo-β-1,4-glucanases in the saccharification process, are potential biocatalysts for the conversion of plant biomass to free glucose residues. RESULTS: In the present study, we used RNA-Seq and genomic data to identify the major β-glucosidase expressed by T. harzianum under biomass degradation conditions. We mapped and quantified the expression of all of the β-glucosidases  from glycoside hydrolase families 1 and 3, and we identified the enzyme with the  highest expression under these conditions. The target gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein (rThBgl) was purified with high yields. rThBgl was characterized using a comprehensive set of biochemical, spectroscopic, and hydrodynamic techniques. Finally, we determined the crystallographic structure of the recombinant protein  at a resolution of 2.6 Å. CONCLUSIONS: Using a rational approach, we investigated the biochemical characteristics and determined the three-dimensional protein structure of a β-glucosidase that is highly expressed by T. harzianum under biomass degradation  conditions. The methodology described in this manuscript will be useful for the bio-prospection of key enzymes, including cellulases and other accessory enzymes, for the development and/or improvement of enzymatic cocktails designed to produce ethanol from plant biomass. 
26843427	 Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated  by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of  a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, 'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/). 
26959367	 Super-shedder cattle are a major disseminator of E. coli O157:H7 into the environment, and the terminal rectum has been proposed as the primary E. coli O157:H7 colonization site. This study aimed to identify host factors that are associated with the super-shedding process by comparing transcriptomic profiles in rectal tissue collected from 5 super-shedder cattle and 4 non-shedder cattle using RNA-Seq. In total, 17,859 ± 354 genes and 399 ± 16 miRNAs were detected, and 11,773 genes were expressed in all animals. Fifty-eight differentially expressed (DE) genes (false discovery rate < 0.05) including 11 up-regulated and  47 down-regulated (log 2 (fold change) ranged from -5.5 to 4.2), and 2 up-regulated DE miRNAs (log 2 (fold change) = 2.1 and 2.5, respectively) were identified in super-shedders compared to non-shedders. Functional analysis of DE  genes revealed that 31 down-regulated genes were potentially associated with reduced innate and adaptive immune functions in super-shedders, including 13 lymphocytes membrane receptors, 3 transcription factors and 5 cytokines, suggesting the decreased key host immune functions in the rectal tissue of super-shedders, including decreased quantity and migration of immune cells such as lymphocytes, neutrophils and dendritic cells. The up-regulation of bta-miR-29d-3p and the down regulation of its predicted target gene, regulator of G-protein signaling 13, suggested a potential regulatory role of this miRNA in decreased migration of lymphocytes in super-shedders. Based on these findings, the rectal tissue of super-shedders may inherently exhibit less effective innate  and adaptive immune protection. Further study is required to confirm if such effect on host immunity is due to the nature of the host itself or due to actions mediated by E. coli O157:H7. 
26925056	 LexA is a well-established transcriptional repressor of SOS genes induced by DNA  damage in Escherichia coli and other bacterial species. However, LexA in the cyanobacterium Synechocystis sp. PCC 6803 has been suggested not to be involved in SOS response. In this study, we performed RNA-seq analysis of the wild-type strain and the lexA-disrupted mutant to obtain the comprehensive view of LexA-regulated genes in Synechocystis. Disruption of lexA positively or negatively affected expression of genes related to various cellular functions such as phototactic motility, accumulation of the major compatible solute glucosylglycerol and subunits of bidirectional hydrogenase, photosystem I, and phycobilisome complexes. We also observed increase in the expression level of genes related to iron and manganese uptake in the mutant at the later stage of cultivation. However, none of the genes related to DNA metabolism were affected by disruption of lexA. DNA gel mobility shift assay using the recombinant LexA protein suggested that LexA binds to the upstream region of pilA7, pilA9, ggpS, and slr1670 to directly regulate their expression, but changes in the expression  level of photosystem I genes by disruption of lexA is likely a secondary effect. 
26740583	 Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based  approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNA(Pro14) D-loop  or within the tRNA(Lys43) anticodon. This highly selective target discrimination  occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth. 
26852325	 BACKGROUND: Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be  responsible for DHA production in marine bacteria. RESULTS: Five pfa genes from strain 34H were confirmed to be responsible for DHA  formation by heterogeneous expression in Escherichia coli. The complexity of fatty acid profile of this strain was revealed by GC and GC-MS. Treatment of cells with cerulenin resulted in significantly reduced level of C16 monounsaturated fatty acid (C16:1(Δ9t), C16:1(Δ7)). In contrast, the amount of saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and 3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA sequencing (RNA-Seq) revealed the altered gene expression pattern when C. psychrerythraea cells were treated with cerulenin. Genes involved in polyketide synthase pathway and fatty acid biosynthesis pathway were not obviously affected  by cerulenin treatment. In contrast, several genes involved in fatty acid degradation or β-oxidation pathway were dramatically reduced at the transcriptional level. CONCLUSIONS: Genes responsible for DHA formation in C. psychrerythraea was first  cloned and characterized. We revealed the complexity of fatty acid profile in this DHA-producing strain. Cerulenin could substantially change the fatty acid composition by affecting the fatty acid degradation at transcriptional level. Acyl-CoA dehydrogenase gene family involved in the first step of β-oxidation pathway may be important to the selectivity of degraded fatty acids. In addition, inhibition of FabB protein by cerulenin may lead to the accumulation of malonyl-CoA, which is the substrate for DHA formation. 
26541162	 Tuberculosis (TB) remains to be a major global health problem, with about 9 million new cases and 1.4 million deaths in 2011. For the control of tuberculosis as well as other infectious diseases, WHO recommended "ASSURED" (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to the end user) diagnostic tools that can easily be maintained and used in developing countries. Aptamers are promising tools for developing point-of-care diagnostic assays for TB. In this study, ssDNA aptamers that recognize Mycobacterium tuberculosis H37Ra were selected by systematic evolution  of ligands by exponential enrichment (SELEX). For this purpose, two different selection protocols, ultrafiltration and centrifugation, were applied. A total of 21 TB specific aptamers were selected. These aptamers exhibited "G-rich" regions  on the 3' terminus of the aptamers, including a motif of "TGGGG," "GTGG," or "CTGG." Binding capability of selected aptamers were investigated by quantitative PCR and Mtb36 DNA aptamer was found the most specific aptamer to M. tuberculosis  H37Ra. The dissociation constant (K d) of Mtb36 aptamer was calculated as 5.09 ± 1.43 nM in 95% confidence interval. Relative binding ratio of Mtb36 aptamer to M. tuberculosis H37Ra over Mycobacterium bovis and Escherichia coli was also determined about 4 times and 70 times more, respectively. Mtb36 aptamer  is highly selective for M. tuberculosis, and it can be used in an aptamer-based biosensor for the detection of M. tuberculosis. 
26472673	 The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was elucidated through global transcriptomic analysis. Around 375 genes showed difference of more than 2-fold in 3-HP-treated samples. Further analysis revealed that the toxicity effect of 3-HP was due to the cation and anion components of this acid and some effects-specific to 3-HP. Genes related to the oxidative stress, DNA protection, and repair were upregulated in treated cells due to the lowered cytoplasmic pH caused by accumulated cations. 3-HP-treated E. coli used the arginine acid tolerance mechanism to increase the cytoplasmic pH. Additionally, the anion effects were manifested as imbalance in the osmotic pressure. Analysis of top ten highly upregulated genes suggests the formation of  3-hydroxypropionaldehyde under 3-HP stress. The transcriptomic analysis shed light on the global genetic reprogramming due to 3-HP stress and suggests strategies for increasing the tolerance of E. coli toward 3-HP. 
26635367	 The Rcs phosphorelay is a two-component signal transduction system that is induced by cell envelope stress. RcsB, the response regulator of this signaling system, is a pleiotropic transcription regulator, which is involved in the control of various stress responses, cell division, motility, and biofilm formation. RcsB regulates transcription either as a homodimer or together with auxiliary regulators, such as RcsA, BglJ, and GadE in Escherichia coli. In this study, we show that RcsB in addition forms heterodimers with MatA (also known as  EcpR) and with DctR. Our data suggest that the MatA-dependent transcription regulation is mediated by the MatA-RcsB heterodimer and is independent of RcsB phosphorylation. Furthermore, we analyzed the relevance of amino acid residues of the active quintet of conserved residues, and of surface-exposed residues for activity of RcsB. The data suggest that the activity of the phosphorylation-dependent dimers, such as RcsA-RcsB and RcsB-RcsB, is affected by mutation of residues in the vicinity of the phosphorylation site, suggesting that a phosphorylation-induced structural change modulates their activity. In contrast, the phosphorylation-independent heterodimers BglJ-RcsB and MatA-RcsB are affected by only very few mutations. Heterodimerization of RcsB with various  auxiliary regulators and their differential dependence on phosphorylation add an  additional level of control to the Rcs system that is operating at the output level. 
26733341	 BACKGROUND: Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. RESULTS: To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 μmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 μmol m(-2) s(-1)). When grown under continuous light at 920 μmol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the  light-stress treatments examined. CONCLUSIONS: We identified genes encoding CrtE and CrtB in E. gracilis and found  that their protein products catalyze the early steps of carotenoid biosynthesis.  Further, we found that the response of the carotenoid biosynthetic pathway to light stress in E. gracilis is controlled, at least in part, by the level of crtB transcription. This is the first functional analysis of crtE and crtB in Euglena. 
26686283	 Green leaf volatiles (GLVs) are C6-aliphatic aldehydes/alcohols/acetates, and biosynthesized from the central precursor fatty acid 13-hydroperoxides by 13-hydroperoxide lyases (HPLs) in various plant species. While GLVs have been implicated as defense compounds in plants, GLVs give characteristic grassy note to a bouquet of aroma in green tea, which is manufactured from young leaves of Camellia sinensis. Here we identify three HPL-related genes from C. sinensis via  RNA-Sequencing (RNA-Seq) in silico, and functionally characterized a candidate gene, CYP74B24, as a gene encoding tea HPL. Recombinant CYP74B24 protein heterologously expressed in Escherichia coli specifically produced (Z)-3-hexenal  from 13-HPOT with the optimal pH 6.0 in vitro. CYP74B24 gene was expressed throughout the aerial organs in a rather constitutive manner and further induced  by mechanical wounding. Constitutive expression of CYP74B24 gene in intact tea leaves might account for low but substantial and constitutive formation of a subset of GLVs, some of which are stored as glycosides. Our results not only provide novel insights into the biological roles that GLVs play in tea plants, but also serve as basis for the improvement of aroma quality in tea manufacturing processes. 
26175046	 In order to gain deeper insight into the functions and dynamics of RNA in cells,  the development of methods for imaging multiple RNAs simultaneously is of paramount importance. Here, we describe a modular approach to image RNA in living cells using an RNA aptamer that binds to dinitroaniline, an efficient general contact quencher. Dinitroaniline quenches the fluorescence of different fluorophores when directly conjugated to them via ethylene glycol linkers by forming a non-fluorescent intramolecular complex. Since the binding of the RNA aptamer to the quencher destroys the fluorophore-quencher complex, fluorescence increases dramatically upon binding. Using this principle, a series of fluorophores were turned into fluorescent turn-on probes by conjugating them to dinitroaniline. These probes ranged from fluorescein-dinitroaniline (green) to TexasRed-dinitroaniline (red) spanning across the visible spectrum. The dinitroaniline-binding aptamer (DNB) was generated by in vitro selection, and was found to bind all probes, leading to fluorescence increase in vitro and in living cells. When expressed in E. coli, the DNB aptamer could be labelled and visualized with different-coloured fluorophores and therefore it can be used as a genetically encoded tag to image target RNAs. Furthermore, combining contact-quenched fluorogenic probes with orthogonal DNB (the quencher-binding RNA aptamer) and SRB-2 aptamers (a fluorophore-binding RNA aptamer) allowed dual-colour imaging of two different fluorescence-enhancing RNA tags in living cells, opening new avenues for studying RNA co-localization and trafficking. 
26438818	 Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation,  and mRNA stability by binding to the 5' untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5' fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the  pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp  translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this  is the first example in which prior mRNA processing is required for CsrA-mediated regulation.IMPORTANCE: CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed  by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity. 
26573804	 Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia coli cells that were highly fluorescent due to the expression of RNA aptamers that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two  different aptamers, named ZT-26 and ZT-324, were identified by this method and compared to the fluorescence-signaling properties of Spinach, a previously reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence  over Spinach only in vitro. However, aptamer ZT-324 is 36% brighter than Spinach  when expressed in E. coli. The FACS-based selection strategy presented here is attractive for deriving fluorescent RNA aptamers that function in cells as it directly selects for cells with a high level of fluorescence due to the expression of the RNA aptamer. 
26572487	 BACKGROUND: Resistin (Retn) is a cytokine which has a controversial physiological role regarding its involvement with obesity and type II diabetes mellitus. Recently, murine Retn was found to be a possibly potential regulator of hematopoiesis in mice shown in the screening results of a set of gene chips which mapped the expression level of murine genes during regeneration of impaired bone  marrow (BM) by 5-fluorouracil. RESULTS: Recombinant mice Retn was expressed in Escherichia coli and purified using ion exchange chromatography. Totally 11.4 mg rmRetn was obtained from 500 ml culture with endotoxin level less than 1.0 EU/ug. The purity of recombinant murine Resistin reached to at least 97.6% via SDS-PAGE analysis and HPLC. The protein possessed chemotaxis effects in the mouse aortic endothelial cells in vitro in transwell analysis. In vitro, rmRetn could up regulate the CFU number of mice BM and after rmRetn was administered, the cell number of murine bone marrow  was significantly increased in vivo after chemotherapy. Finally, rmRetn was found able to protect mice from the chemotoxicity of 5-fluorouracil. CONCLUSIONS: The discovery demonstrated a new function of murine Retn and suggested that it could potentially accelerate bone marrow regeneration post chemotherapy. 
26563610	 Engineering of efflux pumps is a promising way to improve host's tolerance to biofuels such as medium-chain alcohols (CmOHs); however, this strategy is restricted by poor understanding of the efflux pumps engaged in extrusion of solvents. In this study, several Escherichia coli mutants of multidrug transporters were evaluated for isoprenol tolerance. Susceptible phenotypes were  observed in the mutants with individual deletion of six transporters, AcrD, EmrAB, MacAB, MdtBC, MdtJI and YdiM, whereas inactivation of AcrAB transporter resulted in an improved tolerance to isoprenol and other CmOHs. AcrAB is the major transporter forming tripartite transperiplasmic complex with outer membrane channel TolC for direct extrusion of toxic molecules in E. coli. The AcrAB inactivation enables to enhance TolC availability for the multidrug transporters  associated with extrusion of CmOHs and increase the tolerance to CmOHs including  isoprenol. It is assumed that outer membrane channel TolC plays an important role in extrusion of isoprenol and other CmOHs. 
26332955	 The two-component system (TCS) is a sophisticated bacterial signal transduction system for regulation of genome transcription in response to environmental conditions. The EnvZ-OmpR system is one of the well-characterized TCS of Escherichia coli, responding to changes in environmental osmolality. Regulation has largely focused on the differential expression of two porins, OmpF and OmpC,  which transport small molecules across the outer membrane. Recently, it has become apparent that OmpR serves a more global regulatory role and regulates additional targets. To identify the entire set of regulatory targets of OmpR, we  performed the genomic SELEX screening of OmpR-binding sites along the E. coli genome. As a result, more than 30 novel genes have been identified to be under the direct control of OmpR. One abundant group includes the genes encoding a variety of membrane-associated transporters that mediate uptake or efflux of small molecules, while another group encodes a set of transcription regulators, raising a concept that OmpR is poised to control a diverse set of responses by altering downstream transcriptional regulators. 
25998249	 Culture methods for the detection of indicator bacteria are currently used for detection of waterborne bacteria. The need for an increased range of analyzed bacteria coupled with the obtainment of rapid and early results justify the development of a DNA microarray for the identification of waterborne pathogens. This DNA microarray has 16 implanted probes with a median size of 147 bases, targeting 12 different parameters, including all mandatory indicator microorganisms, such as Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus, total and fecal coliforms and enterococci. The validation performed with DNA extracted from pure microbial cultures showed the suitability of the probes for detection of the target microorganism. To overcome  the high dilution of water samples it was included either a prior culture step of bacterial contaminants retained after filtering 100 ml of water, or a 10-fold increase in the volume of filtered water, that resulted in the increase of the detected bacteria. The analysis of complex environmental water samples using culture methods and the DNA microarray revealed that the latter detected the same parameters plus other bacteria tested only in the DNA microarray. The results show that this DNA microarray may be a useful tool for water microbiological surveillance. 
26427325	 Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability  of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins  is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin. 
26209663	 Cyanobacteria are photosynthetic cell factories that use solar energy to convert  CO2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to  identify neutral sites from which no transcripts are expressed. We characterized  the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the Pcpc560 or the Ptrc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher  than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with  the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria. 
26078123	 The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes  with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization  assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different  pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. 
26237225	 High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative methylation analysis using ARM-seq provides the first detailed, transcriptome-scale map of these modifications and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-seq demonstrates that tRNA fragments accurately recapitulate the m(1)A modification state for well-characterized yeast tRNAs and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state and may reveal new roles for tRNA fragments as biomarkers or signaling molecules. 
28348809	 Leucine-responsive regulatory protein (Lrp) is a transcriptional regulator for the genes involved in transport, biosynthesis and catabolism of amino acids in Escherichia coli. In order to identify the whole set of genes under the direct control of Lrp, we performed Genomic SELEX screening and identified a total of 314 Lrp-binding sites on the E. coli genome. As a result, the regulation target of Lrp was predicted to expand from the hitherto identified genes for amino acid  metabolism to a set of novel target genes for utilization of amino acids for protein synthesis, including tRNAs, aminoacyl-tRNA synthases and rRNAs. Northern  blot analysis indicated alteration of mRNA levels for at least some novel targets, including the aminoacyl-tRNA synthetase genes. Phenotype MicroArray of the lrp mutant indicated significant alteration in utilization of amino acids and peptides, whilst metabolome analysis showed variations in the concentration of amino acids in the lrp mutant. From these two datasets we realized a reverse correlation between amino acid levels and cell growth rate: fast-growing cells contain low-level amino acids, whilst a high level of amino acids exists in slow-growing cells. Taken together, we propose that Lrp is a global regulator of  transcription of a large number of the genes involved in not only amino acid transport and metabolism, but also amino acid utilization. 
26131907	 Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, "primer-dependent initiation," (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E.  coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory  mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5' ends (5' RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V.  cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´. 
26157798	 Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and  regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake  on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNA(fMet) and tRNA(Lys), we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device. 
25759326	 Mogrosides, the major bioactive components isolated from the fruits of Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as high-potency sweeteners and possess a variety of notable pharmacological activities. Mogrosides are synthesized from 2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase (CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However, the relevant genes have not been characterized to date. In this study, we report  successful identification of SgCbQ and UGT74AC1, which were previously predicted  via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis of the fruits of S. grosvenorii. SgCbQ was functionally characterized by expression in the lanosterol synthase-deficient yeast strain GIL77 and was found  to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously expressed in Escherichia coli as a His-tag protein and it showed specificity for  mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE by in vitro enzymatic activity assays. This study reports the identification of CbQ and glycosyltransferase from S. grosvenorii for the first time. The results also suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene  saponins. 
26020634	 Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized.  Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS  with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC).  Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera. 
25765651	 Identification of transcription units (TUs) encoded in a bacterial genome is essential to elucidation of transcriptional regulation of the organism. To gain a detailed understanding of the dynamically composed TU structures, we have used four strand-specific RNA-seq (ssRNA-seq) datasets collected under two experimental conditions to derive the genomic TU organization of Clostridium thermocellum using a machine-learning approach. Our method accurately predicted the genomic boundaries of individual TUs based on two sets of parameters measuring the RNA-seq expression patterns across the genome: expression-level continuity and variance. A total of 2590 distinct TUs are predicted based on the  four RNA-seq datasets. Among the predicted TUs, 44% have multiple genes. We assessed our prediction method on an independent set of RNA-seq data with longer  reads. The evaluation confirmed the high quality of the predicted TUs. Functional enrichment analyses on a selected subset of the predicted TUs revealed interesting biology. To demonstrate the generality of the prediction method, we have also applied the method to RNA-seq data collected on Escherichia coli and achieved high prediction accuracies. The TU prediction program named SeqTU is publicly available at https://code.google.com/p/seqtu/. We expect that the predicted TUs can serve as the baseline information for studying transcriptional  and post-transcriptional regulation in C. thermocellum and other bacteria. 
25666157	 The larvae of the common green bottle fly (Lucilia sericata) produce antibacterial secretions that have a therapeutic effect on chronic and nonhealing wounds. Recent developments in insect biotechnology have made it possible to use  these larvae as a source of novel anti-infectives. Here, we report the application of next-generation RNA sequencing (RNA-Seq) to characterize the transcriptomes of the larval glands, crop, and gut, which contribute to the synthesis of antimicrobial peptides (AMPs) and proteins secreted into wounds. Our data confirm that L. sericata larvae have adapted in order to colonize microbially contaminated habitats, such as carrion and necrotic wounds, and are protected against infection by a diverse spectrum of AMPs. L. sericata AMPs include not only lucifensin and lucimycin but also novel attacins, cecropins, diptericins, proline-rich peptides, and sarcotoxins. We identified 47 genes encoding putative AMPs and produced 23 as synthetic analogs, among which some displayed activities against a broad spectrum of microbial pathogens, including Pseudomonas aeruginosa, Proteus vulgaris, and Enterococcus faecalis. Against Escherichia coli (Gram negative) and Micrococcus luteus (Gram positive), we found mostly additive effects but also synergistic activity when selected AMPs were tested in combination. The AMPs that are easy to synthesize are currently being produced in bulk to allow their evaluation as novel anti-infectives that can be formulated in hydrogels to produce therapeutic wound dressings and adhesive bandages. 
25343512	 Escherichia coli spans a genetic continuum from enteric strains to several phylogenetically distinct, atypical lineages that are rare in humans, but more common in extra-intestinal environments. To investigate the link between gene regulation, phylogeny and diversification in this species, we analyzed global gene expression profiles of four strains representing distinct evolutionary lineages, including a well-studied laboratory strain, a typical commensal (enteric) strain and two environmental strains. RNA-Seq was employed to compare the whole transcriptomes of strains grown under batch, chemostat and starvation conditions. Highly differentially expressed genes showed a significantly lower nucleotide sequence identity compared with other genes, indicating that gene regulation and coding sequence conservation are directly connected. Overall, distances between the strains based on gene expression profiles were largely dependent on the culture condition and did not reflect phylogenetic relatedness.  Expression differences of commonly shared genes (all four strains) and E. coli core genes were consistently smaller between strains characterized by more similar primary habitats. For instance, environmental strains exhibited increased expression of stress defense genes under carbon-limited growth and entered a more pronounced survival-like phenotype during starvation compared with other strains, which stayed more alert for substrate scavenging and catabolism during no-growth  conditions. Since those environmental strains show similar genetic distance to each other and to the other two strains, these findings cannot be simply attributed to genetic relatedness but suggest physiological adaptations. Our study provides new insights into ecologically relevant gene-expression and underscores the role of (differential) gene regulation for the diversification of the model bacterial species. 
25568260	 YedVW is one of the uncharacterized two-component systems (TCSs) of Escherichia coli. In order to identify the regulation targets of YedVW, we performed genomic  SELEX (systematic evolution of ligands by exponential enrichment) screening using phosphorylated YedW and an E. coli DNA library, and identified YedW-binding sites within three intergenic spacers, yedW-hiuH, cyoA-ampG and cusR-cusC, along the E. coli genome. Using a reporter assay system, we found that transcription of hiuH,  encoding 5-hydroxyisourate hydrolase, was induced at high concentrations of either Cu(2+) or H₂O₂. Cu(2+)-dependent expression of hiuH was observed in the yedWV knockout mutant, but was reduced markedly in the cusRS-null mutant. However, H₂O₂-induced hiuH expression was observed in the cusRS-null mutant, but  not in the yedWV-null mutant. Gel mobility shift and DNase I footprinting analyses showed binding of both YedW and CusR to essentially the same sequence within the hiuH promoter region. Taken together, we concluded that YedVW and CusSR formed a unique cooperative TCS pair by recognizing and regulating the same targets, but under different environmental conditions - YedVW played a role in H₂O₂ response regulation, whilst CusSR played a role in Cu(2+) response regulation. 
25706641	 Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3  and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a  suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation. 
24856730	 Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of  multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by traditional phosphoramidite column-based approaches. Here, we describe a novel method to amplify oligos from microarray chips for direct use in MAGE to perturb thousands  of genomic sites simultaneously. We demonstrated the feasibility of large-scale mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for  a variety of applications in synthetic biology and metabolic engineering. 
25790494	 The binding site(s) on the Escherichia coli genome was determined for an uncharacterized AraC/XylS superfamily transcription factor YeaM by using the in vitro genomic SELEX screening system. The only one clear binding target of YeaM was found to locate in the spacer between the divergently transcribed yeaM and yeaN genes. After the phenotype microarray analysis, the major facilitator superfamily transporter YeaN was found to confer E. coli the resistance to 2-nitroimidazole, the antibacterial and antifungal antibiotic, suggesting that YeaN plays a role in 2-nitromidazole efflux. Purified YeaM bound to three sites within this yeaM-yeaN spacer region. Several lines of in vitro and in vivo evidence indicate that YeaM regulates transcription of both the yeaM gene itself  and the yeaNO operon. Taken together we propose to rename yeaN to nimT (nitroimidazole transporter) and yeaM to nimR (regulator of nimT). 
25841381	 Anthrax toxin excreted by Bacillus anthracis is the key causative agent of infectious anthrax disease. In the present study, we targeted the binding of PA to the ATR/TEM8 Von Willebrand factor type A (VWA) domain, which we cloned into Escherichia coli and purified to homogeneity under denaturing conditions. To develop an anthrax toxin inhibitor, we selected and identified short single strand RNA aptamers (approximately 30mer) consisting of different sequences of nucleic acids with a high binding affinity in the 100 nanomolar range against the recombinant ATR/TEM8 VWA domain using systematic evolution of ligands by exponential enrichment (SELEX). Five candidate aptamers were further characterized by several techniques including secondary structural analysis. The  inhibitor efficiency (IC50) of one of the aptamers toward anthrax toxin was approximately 5μM in macrophage RAW 264.7 cells, as determined from cytotoxicity  analysis by MTT assay. We believe that the candidate aptamers should be useful for blocking the binding of PA to its receptor in order to neutralize anthrax toxin. 
25266388	 While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we  describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional  start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA  candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification  of transcription start sites, promoters, and novel transcripts in E. coli and is  easily accessible, together with the cDNA coverage plots, in an online genome browser. 
25406449	 Sulfur makes up 1 % of the dry mass of bacteria, and it is an abundant element (0.1 %) on earth. Sulfur in the environment is, however, mostly in oxidized forms and inaccessible to living organisms. At present, the entire assimilation pathway of external sulfur to sulfur-containing biomolecules and its regulation in Escherichia coli remain poorly understood, except for the metabolic pathway of cysteine synthesis, the first-step metabolite of sulfur assembly. During the search for regulation targets of uncharacterized transcription factors by Genomic SELEX screening, we found that the hitherto uncharacterized YdcN regulates a set  of genes involved in the utilization of sulfur, including the generation of sulfate and its reduction, the synthesis of cysteine, the synthesis of enzymes containing Fe-S as cofactors, and the modification of tRNA with use of sulfur-containing substrates. Taking these findings together, we propose renaming YdcN as SutR (regulator of sulfur utilization). 
25456064	 Fusarium spp. cause many diseases in farming systems and can produce diverse mycotoxins that can easily impact humans and animals through the ingestion of food and feed. Among these mycotoxins, deoxynivalenol (DON) and nivalenol (NIV) are considered the most important hazards because they can rapidly diffuse into cells and block eukaryotic ribosomes, leading to inhibition of the translation system. Conversely, the effects of DON and NIV mycotoxins on bacteria remain unclear. We employed RNA-seq technology to obtain information regarding the biological responses of bacteria and putative bacterial mechanisms of resistance  to DON and NIV mycotoxins. Most differentially expressed genes down-regulated in  response to these mycotoxins were commonly involved in phenylalanine metabolism,  glyoxylate cycle, and cytochrome o ubiquinol oxidase systems. In addition, we generated an overall network of 1028 up-regulated genes to identify core genes under DON and NIV conditions. The results of our study provide a snapshot view of the transcriptome of Escherichia coli K-12 under DON and NIV conditions. Furthermore, the information provided herein will be useful for development of methods to detect DON and NIV. 
25496196	 BACKGROUND: Most semiconductor nanoparticles used in biomedical applications are  made of heavy metals and involve synthetic methods that require organic solvents  and high temperatures. This issue makes the development of water-soluble nanoparticles with lower toxicity a major topic of interest. In a previous work our group described a biomimetic method for the aqueous synthesis of CdTe-GSH Quantum Dots (QDs) using biomolecules present in cells as reducing and stabilizing agents. This protocol produces nanoparticles with good fluorescent properties and less toxicity than those synthesized by regular chemical methods.  Nevertheless, biomimetic CdTe-GSH nanoparticles still display some toxicity, so it is important to know in detail the effects of these semiconductor nanoparticles on cells, their levels of toxicity and the strategies that cells develop to overcome it. RESULTS: In this work, the response of E. coli exposed to different sized-CdTe-GSH QDs synthesized by a biomimetic protocol was evaluated through transcriptomic, biochemical, microbiological and genetic approaches. It was determined that: i) red QDs (5 nm) display higher toxicity than green (3 nm), ii) QDs mainly induce expression of genes involved with Cd+2 stress (zntA and znuA) and tellurium does not contribute significantly to QDs-mediated toxicity since cells incorporate low levels of Te, iii) red QDs also induce genes related to oxidative stress response and membrane proteins, iv) Cd2+ release is higher in red QDs, and v) QDs render the cells more sensitive to polymyxin B. CONCLUSION: Based on the results obtained in this work, a general model of CdTe-GSH QDs toxicity in E. coli is proposed. Results indicate that bacterial toxicity of QDs is mainly associated with cadmium release, oxidative stress and loss of membrane integrity. The higher toxicity of red QDs is most probably due to higher cadmium content and release from the nanoparticle as compared to green  QDs. Moreover, QDs-treated cells become more sensitive to polymyxin B making these biomimetic QDs candidates for adjuvant therapies against bacterial infections. 
25464340	 Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify  the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions  to reduce arsenate to arsenite in the outer cell layer of the root, facilitating  efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1  single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing  food such as rice. 
25185503	 Peptidoglycan is a highly complex and essential macromolecule of bacterial outer  cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of  this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification. 
25364902	 The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P) does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro) and/or PPG (Pro-Pro-Gly)-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of  S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects  in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the  proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids), only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution. 
25230163	 BACKGROUND: Perturbations of the intestinal microbiome, termed dysbiosis, are linked to intestinal inflammation. Isolation of adherent-invasive Escherichia coli (AIEC) from intestines of patients with Crohn's disease (CD), dogs with granulomatous colitis, and mice with acute ileitis suggests these bacteria share  pathoadaptive virulence factors that promote inflammation. METHODS: To identify genes associated with AIEC, we sequenced the genomes of phylogenetically diverse AIEC strains isolated from people with CD (4), dogs with granulomatous colitis (2), and mice with ileitis (2) and 1 non-AIEC strain from CD ileum and compared them with 38 genome sequences of E. coli and Shigella. We then determined the prevalence of AIEC-associated genes in 49 E. coli strains from patients with CD and controls and correlated genotype with invasion of intestinal epithelial cells, persistence within macrophages, AIEC pathotype, and  growth in standardized conditions. RESULTS: Genes encoding propanediol utilization (pdu operon) and iron acquisition (yersiniabactin, chu operon) were overrepresented in AIEC relative to nonpathogenic E. coli. PduC (propanediol dehydratase) was enriched in CD-derived  AIEC, correlated with increased cellular invasion, and persistence in vitro and was increasingly expressed in fucose-containing media. Growth of AIEC required iron, and the presence of chuA (heme acquisition) correlated with persistence in  macrophages. CD-associated AIEC with lpfA 154 (long polar fimbriae) demonstrated  increased invasion of epithelial cells and translocation across M cells. CONCLUSIONS: Our findings provide novel insights into the genetic basis of the AIEC pathotype, supporting the concept that AIEC are equipped to exploit and promote intestinal inflammation and reveal potential targets for intervention against AIEC and inflammation-associated dysbiosis. 
25234108	 The availability of a large number of sequenced bacterial genomes facilitates in-depth studies about why genes (operons) in a bacterial genome are globally organized the way they are. We have previously discovered that (the relative) transcription- activation frequencies among different biological pathways encoded in a genome have a dominating role in the global arrangement of operons. One complicating factor in such a study is that some operons may be involved in multiple pathways with different activation frequencies. A quantitative model has been developed that captures this information, which tends to be minimized by the current global arrangement of operons in a bacterial (and archaeal) genome compared to possible alternative arrangements. A study is carried out here using  this model on a collection of 52 closely related E. coli genomes, which revealed  interesting new insights about how bacterial genomes evolve to optimally adapt to their environments through adjusting the (relative) genomic locations of the encoding operons of biological pathways once their utilization and hence transcription activation frequencies change, to maintain the above energy-efficiency property. More specifically we observed that it is the frequencies of the transcription activation of pathways relative to those of the  other encoded pathways in an organism as well as the variation in the activation  frequencies of a specific pathway across the related genomes that play a key role in the observed commonalities and differences in the genomic organizations of genes (and operons) encoding specific pathways across different genomes. 
25128614	 The mechanism of heat shock response of Escherichia coli can be explored to program novel biological functions. In this study, the strongest heat shock promoters were identified by microarray experiments conducted at different temperatures (37°C and 45°C, 5min). The promoters of the genes ibpA, dnaK and fxsA were selected and validated by RT-qPCR. These promoters were used to construct and characterize stress probes using green fluorescence protein (GFP).  Cellular stress levels were evaluated in experiments conducted at different shock temperatures during several exposure times. It was concluded that the strength of the promoter is not the only relevant factor in the construction of an efficient  stress probe. Furthermore, it was found to be crucial to test and optimize the ribosome binding site (RBS) in order to obtain translational efficiency that balances the transcription levels previously verified by microarrays and RT-qPCR. These heat shock promoters can be used to trigger in situ gene expression of newly constructed biosynthetic pathways. 
25063650	 Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased  furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This  increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural. 
24771245	 An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses  a partially cyclized pentose phosphate pathway leading to an increased NADPH per  glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation.  DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level  in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmolMHB  h(-1)  gcdw (-1) ) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h(-1)  gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1)  gcdw (-1) ) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference  strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of  NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol dehydrogenase. The differences in respiration rates can explain the suggested higher ArcAB activity in the reference strain. 
24852346	 Full-length cDNA encoding two leptin sequences (tLepA and tLepB) and one leptin receptor sequence (tLepR) were identified in tilapia (Oreochromis niloticus). The full-length cDNA of tLepR was 3423bp, encoding a protein of 1140 amino acid (aa)  which contained all functionally important domains conserved among vertebrate leptin receptors. The cDNAs of tLepA and tLepB were 486bp and 459bp in length, encoding proteins of 161 aa and 152 aa, respectively. Modeling the three-dimensional structures of tLepA and tLepB predicted strong conservation of  tertiary structure with that of human leptin, comprised of four helixes. Using synteny, the tLeps were found near common genes, such as IMPDH1 and LLRC4. The cDNA for tLepA and tLepB was cloned and synthetic cDNA optimized for expression in Escherichia coli was prepared according to the cloned sequence. The tLepA- and tLepB-expressing plasmids were transformed into E. coli and expressed as recombinant proteins upon induction with nalidixic acid, found almost entirely in insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and purified to homogeneity by anion-exchange chromatography. In the case of tLepA, the fraction eluted contained a mixture of monomers and dimers. The purified tLepA and tLepB monomers and tLepA dimer showed a single band of ∼15kDa on an SDS-polyacrylamide gel in the presence of reducing agent, whereas the tLepA dimer showed one band of ∼30kDa in the absence of reducing agent, indicating its formation by S-S bonds. The three tLeps were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor (hLepR), but their activity was four orders of magnitude lower than that of mammalian leptin. Furthermore, the three tLeps were biologically active in promoting STAT-LUC activation in COS7 cells transfected with the identified tLepR but not in cells transfected with hLepR. tLepA was more active than tLepB. Low or no activity likely resulted from low identity (9-22%) to mammalian leptins. In an in vivo experiment in which tilapia were fed ad libitum  or fasted, there was no significant difference in the expressions of tLepA, tLepB or tLepR in the brain between the two groups examined both by real-time PCR and RNA next generation sequencing. In conclusion, in the present report we show novel, previously unknown sequences of tilapia leptin receptor and two leptins and prepare two biologically active recombinant leptin proteins. 
25073429	 Notothenia coriiceps, a typical Antarctic notothenioid teleost, has evolved to adapt to the extreme Antarctic marine environment. We previously reported an extensive analysis of the Antarctic notothenioid transcriptome. In this study, we focused on a key component of the innate immune system, the Toll-like receptors (TLRs). We cloned the full-length sequence of 12 TLRs of N. coriiceps. The N. coriiceps transcriptome for TLR homologue (ncTLR) genes encode a typical TLR structure, with multiple extracellular leucine-rich regions and an intracellular  Toll/IL-1 receptor (TIR) domain. Using phylogenetic analysis, we established that all of the cloned ncTLR genes could be classified into the same orthologous clade with other teleost TLRs. ncTLRs were widely expressed in various organs, with the highest expression levels observed in immune-related tissues, such as the skin, spleen, and kidney. A subset of the ncTLR genes was expressed at higher levels in fish exposed to pathogen-mimicking agonists, heat-killed Escherichia coli, and polyinosinic-polycytidylic acid (poly(I:C)). However, the mechanism involved in the upregulation of TLR expression following pathogen exposure in fish is currently unknown. Further research is required to elucidate these mechanisms and to thereby increase our understanding of vertebrate immune system evolution. 
25237058	 Escherichia coli endoribonuclease E has a major influence on gene expression. It  is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5'-sensor mutant of RNase E in vitro that, contrary to current models, 5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5'-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5'-monophosphorylated ends was also revealed. 
25035238	 For whole-cell aptamers selection, cells surface situation has great impact on single-stranded (ssDNA) binding and aptamers selection. In this work, both Lactobacillus acidophilus and Escherichia coli as well as their protoplasts were  as cells targets, their interaction with ssDNA library were evaluated based on capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) with UV and LIF detection. Our results demonstrated that protoplasts without cells wall had apparently stronger interaction with ssDNA library than bacteria,  the protoplasts-ssDNA complex could be observed clearly with CZE-LIF. Furthermore, E. coli pretreated by four organic solvents (methanol, ethanol, formaldehyde and glutaraldehyde) showed binding difference with ssDNA library, which could be identified with ACE-UV. Binding constants indicated the interaction of E. coli with ssDNA library were in the order of E. coli protoplasts>methanol (ethanol) treated E. coli>formaldehyde (glutaraldehyde) treated E. coli≈E. coli. Above results suggest that cells surface situation determines their binding affinity with ssDNA, which should be considered in whole-cell aptamers selection and aptamers further application. Capillary electrophoresis is a preferable technique for interaction evaluation of composite targets binding with ssDNA library. 
25162599	 The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving > 100 genes) against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport  them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers. 
24952731	 Many contaminated sites are characterized by the presence of different metals, thus increasing the complexity of toxic responses in exposed organisms. Within toxicogenomics, transcriptomics can be approached through the use of microarrays  aimed at producing a genetic fingerprint for the response of model organisms to the presence of chemicals. We studied temporal changes in the early gene expression profiles of Escherichia coli cells exposed to three metal doses of a polymetallic solution over three exposure times, through the application of cDNA  microarray technology. In the absence of metals, many genes belonging to a variety of cellular functions were up- and down-regulated over time. At the lowest metal dose, an activation of metal-specific transporters (Cus and ZraP proteins) and a mobilization of glutathione transporters involved in metal sequestration and trafficking was observed over time; this metal dose resulted in the generation of ROS capable of stimulating the transcription of Mn-superoxide dismutase, the assembly of Fe-S clusters and the synthesis of cysteine. At the intermediate dose, an overexpression of ROS scavengers (AhpF, KatG, and YaaA) and heat shock proteins (ClpP, HslV, DnaK, and IbpAB) was observed. Finally, at the highest dose, E. coli cells showed a repression of genes related with DNA mutation correctors (MutY glycopeptidases). 
24837290	 The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and  TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature. 
25036633	 Adenomatous polyposis coli (APC) is a microtubule plus-end scaffolding protein important in biology and disease. APC is implicated in RNA localization, although the mechanisms and functional significance remain unclear. We show APC is an RNA-binding protein and identify an RNA interactome by HITS-CLIP. Targets were highly enriched for APC-related functions, including microtubule organization, cell motility, cancer, and neurologic disease. Among the targets is β2B-tubulin,  known to be required in human neuron and axon migration. We show β2B-tubulin is synthesized in axons and localizes preferentially to dynamic microtubules in the  growth cone periphery. APC binds the β2B-tubulin 3' UTR; experiments interfering  with this interaction reduced β2B-tubulin mRNA axonal localization and expression, depleted dynamic microtubules and the growth cone periphery, and impaired neuron migration. These results identify APC as a platform binding functionally related protein and RNA networks, and suggest a self-organizing model for the microtubule to localize synthesis of its own subunits. 
24763818	 Nucleic acid aptamers have long demonstrated the capacity to bind cells with high affinity so that they have been utilized to diagnose various important pathogens. In this study, a DNA aptamer library was on initial efforts developed to act as a specific reporter for rapid detection of enter toxigenic Escherichia coli (ETEC)  K88 combined with immuno-magnetic separation (IMS). During a Whole-cell Systematic Evolution of Ligands by Exponential Enrichment (CELL-SELEX) procedure, the last selection pool against ETEC K88, which is named "DNA aptamer library" here, was selected and subsequently identified by flow cytometric analysis and confocal imaging. A K88 monoclonal antibody (mAb) with high affinity (K(aff): 1.616 ± 0.033 × 10(8) M(-1)) against K88 fimbrial protein was prepared, biotinylated and conjugated to streptavidin-coated magnetic beads (MBs). After the bacteria were effectively captured and enriched from the complex sample by immuno-magnetic beads (IMBs), 5'-FITC modified aptamer library was directly bound to target cells as a specific reporter for its detection. The detection system showed clearly high specificity and sensitivity with the detection limit of 1.1 × 10(3) CFU/ml in pure culture and 2.2 × 10(3) CFU/g in artificially contaminated fecal sample. The results also indicated that fluorophore-lablled DNA aptamer library as specific reporter could generate more reliable signals than individual aptamer with best affinity against target cells and implied it would have great applied potential in directly reporting bacteria from complex samples combined with IMS technology. 
24926786	 Escherichia coli O157∶H7 is a mesophilic food-borne pathogen. We investigated the growth kinetics of E. coli O157∶H7 Sakai during an abrupt temperature downshift from 35°C to either 20°C, 17°C, 14°C or 10°C; as well as the molecular mechanisms enabling growth after cold stress upon an abrupt downshift from 35°C to 14°C in an integrated transcriptomic and proteomic analysis. All downshifts caused a lag  period of growth before growth resumed at a rate typical of the post-shift temperature. Lag and generation time increased with the magnitude of the shift or with the final temperature, while relative lag time displayed little variation across the test range. Analysis of time-dependent molecular changes revealed, in  keeping with a decreased growth rate at lower temperature, repression of genes and proteins involved in DNA replication, protein synthesis and carbohydrate catabolism. Consistent with cold-induced remodelling of the bacterial cell envelope, alterations occurred in the expression of genes and proteins involved in transport and binding. The RpoS regulon exhibited sustained induction confirming its importance in adaptation and growth at 14°C. The RpoE regulon was  transiently induced, indicating a potential role for this extracytoplasmic stress response system in the early phase of low temperature adaptation during lag phase. Interestingly, genes previously reported to be amongst the most highly up-regulated under oxidative stress were consistently down-regulated. This comprehensive analysis provides insight into the molecular mechanisms operating during adaptation of E. coli to growth at low temperature and is relevant to its  physiological state during chilling in foods, such as carcasses. 
24814025	 cAMP receptor protein (CRP) is the best characterized global regulator of Escherichia coli. After genomic SELEX screening, a total of minimum 378 promoters have been identified as its regulation targets on the E. coli genome. Among a number of promoters carrying two CRP-binding sites, several promoters carry two CRP-binding sites, one upstream but another downstream of transcription initiation sites. The regulatory role of downstream CRP site remains unsolved. Using the pck gene encoding phosphoenolpyruvate carboxykinase as a model promoter, we analyzed the role of CRP-associated downstream of the transcription  initiation site. Gel shift assay and AFM observation indicate that CRP binds to both the promoter-distal site (CRP box-1) at -90.5 and the site (CRP box-2) at +13.5 downstream of transcription initiation site. The binding affinity is higher for CRP box-1. Roles of two CRP sites were examined using in vitro transcription  assay and in vivo reporter assay. In both cases, transcription repression was observed in the presence of high concentrations of CRP. Taken together, we propose that cAMP-CRP associated at downstream CRP box-2 plays as a repressor for pck transcription only in the presence of high levels of cAMP-CRP. 
24659771	 The mraZ and mraW genes are highly conserved in bacteria, both in sequence and in their position at the head of the division and cell wall (dcw) gene cluster. Located directly upstream of the mraZ gene, the Pmra promoter drives the transcription of mraZ and mraW, as well as many essential cell division and cell  wall genes, but no regulator of Pmra has been found to date. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin and MraW is known to methylate the 16S rRNA, mraZ and mraW null mutants have no detectable phenotypes. Here we show that overproduction of Escherichia coli MraZ inhibited cell division and was lethal in rich medium at high induction levels and in minimal medium at low induction levels. Co-overproduction of MraW suppressed MraZ toxicity, and loss of MraW enhanced MraZ toxicity, suggesting that MraZ and MraW have antagonistic functions. MraZ-green fluorescent protein localized to the nucleoid, suggesting that it binds DNA. Consistent with this idea, purified MraZ directly bound a region of DNA containing three direct repeats between Pmra and the mraZ gene. Excess MraZ reduced the expression of an  mraZ-lacZ reporter, suggesting that MraZ acts as a repressor of Pmra, whereas a DNA-binding mutant form of MraZ failed to repress expression. Transcriptome sequencing (RNA-seq) analysis suggested that MraZ also regulates the expression of genes outside the dcw cluster. In support of this, purified MraZ could directly bind to a putative operator site upstream of mioC, one of the repressed  genes identified by RNA-seq. 
24338599	 Escherichia coli is used intensively for recombinant protein production, but one  key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA  microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously  reported, the classical heat-shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis-related and energy-synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium.  Additionally, putative genes represented over one-third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate  that IB formation is complex, stimulates the heat-shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses. 
24645791	 In Gram-negative bacteria, N-acylhomoserine lactone (HSL) is used as a signal in  cell-cell communication and quorum sensing (QS). The model prokaryote Escherichia coli lacks the system of HSL synthesis, but is capable of monitoring HSL signals  in environment. Transcription factor SdiA for cell division control is believed to play a role as a HSL sensor. Using a collection of 477 species of chemically synthesized HSL analogues, we identified three synthetic signal molecules (SSMs)  that bind in vitro to purified SdiA. After SELEX-chip screening of SdiA-binding DNA sequences, a striking difference was found between these SSMs in the pattern  of regulation target genes on the E. coli genome. Based on Northern blot analysis in vivo, a set of target genes were found to be repressed by SdiA in the absence  of effectors and derepressed by the addition of SSMs. Another set of genes were,  however, expressed in the absence of effector ligands but repressed by the addition of SSMs. Taken together, we propose that the spectrum of taget gene selection by SdiA is modulated in multiple modes depending on the interacting HSL-like signal molecules. 
24663182	 This study was conducted to characterize the effects of infection with a pathogenic F-18 Escherichia coli and 3 different plant extracts on gene expression of ileal mucosa in weaned pigs. Weaned pigs (total = 64, 6.3 ± 0.2 kg  BW, and 21-d old) were housed in individual pens for 15 d, 4 d before and 11 d after the first inoculation (d 0). Treatments were in a 2 × 4 factorial arrangement: with or without an F-18 E. coli challenge and 4 diets (a nursery basal, control diet [CON], 10 ppm of capsicum oleoresin [CAP], garlic botanical [GAR], or turmeric oleoresin [TUR]). Results reported elsewhere showed that the plant extracts reduced diarrhea in challenged pigs. Total RNA (4 pigs/treatment)  was extracted from ileal mucosa of pigs at d 5 post inoculation. Double-stranded  cDNA was amplified, labeled, and further hybridized to the microarray, and data were analyzed in R. Differential gene expression was tested by fitting a mixed linear model in a 2 × 4 factorial ANOVA. Bioinformatics analysis was conducted by DAVID Bioinformatics Resources 6.7 (DAVID; National Institute of Allergy and Infectious Diseases [NIAID, NIH], http://david.abcc.ncifcrf.gov). The E. coli infection altered (P < 0.05) the expression of 240 genes in pigs fed the CON (148 up- and 92 down-regulated). Compared with the infected CON, feeding CAP, GAR, or  TUR altered (P < 0.05) the expression of 52 genes (18 up, 34 down), 117 genes (34 up- and 83 down-regulated), or 84 genes (16 up- and 68 down-regulated), respectively, often counteracting the effects of E. coli. The E. coli infection up-regulated (P < 0.05) the expression of genes related to the activation of immune response and complement and coagulation cascades, but down-regulated (P <  0.05) the expression of genes involved in protein synthesis and accumulation. Compared with the CON, feeding CAP and GAR increased (P < 0.05) the expression of genes related to integrity of membranes in infected pigs, indicating enhanced gut mucosa health. Moreover, feeding all 3 plant extracts reduced (P < 0.05) the expression of genes associated with antigen presentation or other biological processes of immune responses, indicating they attenuated overstimulation of immune responses caused by E. coli. These findings may explain why diarrhea was reduced and clinical immune responses were ameliorated in infected pigs fed plant extracts. In conclusion, plant extracts altered the expression of genes in ileal  mucosa of E. coli-infected pigs, perhaps leading to the reduction in diarrhea reported previously. 
24509276	 OBJECTIVES: This study coupled 2 strategies-trait extremes and genome-wide pooling-to discover a novel blood pressure (BP) locus that encodes a previously uncharacterized thiamine transporter. BACKGROUND: Hypertension is a heritable trait that remains the most potent and widespread cardiovascular risk factor, although details of its genetic determination are poorly understood. METHODS: Representative genomic deoxyribonucleic acid (DNA) pools were created from male and female subjects in the highest- and lowest-fifth percentiles of BP  in a primary care population of >50,000 patients. The peak associated single-nucleotide polymorphisms were typed in individual DNA samples, as well as  in twins/siblings phenotyped for cardiovascular and autonomic traits. Biochemical properties of the associated transporter were evaluated in cellular assays. RESULTS: After chip hybridization and calculation of relative allele scores, the  peak associations were typed in individual samples, revealing an association between hypertension, systolic BP, and diastolic BP and the previously uncharacterized solute carrier SLC35F3. The BP genetic association at SLC35F3 was validated by meta-analysis in an independent sample from the original source population, as well as the International Consortium for Blood Pressure Genome-Wide Association Studies (across North America and western Europe). Sequence homology to a putative yeast thiamine (vitamin B1) transporter prompted  us to express human SLC35F3 in Escherichia coli, which catalyzed [(3)H]-thiamine  uptake. SLC35F3 risk-allele homozygotes (T/T) displayed decreased erythrocyte thiamine content on microbiological assay. In twin pairs, the SLC35F3 risk allele predicted heritable cardiovascular traits previously associated with thiamine deficiency, including elevated cardiac stroke volume with decreased vascular resistance, and elevated pressor responses to environmental (cold) stress. Allelic expression imbalance confirmed that cis variation at the human SLC35F3 locus influenced expression of that gene, and the allelic expression imbalance peak coincided with the hypertension peak. CONCLUSIONS: Novel strategies were coupled to position a new hypertension-susceptibility locus, uncovering a previously unsuspected thiamine transporter whose genetic variants predicted several disturbances in cardiac and  autonomic function. The results have implications for the pathogenesis and treatment of systemic hypertension. 
24280049	 Microbial cells have many binding moieties on their surface for binding to their  specific bioreceptors. The whole-cell SELEX process enables the isolation of various aptamers that can bind to different components on the cell surface such as proteins, polysaccharides, or flagella with high affinity and specificity. Here, we examine the binding capacity of an aptamer mixture (aptamer cocktail) composed of various combinations of 3 different DNA aptamers isolated from Escherichia coli and compare it with one of the single aptamers using fluorescence-tagged aptamers. The aptamer mixtures showed higher fluorescence signal than did any single aptamer used, which suggests that use of aptamer mixtures can enhance the sensitivity of detection of microbial cells. To further  evaluate this effect, the signal enhancement and improvement of sensitivity provided by combinatorial use of aptamers were examined in an electrochemical detection system. With regard to current decreases, the aptamer cocktail immobilized on gold electrodes performed better than a single aptamer immobilized on gold electrodes did. Consequently, the detection limit achieved using the aptamers individually was approximately 18 times that when the 3 aptamers were used in combination. These results support the use of aptamer cocktails for detection of complex targets such as E. coli with enhanced sensitivity. 
24655930	 Knobloch JK, Niemann S, Kohl TA, Lindner U, Nitschke M, Sayk F, Solbach W. 
24138539	 Triclosan is an active agent that is commonly found in biocide formulations which are used by the food industry to control microbial contamination. The aim of this study was to use microarray analysis to compare gene expression between a triclosan-susceptible Escherichia coli O157:H19 isolate (minimum inhibitory concentration [MIC] 6.25 μg/ml) and its in vitro generated triclosan-tolerant mutant (MIC >8,000 μg/ml). Gene expression profiling was performed on the wild-type and mutant isogenic pairs after 30 min exposure to the parent MIC for triclosan and an untreated control. Microarray analysis was carried out using the Affymetrix GeneChip E. coli Genome 2.0 Array, and differential expression of genes was analyzed using the pumaDE method in Bioconductor R software. Wild-type  gene expression was found to be significantly different from the triclosan-tolerant mutant for a large number of genes, even in the absence of triclosan exposure. Significant differences were observed in the expression of a  number of pathway genes involved in metabolism, transport, and chemotaxis. In particular, gene expression in the triclosan-tolerant mutant was highly up-regulated for 33 of 38 genes belonging to the flagellar assembly pathway. The  presence of extended flagella in the mutant isolate was confirmed visually by transmission electron microscopy, although no significant difference was observed in the motility of the parent and mutant at low levels of triclosan. Data from this study show that at a transcriptomic level, a triclosan-tolerant E. coli O157:H19 mutant is significantly different from the wild-type strain in a number  of different pathways, providing an increased understanding of triclosan tolerance. 
24559310	 A rapid strategy for the covalent immobilization of DNA onto silicon-based materials using the UV-initiated radical thiol-ene reaction is presented in this  study. Following this approach, thiol- and alkene-modified oligonucleotide probes were covalently attached in microarray format, reaching immobilization densities  around 6 pmol·cm(-2). The developed methodology presents the advantages of spatially controlled probe anchoring (using a photomask), direct attachment without using cross-linkers (one-pot fashion), and short irradiation times (20 min). Using the described strategy, hybridization efficiencies up to 65% with full complementary strands were reached. The approach was evaluated by scoring single-base pair mismatches with discrimination ratios around 15. Moreover, the efficacy of the proposed DNA detection scheme is further demonstrated through the assay on a genomic target of bacterial Escherichia coli. 
24604203	 Little is known about how human genetic variation affects the responses to environmental stimuli in the context of complex diseases. Experimental and computational approaches were applied to determine the effects of genetic variation on the induction of pathogen-responsive genes in human dendritic cells. We identified 121 common genetic variants associated in cis with variation in expression responses to Escherichia coli lipopolysaccharide, influenza, or interferon-β (IFN-β). We localized and validated causal variants to binding sites of pathogen-activated STAT (signal transducer and activator of transcription) and IRF (IFN-regulatory factor) transcription factors. We also identified a common variant in IRF7 that is associated in trans with type I IFN induction in response to influenza infection. Our results reveal common alleles that explain interindividual variation in pathogen sensing and provide functional annotation for genetic variants that alter susceptibility to inflammatory diseases. 
24603758	 The promoter selectivity of Escherichia coli RNA polymerase is determined by the  sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a  specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of "constitutive promoters" that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each  harboring one base replacement, within the model promoter with the conserved -35  and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent "inducible promoters".  One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or  -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various  stressful conditions in nature. 
24594867	 The present study was undertaken to investigate growth kinetics and time-dependent change in global expression of Escherichia coli O157∶H7 Sakai upon an abrupt downshift in water activity (aw). Based on viable count data, shifting  E. coli from aw 0.993 to aw 0.985 or less caused an apparent loss, then recovery, of culturability. Exponential growth then resumed at a rate characteristic for the aw imposed. To understand the responses of this pathogen to abrupt osmotic stress, we employed an integrated genomic and proteomic approach to characterize  its cellular response during exposure to a rapid downshift but still within the growth range from aw 0.993 to aw 0.967. Of particular interest, genes and proteins with cell envelope-related functions were induced during the initial loss and subsequent recovery of culturability. This implies that cells undergo remodeling of their envelope composition, enabling them to adapt to osmotic stress. Growth at low aw, however, involved up-regulating additional genes and proteins, which are involved in the biosynthesis of specific amino acids, and carbohydrate catabolism and energy generation. This suggests their important role in facilitating growth under such stress. Finally, we highlighted the ability of  E. coli to activate multiple stress responses by transiently inducing the RpoE and RpoH regulons to control protein misfolding, while simultaneously activating  the master stress regulator RpoS to mediate long-term adaptation to hyperosmolality. This investigation extends our understanding of the potential mechanisms used by pathogenic E. coli to adapt, survive and grow under osmotic stress, which could potentially be exploited to aid the selection and/or development of novel strategies to inactivate this pathogen. 
24375106	 The holE gene is an enterobacterial ORFan gene (open reading frame [ORF] with no  detectable homology to other ORFs in a database). It encodes the θ subunit of the DNA polymerase III core complex. The precise function of the θ subunit within this complex is not well established, and loss of holE does not result in a noticeable phenotype. Paralogs of holE are also present on many conjugative plasmids and on phage P1 (hot gene). In this study, we provide evidence indicating that θ (HolE) exhibits structural and functional similarities to a family of nucleoid-associated regulatory proteins, the Hha/YdgT-like proteins that are also encoded by enterobacterial ORFan genes. Microarray studies comparing the transcriptional profiles of Escherichia coli holE, hha, and ydgT mutants revealed highly similar expression patterns for strains harboring holE and ydgT alleles. Among the genes differentially regulated in both mutants were genes of the tryptophanase (tna) operon. The tna operon consists of a transcribed leader region, tnaL, and two structural genes, tnaA and tnaB. Further experiments with transcriptional lacZ fusions (tnaL::lacZ and tnaA::lacZ) indicate that HolE  and YdgT downregulate expression of the tna operon by possibly increasing the level of Rho-dependent transcription termination at the tna operon's leader region. Thus, for the first time, a regulatory function can be attributed to HolE, in addition to its role as structural component of the DNA polymerase III complex. 
24586182	 Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from  both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between  molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems. 
24498290	 Closure of an epithelium opening is a critical morphogenetic event for development. An excellent example for this process is the transient closure of embryonic eyelid. Eyelid closure requires shape change and migration of epithelial cells at the tip of the developing eyelids, and is dictated by numerous signaling pathways. Here we evaluated gene expression in epithelial cells isolated from the tip (leading edge, LE) and inner surface epithelium (IE)  of the eyelid from E15.5 mouse fetuses by laser capture microdissection (LCM). We showed that the LE and IE cells are different at E15.5, such that IE had higher expression of muscle specific genes, while LE acquired epithelium identities. Despite their distinct destinies, these cells were overall similar in expression  of signaling components for the "eyelid closure pathways". However, while the LE  cells had more abundant expression of Fgfr2, Erbb2, Shh, Ptch1 and 2, Smo and Gli2, and Jag1 and Notch1, the IE cells had more abundant expression of Bmp5 and  Bmpr1a. In addition, the LE cells had more abundant expression of adenomatosis polyposis coli down-regulated 1 (Apcdd1), but the IE cells had high expression of Dkk2. Our results suggest that the functionally distinct LE and IE cells have also differential expression of signaling molecules that may contribute to the cell-specific responses to morphogenetic signals. The expression pattern suggests that the EGF, Shh and NOTCH pathways are preferentially active in LE cells, the BMP pathways are effective in IE cells, and the Wnt pathway may be repressed in LE and IE cells via different mechanisms. 
24454682	 The type III protein secretion system is an important pathogenicity factor of enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes encoding this apparatus are located on a pathogenicity island (the locus of enterocyte effacement) and are transcriptionally activated by the master regulator Ler. In each pathotype Ler is also known to regulate genes located elsewhere on the chromosome, but the full extent of the Ler regulon is unclear, especially for enteropathogenic E. coli. The Ler regulon was defined for two strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic) in mid and late log phases of growth by DNA microarray analysis of the transcriptomes of wild-type and ler mutant versions of each strain. In both strains the Ler regulon is focused on the locus of enterocyte effacement - all major transcriptional units of which are activated by Ler, with the sole exception of the LEE1 operon during mid-log phase growth in E2348/69. However, the Ler regulon does extend more widely and also includes unlinked pathogenicity  genes: in E2348/69 more than 50 genes outside of this locus were regulated, including a number of known or potential pathogenicity determinants; in EDL933 only 4 extra-LEE genes, again including known pathogenicity factors, were activated. In E2348/69, where the Ler regulon is clearly growth phase dependent,  a number of genes including the plasmid-encoded regulator operon perABC, were found to be negatively regulated by Ler. Negative regulation by Ler of PerC, itself a positive regulator of the ler promoter, suggests a negative feedback loop involving these proteins. 
24839553	 Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to  identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but  not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells. 
24318882	 The ability to map transcription start sites is critical for studies of gene regulation and for identification of novel RNAs. Conventional RNA-seq is often insufficient for identification of transcription start sites due to low coverage  and/or RNA processing events. We have developed a highly sensitive, genome-scale  method for detection of transcription start sites in bacteria. This method uses deep sequencing of cDNA libraries to identify transcription start sites with strand specificity at nucleotide resolution. Here, we describe the application of this method for transcription start site identification in Escherichia coli. 
24356773	 Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria. 
24097971	 In aerobically growing cells, in which reactive oxygen species are produced, the  guanine base is oxidized to 8-oxo-7,8-dihydroguanine, which can pair with adenine as well as cytosine. This mispairing causes alterations in gene expression, and cells possess mechanisms to prevent such outcomes. In Escherichia coli, 8-oxo-7,8-dihydroguanine-related phenotypic suppression of lacZ amber is enhanced by mutations in genes related to the prevention of abnormal protein synthesis under oxidative stress. A genome-wide search for the genes responsible, followed  by DNA sequence determination, revealed that specific amino acid changes in guanylate kinase and in the β and β' subunits of RNA polymerase cause elevated levels of phenotypic suppression, specifically under aerobic conditions. The involvement of the DnaB, DnaN, and MsbA proteins, which are involved in DNA replication and in preserving the membrane structure, was also noted. Interactions of these proteins with each other and also with other molecules may  be important for preventing errors in gene expression. 
23918183	 The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x  intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated. 
23274138	 Riboswitches are cis-acting mRNA elements that regulate gene expression in response to ligand binding. Recently, a class of riboswitches was proposed to respond to the molybdenum cofactor (Moco), which serves as a redox center for metabolic enzymes. The 5' leader of the Escherichia coli moaABCDE transcript exemplifies this candidate riboswitch class. This mRNA encodes enzymes for Moco biosynthesis, and moaA expression is feedback inhibited by Moco. Previous RNA-seq analyses showed that moaA mRNA copurified with the RNA binding protein CsrA (carbon storage regulator), suggesting that CsrA binds to this RNA in vivo. Among its global regulatory roles, CsrA represses stationary phase metabolism and activates central carbon metabolism. Here, we used gel mobility shift analysis to determine that CsrA binds specifically and with high affinity to the moaA 5' mRNA leader. Northern blotting and studies with a series of chromosomal lacZ reporter  fusions showed that CsrA posttranscriptionally activates moaA expression without  altering moaA mRNA levels, indicative of translation control. Deletion analyses,  nucleotide replacement studies and footprinting with CsrA-FeBABE identified two sites for CsrA binding. Toeprinting assays suggested that CsrA binding causes changes in moaA RNA structure. We propose that the moaA mRNA leader forms an aptamer, which serves as a target of posttranscriptional regulation by at least two different factors, Moco and the protein CsrA. While we are not aware of similar dual posttranscriptional regulatory mechanisms, additional examples are likely to emerge. 
24232625	 A spectral-multiplexed fluorescence contact imaging microsystem for DNA analysis  is presented. The microsystem integrates a filterless CMOS Color PhotoGate (CPG)  sensor that exploits the polysilicon gate as an optical filter, and therefore does not require an external color filter. The CPG is applied to fluorescence-based transduction in a spectrally multiplexed format by differentiating among multiple emission bands, hence replacing the functionality  of a bank of emission filters. A microsystem has been quantitatively modeled and  prototyped based on the CPG fabricated in a standard 0.35 μm CMOS technology. The multi-color imaging capability of the microsystem in analyzing DNA targets has been validated in the detection of marker gene sequences for spinal muscular atropy disease and Escherichia coli (E. coli). Spectral-multiplexing enables the  two DNA targets to be simultaneously detected with a measured detection limits of 240 nM and 210 nM for the two target concentrations at a sample volume of 10 μL for the green and red transduction channels, respectively. 
23913318	 ModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in Escherichia coli. ModE is activated by binding molybdate and acts as both an activator and a repressor. By  genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as morA and morB, located on antisense strands of yghW and torY, respectively. The morA gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the morA 5' untranslated region exhibited an inhibitory influence on colony formation of E. coli K-12. 
23925128	 Cancerous and aging cells have long been thought to be impacted by transcription  errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies  in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through ∼10(3) nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate,  and (ii) increased backtracking of the RNA polymerase that decreases a chance of  error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity. 
24086278	 Message RNA (mRNA) carries a large number of local secondary structures, with structural stability to participate in the regulations of gene expression. A worthy question is how the local structural stability is maintained under the constraint that multiple selective pressures are imposed on mRNA local regions. Here, we performed the first genome-wide study of natural selection operating on  high structural stability regions (HSRs) of mRNAs in Escherichia coli. We found that HSR tends to adjust the folded conformation to reduce the harm of mutations, showing a high level of mutational robustness. Moreover, guanine preference in HSR was observed, supporting the hypothesis that the selective constraint for high structural stability may partly account for the high percentage of G content in Escherichia coli genome. Notably, we found a substantially reduced synonymous  substitution rate in HSRs compared with that in their adjacent regions. Surprisingly and interestingly, the non-key sites in HSRs, which have slight effect on structural stability, have synonymous substitution rate equivalent to background regions. To explain this result, we identified compensatory mutations  in HSRs based on structural stability, and found that a considerable number of synonymous mutations occur to restore the structural stability decreased heavily  by the mutations on key sites. Overall, these results suggest a significant role  of local structural stability as a selective force operating on mRNA, which furthers our understanding of the constraints imposed on protein-coding RNAs. 
23707622	 To regulate stress responses and virulence, bacteria use small regulatory RNAs (sRNAs). These RNAs can up or down regulate target mRNAs through base pairing by  influencing ribosomal access and RNA decay. A large class of these sRNAs, called  trans-encoded sRNAs, requires the RNA binding protein Hfq to facilitate base pairing between the regulatory RNA and its target mRNA. The resulting network of  regulation is best characterized in Escherichia coli and Salmonella typhimurium,  but the importance of Hfq dependent sRNA regulation is recognized in a diverse population of bacteria. In this review we present the approaches and methods used to discover Hfq binding RNAs, characterize their interactions and elucidate their functions. 
23866139	 Despite the widespread use of monochloramine in drinking water treatment, there is surprisingly little information about its mode of action. In this study, DNA microarrays were used to investigate the global gene expression of Escherichia coli cells exposed to sub-lethal concentrations of monochloramine, with a focus on temporal dynamics. Genes induced by monochloramine were associated with several stress response functions, including oxidative stress, DNA repair, multidrug efflux, biofilm formation, antibiotic resistance, and cell wall repair. The diversity of functional associations supports a model of monochloramine action involving multiple cellular targets including cell membranes, nucleic acids, and proteins. These data suggest that E. coli responds to monochloramine exposure by activating diverse defense responses rather than a single antioxidant system and the exposure may also induce biofilm formation. The induction of multidrug efflux pumps and specific antibiotic resistance genes further suggests  that exposure to monochloramine may contribute to reduced susceptibility to some  antibiotics. 
24040140	 Despite the fact that much of the global microbial biosphere is believed to exist in high pressure environments, the effects of hydrostatic pressure on microbial physiology remain poorly understood. We use a genome-wide screening approach, combined with a novel high-throughput high-pressure cell culture method, to investigate the effects of hydrostatic pressure on microbial physiology in vivo.  The Keio collection of single-gene deletion mutants in Escherichia coli K-12 was  screened for growth at a range of pressures from 0.1 MPa to 60 MPa. This led to the identification of 6 genes, rodZ, holC, priA, dnaT, dedD and tatC, whose products were required for growth at 30 MPa and a further 3 genes, tolB, rffT and iscS, whose products were required for growth at 40 MPa. Our results support the  view that the effects of pressure on cell physiology are pleiotropic, with DNA replication, cell division, the cytoskeleton and cell envelope physiology all being potential failure points for cell physiology during growth at elevated pressure. 
23876479	 The human interferon-gamma (IFN-γ) is a potential drug candidate for treating various diseases due to its immunomodulatory properties. The efficient production of this protein can be achieved through a popular industrial host, Chinese hamster ovary (CHO) cells. However, recombinant expression of foreign proteins is typically suboptimal possibly due to the usage of non-native codon patterns within the coding sequence. Therefore, we demonstrated the application of a recently developed codon optimization approach to design synthetic IFN-γ coding sequences for enhanced heterologous expression in CHO cells. For codon optimization, earlier studies suggested to establish the target usage distribution pattern in terms of selected design parameters such as individual codon usage (ICU) and codon context (CC), mainly based on the host's highly expressed genes. However, our RNA-Seq based transcriptome profiling indicated that the ICU and CC distribution patterns of different gene expression classes in CHO cell are relatively similar, unlike other microbial expression hosts, Escherichia coli and Saccharomyces cerevisiae. This finding was further corroborated through the in vivo expression of various ICU and CC optimized IFN-γ in CHO cells. Interestingly, the CC-optimized genes exhibited at least 13-fold increase in expression level compared to the wild-type IFN-γ while a maximum of 10-fold increase was observed for the ICU-optimized genes. Although design criteria based on individual codons, such as ICU, have been widely used for gene  optimization, our experimental results suggested that codon context is relatively more effective parameter for improving recombinant IFN-γ expression in CHO cells. 
24058617	 Bile salts exhibit potent antibacterial properties, acting as detergents to disrupt cell membranes and as DNA-damaging agents. Although bacteria inhabiting the intestinal tract are able to resist bile's antimicrobial effects, relatively  little is known about how bile influences virulence of enteric pathogens. Escherichia coli O157:H7 is an important pathogen of humans, capable of causing severe diarrhea and more serious sequelae. In this study, the transcriptome response of E. coli O157:H7 to bile was determined. Bile exposure induced significant changes in mRNA levels of genes related to virulence potential, including a reduction of mRNA for the 41 genes making up the locus of enterocyte  effacement (LEE) pathogenicity island. Bile treatment had an unusual effect on mRNA levels for the entire flagella-chemotaxis regulon, resulting in two- to four-fold increases in mRNA levels for genes associated with the flagella hook-basal body structure, but a two-fold decrease for "late" flagella genes associated with the flagella filament, stator motor, and chemotaxis. Bile salts also caused increased mRNA levels for seventeen genes associated with iron scavenging and metabolism, and counteracted the inhibitory effect of the iron chelating agent 2,2'-dipyridyl on growth of E. coli O157:H7. These findings suggest that E. coli O157:H7 may use bile as an environmental signal to adapt to  changing conditions associated with the small intestine, including adaptation to  an iron-scarce environment. 
23806640	 RNA-seq experiments are now routinely used for the large scale sequencing of transcripts. In bacteria or archaea, such deep sequencing experiments typically produce 10-50 million fragments that cover most of the genome, including intergenic regions. In this context, the precise delineation of the non-coding elements is challenging. Non-coding elements include untranslated regions (UTRs)  of mRNAs, independent small RNA genes (sRNAs) and transcripts produced from the antisense strand of genes (asRNA). Here we present a computational pipeline (DETR'PROK: detection of ncRNAs in prokaryotes) based on the Galaxy framework that takes as input a mapping of deep sequencing reads and performs successive steps of clustering, comparison with existing annotation and identification of transcribed non-coding fragments classified into putative 5' UTRs, sRNAs and asRNAs. We provide a step-by-step description of the protocol using real-life example data sets from Vibrio splendidus and Escherichia coli. 
23255247	 The Chrysanthemum lavandulifolium extract, which includes chrysoeriol, sudachitin, and acacetin, has excellent antibiotic effects on Escherichia coli O157:H7 (E. coli O157). A notable point is that the antibiotic targets of the herb extract are similar to the targets of commonly used antibiotic drugs, including bacterial cell wall biosynthesis, bacterial protein synthesis, and bacterial DNA replication and repair. In addition, the herbal antibiotic inhibits the etiological factors that contribute to the pathogenic property. The herbal sample was extracted and fractionated and then inoculated through a disk diffusion method to confirm its antibiotic effect against E. coli O157. Total RNA was isolated from the affected bacterial cells, and its expression level was analyzed through a microarray analysis. To confirm the accuracy of the microarray data, a real-time PCR was performed. Three active compounds, chrysoeriol, sudachitin, and acacetin, were identified with a high-performance liquid chromatography-electrospray ionization/mass spectrometry chromatogram, and the disk diffusion study confirmed that chrysoeriol and sudachitin contribute to the  antibiotic properties of the herb extract. The results demonstrate that the multi-target efficacy of the herbal sample may indicate the potential for the development of more effective and safer drugs that will act as substitutes for existing antibiotics. 
23765265	 Phenylalanine ammonia-lyase (PAL) catalyzes the initial step of phenylpropanoid biosynthesis in plants. Five PAL genes (PtrPAL1 to 5) have been identified in Populus trichocarpa. These genes are classified into two subgroups according to their transcript sequence similarity and tissue specificity. However, the regulation of these genes and their protein functions are not well understood. In this study, enzymatic properties of each PtrPALs were characterized based on their recombinant proteins expressed in E.coli. Subcellular localizations of each PtrPALs in stem wood forming tissue were investigated and individual PtrPAL protein abundances in cytosol and membrane protein fractions were measured using  protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Protein/mRNA ratios of PtrPALs were further verified using RNA-Seq and gel-enhanced liquid chromatography mass spectrometry (GeLC-MS). All PtrPALs have similar catalytic properties for the deamination of L-phenylalanine, their major substrate. All PtrPALs have similar subcellular locations in stem wood forming tissue, with major amount in the cytosol (93-96 %) and less in the membrane (4-7 %). However,  the protein/mRNA ratios of subgroup A (PtrPAL2, 4 and 5) are about five times that of subgroup B (PtrPAL1 and 3) in stem wood forming tissue, while all PtrPALs have similar transcript abundances. These results indicate a greater functional significance of subgroup A PtrPALs for stem wood formation, and highlight the role of gene post-transcriptional regulation. 
23770902	 Escherichia coli K-12 was grown to the stationary phase, for maximum physiological resistance, in brain heart infusion (BHI) broth at 37°C. Cells were then heated at 58°C or 60°C to reach a process lethality value \[\mathbf{\left(}{{\mathit{F}}^{\mathit{o}}}_{\mathbf{70}}^{\mathbf{10}}\mathbf{\ right)} \] of 2 or 3 or to a core temperature of 71°C (control industrial cooking temperature). Growth recovery and cell membrane integrity were evaluated immediately after heating, and a global transcription analysis was performed using gene expression microarrays. Only cells heated at 58°C with F(o) = 2 were still able to grow on liquid or solid BHI broth after heat treatment. However, their transcriptome did not differ from that of bacteria heated at 58°C with F(o) = 3 (P value for the false discovery rate [P-FDR] > 0.01), where no growth recovery was observed posttreatment. Genome-wide transcriptomic data obtained at  71°C were distinct from those of the other treatments without growth recovery. Quantification of heat shock gene expression by real-time PCR revealed that dnaK  and groEL mRNA levels decreased significantly above 60°C to reach levels similar  to those of control cells at 37°C (P < 0.0001). Furthermore, despite similar levels of cell inactivation measured by growth on BHI media after heating, 132 and 8 genes were differentially expressed at 71°C compared to 58°C and 60°C at F(o) = 3, respectively (P-FDR < 0.01). Among them, genes such as aroA, citE, glyS, oppB, and asd, whose expression was upregulated at 71°C, may be worth investigating as good biomarkers for accurately determining the efficiency of heat treatments, especially when cells are too injured to be enumerated using growth media. 
23716638	 Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have  not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing  reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper  using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system's ability to identify novel small RNAs, operons and transcription start sites. Our  results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes. 
23899370	 BACKGROUND: Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes  and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. RESULTS: To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts  under different culture conditions and growth phases. CONCLUSIONS: As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play  important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using  directional RNA-seq short reads. 
23651393	 Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK  operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD  and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this  ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides  and its salvage pathway. 
23563322	 Most current metabolic engineering applications rely on the inactivation of unwanted reactions and the amplification of product-oriented reactions. All of the biochemical reactions involved with cellular metabolism are tightly coordinated with the electron flow, which depends on the cellular energy status.  Thus, the cellular metabolic flux can be controlled either by modulation of the electron flow or the regulation of redox reactions. This study analyzed the genome-wide anaerobic fermentation products of 472 Escherichia coli single gene knockouts, which comprised mainly of dehydrogenases, oxidoreductases, and redox-related proteins. Many metabolic pathways that were located far from anaerobic mixed-acid fermentation significantly affected the profiles of lactic acid, succinic acid, acetic acid, formic acid, and ethanol. Unexpectedly, D-lactate overproduction was determined by a single gene deletion in dehydrogenases (e.g., guaB, pyrD, and serA) involved with nucleotide and amino acid metabolism. Furthermore, the combined knockouts of guaB, pyrD, serA, fnr, arcA, or arcB genes, which are involved with anaerobic transcription regulation,  enhanced D-lactate overproduction. These results suggest that the anaerobic fermentation profiles of E. coli can be tuned via the disruption of peripheral dehydrogenases in anaerobic conditions. 
23376462	 H-NS and Hha belong to the nucleoid-associated family of proteins and modulate gene expression in response to environmental stimuli. Genes coding for these proteins can be either chromosomally or plasmid-encoded. In this work, we analyse the regulatory role of the Hha protein encoded in the virulence plasmid of the enterohemorrhagic Escherichia coli O157:H7 (Hha(pO157)). This plasmid is present  in all clinical isolates of E. coli O157:H7 and contributes to virulence. Both, Hha(pO157) and E. coli O157:H7-chromosomal Hha (Hhachr) exhibit a significant degree of similarity. The hha gene from plasmid pO157 is transcribed from its own putative promoter and is overexpressed in a chromosomal hha mutant. As its chromosomal counterpart, Hha(pO157) is able to interact with H-NS. Remarkably, Hha(pO157) targets only a subset of the genes modulated by Hhachr. This has been  evidenced by both assaying the ability of Hha(pO157) to complement expression of  a specific operon (i.e., the haemolysin operon) and by comparing the global transcriptome of the wt strain and its hhap, hhac and hhapc mutant derivatives. Hha(pO157) and Hhachr share some common regulatory features, however they also display specific targeting of some genes and even a different modulatory role in  some others. 
23758654	 Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The  higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It  is noteworthy that at the protein level secretion of TNF-α and IL-1β was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection. 
23524613	 The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 codes for two interacting DNA binding proteins, Cra and KdpE, that coregulate expression of the locus of enterocyte effacement (LEE) genes in a metabolite-dependent manner.  Cra is a transcription factor that uses fluctuations in the concentration of carbon metabolism intermediates to positively regulate virulence of EHEC. KdpE is a response regulator that activates the transcription of homeostasis genes in response to salt-induced osmolarity and virulence genes in response to changes in metabolite concentrations. Here, we probed the transcriptional profiles of the Δcra, ΔkdpE, and Δcra ΔkdpE mutant strains and show that Cra and KdpE share several targets besides the LEE, but both Cra and KdpE also have independent targets. Several genes within O-islands (genomic islands present in EHEC but absent from E. coli K-12), such as Z0639, Z0640, Z3388, Z4267, and espFu (encoding an effector necessary for formation of attaching and effacing lesions on epithelial cells), were directly regulated by both Cra and KdpE, while Z2077 was only regulated by Cra. These studies identified and confirmed new direct targets for Cra and KdpE that included putative virulence factors as well as characterized virulence factors, such as EspFu and EspG. These results map out the role of the two interacting regulators, Cra and KdpE, in EHEC pathogenesis and global gene regulation. 
23738017	 Escherichia coli O157:H7 is an important food-borne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome in humans. pO157_Sal, a novel conjugative plasmid is present in a Chinese O157:H7 outbreak strain Xuzhou21. Here we investigated the phenotypic and transcriptional differences between the wild type strain Xuzhou21 and the pO157_Sal cured mutant strain Xuzhou21m. RNA-Seq analysis found that all 52 ORFs encoded on pO157_Sal were transcribed. One hundred and sixty eight chromosomal and pO157 genes were differentially expressed (≥2 fold difference) between Xuzhou21 and Xuzhou21m. Sixty-seven and 101 genes were up-regulated and down-regulated respectively by pO157_Sal including genes related to stress response, adaption and virulence. The plasmid-cured mutant Xuzhou21m grew slower than wild type Xuzhou21 and pO157_Sal  plasmid complemented strain Xuzhou21c in M9 medium under the condition of high NaCl or presence of sodium deoxycholate (NaDC), corroborating with the RNA-Seq data. Seven differentially expressed genes are associated with NaDC resistance, including the adenine-specific DNA-methyltransferase gene (dam), multidrug efflux system subunit gene mdtA, hyperosmotically inducible periplasmic protein gene osmY and oxidation-reduction related genes while two differentially expressed genes (osmY and pspD) are likely to be related to resistance to osmotic pressure. A number of differentially expressed genes were virulence associated including four genes encoding T3SS effectors from the chromosome and ehxD from pO157. Through complementation of Xuzhou21m with a plasmid construct carrying the pO157_Sal hha homolog we further showed that the pO157_Sal hha represses the expression of T3SS effectors. These findings demonstrated that the plasmid pO157_Sal affects the transcription of the chromosomal and pO157 plasmid genes and contributes to the enhanced ability to resist stress. We conclude that pO157_Sal plays an important role in regulating global gene expression and affects the virulence and adaptation of E. coli O157:H7. 
23717391	 Expression of bacterial genes takes place under the control of RNA polymerase with exchangeable σ-subunits and multiple transcription factors. A typical promoter region contains one or several overlapping promoters. In the latter case promoters have the same or different σ-specificity and are often subjected to different regulatory stimuli. Genes, transcribed from multiple promoters, have on average higher expression levels. However, recently in the genome of Escherichia  coli we found 78 regions with an extremely large number of potential transcription start points (promoter islands, PIs). It was shown that all PIs interact with RNA polymerase in vivo and are able to form transcriptionally competent open complexes both in vitro and in vivo but their transcriptional activity measured by oligonucleotide microarrays was very low, if any. Here we confirmed transcriptional defectiveness of PIs by analyzing the 5'-end specific RNA-seq data, but showed their ability to produce short oligos (9-14 bases). This combination of functional properties indicated a deliberate suppression of transcriptional activity within PIs. According to our data this suppression may be due to a specific conformation of the DNA double helix, which provides an ideal platform for interaction with both RNA polymerase and the histone-like nucleoid protein H-NS. The genomic DNA of E.coli contains therefore several dozen sites optimized by evolution for staying in a heterochromatin-like state. Since almost all promoter islands are associated with horizontally acquired genes, we offer them as specific components of bacterial evolution involved in acquisition  of foreign genetic material by turning off the expression of toxic or useless aliens or by providing optimal promoter for beneficial genes. The putative molecular mechanism underlying the appearance of promoter islands within recipient genomes is discussed. 
23357235	 Aptamers are powerful capturing probes against various targets such as proteins,  small organic compounds, metal ions, and even cells. In this study, we isolated and characterized single-stranded DNA (ssDNA) aptamers against Escherichia coli.  A total of 28 ssDNAs were isolated after 10 rounds of selection using a bacterial cell-SELEX (systematic evolution of ligands by exponential enrichment) process. Other bacterial species (Klebsiella pneumoniae, Citrobacter freundii, Enterobacter aerogenes, and Staphylococcus epidermidis) were used for counter selection to enhance the selectivity of ssDNA aptamers against E. coli. Finally,  four ssDNA aptamers showed high affinity and selectivity to E. coli, The dissociation constants (K(d)) of these four ssDNA aptamers to E. coli were estimated to range from 12.4 to 25.2 nM. These aptamers did not bind to other bacterial species, including four counter cells, but they showed affinity to different E. coli strains. The binding of these four aptamers to E. coli was observed directly by fluorescence microscopy. 
23435884	 The direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food- and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in  nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (general Escherichia coli), and a process  control strain in the microfluidic qPCR system. We preamplified the template DNA  to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA  without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest  that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach. 
23613991	 The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes  known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest. 
23613952	 Bdellovibrio bacteriovorus is an obligate predator of bacteria ubiquitously found in the environment. Its life cycle is composed of two essential phases: a free-living, non-replicative, fast swimming attack phase (AP) wherein the predator searches for prey; and a non-motile, actively dividing growth phase (GP) in which it consumes the prey. The molecular regulatory mechanisms governing the  switch between AP and GP are largely unknown. We used RNA-seq to generate a single-base-resolution map of the Bdellovibrio transcriptome in AP and GP, revealing a specific "AP" transcriptional program, which is largely mutually exclusive of the GP program. Based on the expression map, most genes in the Bdellovibrio genome are classified as "AP only" or "GP only". We experimentally generated a genome-wide map of 140 AP promoters, controlling the majority of AP-specific genes. This revealed a common sigma-like DNA binding site highly similar to the E. coli flagellar genes regulator sigma28 (FliA). Further analyses suggest that FliA has evolved to become a global AP regulator in Bdellovibrio. Our results also reveal a non-coding RNA that is massively expressed in AP. This  ncRNA contains a c-di-GMP riboswitch. We suggest it functions as an intracellular reservoir for c-di-GMP, playing a role in the rapid switch from AP to GP. 
23540252	 In this report a novel wash-free method for multiplexed DNA detection is demonstrated employing target specific probe pairs and switchable lanthanide luminescence technology on a solid-phase array. Four oligonucleotide capture probes, conjugated at 3' to non-luminescent lanthanide ion carrier chelate, were  immobilized as a small array on the bottom of a microtiter plate well onto which  a mix of corresponding detection probes, conjugated at 5' to a light absorbing antenna ligand, were added. In the presence of complementary target nucleic acid  both the spotted capture probe and the liquid-phase detection probe hybridize adjacently on the target. Consequently the two non-luminescent label molecules self-assemble and form a luminescent mixed lanthanide chelate complex. Lanthanide luminescence is thereafter measured without a wash step from the spots by scanning in time-resolved mode. The homogeneous solid-phase array-based method resulted in quantitative detection of synthetic target oligonucleotides with 0.32 nM and 0.60 nM detection limits in a single target and multiplexed assay, respectively, corresponding to 3× SD of the background. Also qualitative detection of PCR-amplified target from Escherichia coli is described. 
23247721	 The shikimate pathway enzymes offer attractive targets for the development of antimetabolites. Glyphosate is an effective antimetabolite that inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the shikimate pathway, thereby resulting in a shortage of the chorismate-derived essential aromatic amino acids. However, little is known about the genome-wide transcriptional responses of bacteria to glyphosate shock. In the current study, a transcriptome  analysis of Escherichia coli (E. coli) exposed to glyphosate identified the differential expression of 1040 genes, which represent 23.2% of the genome. The differentially expressed genes are primarily involved in amino acid metabolism, cell motility, and central carbon metabolism, indicating that the impact of glyphosate on the shikimate pathway also extends to other metabolic pathways. Expectedly, almost all genes encoding the proteins for the shikimate and specific aromatic amino acid pathways were downregulated after the addition of glyphosate. Furthermore, the expression of many energy- and metabolism-related genes was repressed. In contrast, glyphosate treatment induced the coordinated upregulation of at least 50 genes related to cell motility and chemotaxis. The reverse transcription-quantitative real-time PCR (RT-qPCR) data showed that the expression profiles of selected genes from the referred pathways were found to be consistent with the microarray data. The results suggest that the presence of glyphosate during growth induces metabolic starvation, an energy drain and other  non-target effects. 
23316049	 We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammary-associated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide  (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors was analyzed using a  transcription factor protein/DNA array. MUC3 and relevant transcription factors were detected using real-time PCR and/or Western blots. The luciferase assay, EMSA, and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP  induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal 10-mer monomeric peptide, and MUC3 expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides -242 and  -62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection. 
23281480	 The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA  strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic  and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes. 
23301696	 Peptidoglycan (PG), also designated as murein, forms a skeletal mesh within the periplasm of bacterial membrane. PG is a metabolically stable cell architecture in Escherichia coli, but under as yet ill-defined conditions, a portion of PG is  degraded, of which both amino sugar and peptide moieties are either recycled or used as self-generated nutrients for cell growth. At present, the control of PG degradation remains uncharacterized. Using the Genomic SELEX screening system, we identified an uncharacterized transcription factor YcjZ is a repressor of the expression of the initial step enzymes for PG peptide degradation. Under nutrient starvation, the genes encoding the enzymes for PG peptide degradation are derepressed so as to generate amino acids but are tightly repressed at high osmotic conditions so as to maintain the rigid membrane for withstanding the turgor. Taken together, we propose to rename YcjZ as PgrR (regulator of peptide glycan recycling). 
23199033	 Modern risk control and food safety practices involving food-borne bacterial pathogens are benefiting from new genomic technologies for rapid, yet highly specific, strain characterisations. Within the United States Food and Drug Administration (USFDA) Center for Food Safety and Applied Nutrition (CFSAN), optical genome mapping and DNA microarray genotyping have been used for several years to quickly assess genomic architecture and gene content, respectively, for  outbreak strain subtyping and to enhance retrospective trace-back analyses. The application and relative utility of each method varies with outbreak scenario and the suspect pathogen, with comparative analytical power enhanced by database scale and depth. Integration of these two technologies allows high-resolution scrutiny of the genomic landscapes of enteric food-borne pathogens with notable examples including Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica serovars from a variety of food commodities. Moreover, the recent application of whole genome sequencing technologies to food-borne pathogen outbreaks and surveillance has enhanced resolution to the single nucleotide scale. This new wealth of sequence data will support more refined next-generation custom microarray designs, targeted re-sequencing and "genomic signature recognition" approaches involving a combination of genes and single nucleotide polymorphism detection to distil strain-specific fingerprinting to a minimised scale. This paper examines the utility of microarrays and optical mapping in analysing outbreaks, reviews best practices and the limits of these technologies  for pathogen differentiation, and it considers future integration with whole genome sequencing efforts. 
23085385	 The level of supercoiling in the chromosome can affect gene expression. To clarify the basis of supercoiling sensitivity, we analyzed the structural features of nucleotide sequences in the vicinity of promoters for the genes with  expression enhanced and decreased in response to loss of chromosomal supercoiling in Escherichia coli. Fourier analysis of promoter sequences for supercoiling-sensitive genes reveals the tendency in selection of sequences with  helical periodicities close to 10nt for relaxation-induced genes and to 11nt for  relaxation-repressed genes. The helical periodicities in the subsets of promoters recognized by RNA polymerase with different sigma factors were also studied. A special procedure was developed for the study of correlations between the intensities of periodicities in promoter sequences and the expression levels of corresponding genes. Significant correlations of expression with the AT content and with AT periodicities about 10, 11, and 50nt indicate their role in regulation of supercoiling-sensitive genes. 
23472115	 This study demonstrates the effects of simulated microgravity on E. coli K 12 MG1655 grown on LB medium supplemented with glycerol. Global gene expression analysis indicated that the expressions of hundred genes were significantly altered in simulated microgravity conditions compared to that of normal gravity conditions. Under these conditions genes coding for adaptation to stress are up regulated (sufE and ssrA) and simultaneously genes coding for membrane transporters (ompC, exbB, actP, mgtA, cysW and nikB) and carbohydrate catabolic processes (ldcC, ptsA, rhaD and rhaS) are down regulated. The enhanced growth in  simulated gravity conditions may be because of the adequate supply of energy/reducing equivalents and up regulation of genes involved in DNA replication (srmB) and repression of the genes encoding for nucleoside metabolism (dfp, pyrD and spoT). In addition, E. coli cultured in LB medium supplemented with glycerol (so as to protect the cells from freezing temperatures) do not exhibit multiple stress responses that are normally observed when cells are exposed to microgravity in LB medium without glycerol. 
23227988	 Second-generation sequencing (SGS) has become the preferred method for RNA transcriptome profiling of organisms and single cells. However, SGS analysis of transcriptome diversity (including protein-coding transcripts and regulatory non-coding RNAs) is inefficient unless the sample of interest is first depleted of nucleic acids derived from ribosomal RNA (rRNA), which typically account for up to 95% of total intracellular RNA content. Here we describe a novel microscale hydroxyapatite chromatography (HAC) normalization method to remove eukaryotic and prokaryotic high abundant rRNA species, thereby increasing sequence coverage depth and transcript diversity across non-rRNA populations. RNA-seq analysis of Escherichia coli K-12 and human intracellular total RNA showed that HAC-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAC normalization generated rRNA-depleted cDNA libraries comparable to the well-established duplex specific nuclease (DSN) normalization and Ribo-Zero rRNA-depletion methods, thus establishing microscale HAC as an effective, cost saving, and non-destructive alternative normalization technique. 
23035190	 Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (bla(KPC)) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 × 10(5) CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine,  on average, for K. pneumoniae (Norgen). This study presents, for the first time,  the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship. 
23233451	 The FixJ/LuxR family transcription factor CsgD is a master regulator of biofilm formation in Escherichia coli. Previously, we identified more than 10 transcription factors that participate in regulation of the csgD promoter. After  genomic SELEX screening of regulation targets, an uncharacterized TetR-type transcription factor YbjK was found to be involved in regulation of the csgD promoter. In addition, a number of stress-response genes were found to be under the direct control of YbjK. Taken together, we propose to rename it to RcdA (regulator of csgD). One unique feature of RcdA is its mode of DNA binding. Gel shift, DNase-I footprinting, and atomic force microscopic (AFM) analyses indicated that RcdA is a DNA-binding protein with a high level of cooperativity,  with which it covers the entire surface of probe DNA through protein-protein interaction and moreover it induces the formation of aggregates of DNA-RcdA complexes. 
23075417	 The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live Salmonella typhimurium were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable S. typhimurium and a negative selection step against heat killed S. typhimurium and a mixture of related pathogens, including Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. typhimurium down to 600 CFU mL(-1) (equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from other Salmonella species, including S. enteritidis and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform. 
23117822	 A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs, species termed "nanoRNAs," to prime transcription initiation in Escherichia coli. Priming of transcription initiation with nanoRNAs represents a previously undocumented component of transcription start site selection and gene expression. 
22992053	 Selective covalent immobilization of thiolated oligonucleotides onto an epoxy-functionalized silicon-substrate can be achieved via light radiation (365 nm). Following this approach, thiol-modified oligonucleotide probes were covalently attached as microarrays, reaching an immobilization density of 2.5 pmol·cm(-2), with a yield of 53%. The developed method presents the advantages of spatially controlled probe anchoring (by means of using a photomask), direct attachment without using cross-linkers, and short irradiation times (10 min). Hybridization efficiencies up to 65%, with full complementary strands, were reached. The approach was evaluated by scoring single nucleotide polymorphisms with a discrimination ratio around 15. Moreover, sensitive and selective detection of bacterial Escherichia coli was demonstrated. 
22996052	 To expand the chemical array available for DNA sequences in the context of in vitro selection, I present herein the synthesis of five nucleoside triphosphate analogues containing side chains capable of organocatalysis. The synthesis involved the coupling of L-proline-containing residues (dU(tP)TP and dU(cP)TP), a dipeptide (dU(FP)TP), a urea derivative (dU(Bpu)TP), and a sulfamide residue (dU(Bs)TP) to a suitably protected common intermediate, followed by triphosphorylation. These modified dNTPs were shown to be excellent substrates for the Vent (exo(-)) and Pwo DNA polymerases, as well as the Klenow fragment of  E. coli DNA polymerase I, although they were only acceptable substrates for the 9°N(m) polymerase. All of the modified dNTPs, with the exception of dU(Bpu)TP, were readily incorporated into DNA by the polymerase chain reaction (PCR). Modified oligonucleotides efficiently served as templates for PCR for the regeneration of unmodified DNA. Thermal denaturation experiments showed that these modifications are tolerated in the major groove. Overall, these heavily modified dNTPs are excellent candidates for SELEX. 
22790675	 It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were  caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain. 
22885723	 Bacterial attachment and subsequent biofilm formation pose key challenges to the  optimal performance of medical devices. In this study, we determined the attachment of selected bacterial species to hundreds of polymeric materials in a  high-throughput microarray format. Using this method, we identified a group of structurally related materials comprising ester and cyclic hydrocarbon moieties that substantially reduced the attachment of pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli). Coating silicone with these 'hit' materials achieved up to a 30-fold (96.7%) reduction in the surface area covered by bacteria compared with a commercial silver hydrogel coating in vitro, and the same material coatings were effective at reducing bacterial attachment in vivo in a mouse implant infection model. These polymers represent a class of materials that reduce the attachment of bacteria that could not have been predicted to have this property from the current understanding of bacteria-surface interactions. 
22802628	 Phenotypic heterogeneity displayed by a clonal bacterial population permits a small fraction of cells to survive prolonged exposure to antibiotics. Although first described over 60 y ago, the molecular mechanisms underlying this behavior, termed persistence, remain largely unknown. To systematically explore the genetic basis of persistence, we selected a library of transposon-mutagenized Escherichia coli cells for survival to multiple rounds of lethal ampicillin exposure. Application of microarray-based genetic footprinting revealed a large number of loci that drastically elevate persistence frequency through null mutations and domain disruptions. In one case, the C-terminal disruption of methionyl-tRNA synthetase (MetG) results in a 10,000-fold higher persistence frequency than wild type. We discovered a mechanism by which null mutations in transketolase A (tktA) and glycerol-3-phosphate (G3P) dehydrogenase (glpD) increase persistence through  metabolic flux alterations that increase intracellular levels of the growth-inhibitory metabolite methylglyoxal. Systematic double-mutant analyses revealed the genetic network context in which such persistent mutants function. Our findings reveal a large mutational target size for increasing persistence frequency, which has fundamental implications for the emergence of antibiotic tolerance in the clinical setting. 
22522900	 In Escherichia coli, putrescine is metabolized to succinate for use as a carbon and nitrogen source by the putrescine utilization pathway (Puu pathway). One gene in the puu gene cluster encodes a transcription factor, PuuR, which has a helix-turn-helix DNA-binding motif. DNA microarray analysis of an E. coli puuR mutant, in which three amino acid residues in the helix-turn-helix DNA binding motif of PuuR were mutated to alanine to eliminate DNA binding of PuuR, suggested that PuuR is a negative regulator of puu genes. Results of gel shift and DNase I  footprint analyses suggested that PuuR binds to the promoter regions of puuA and  puuD. The binding of wild-type PuuR to a DNA probe containing PuuR recognition sites was diminished with increasing putrescine concentrations in vitro. These results suggest that PuuR regulates the intracellular putrescine concentration by the transcriptional regulation of genes in the Puu pathway, including puuR itself. The puu gene cluster is found in E. coli and closely related enterobacteria, but this gene cluster is uncommon in other bacterial groups. E. coli and related enterobacteria may have gained the Puu pathway as an adaptation  for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations. 
22720940	 The innate immune response plays a major role in defense against mastitis-causing pathogens. Identification of existing variation in innate immune signaling among  cows and the underlying molecular causes for the variation may help in design of  new mastitis control strategies. The dermal fibroblast has been used as a model cell type to explore between-cow variation in the ability of cells to produce IL-8 in response to lipopolysaccharide (LPS) treatment, and this response appears related to an animal's ability to respond to in vivo challenge with LPS or Escherichia coli mastitis. In this study, primary dermal fibroblast cultures of cows and microarray-based genomic analysis were used to investigate the cause(s)  for the variable response to LPS. Fibroblast cultures from 2 cows, one with a low response phenotype (LR(array)) and another with a high response phenotype (HR(array)), were selected from our collection of fibroblast cultures established from 88 cows. The LR(array) fibroblast culture produced approximately 5-fold less IL-8 and IL-6 protein in response to 24-h LPS treatment than the HR(array) fibroblast culture. Genomic analysis of RNA obtained from 3 replicates of the 2 cultures before and after 8-h LPS treatment revealed a combined LPS-induced differential expression of 321 transcripts, indicating the robust response capability of the fibroblast cell. Under basal conditions, the microarray analysis revealed 2-fold less expression of toll-like receptor 4 (TLR4) in the LR(array) fibroblasts compared with the HR(array) fibroblasts, and this was associated with a marked reduction in expression of genes regulated by the TLR4-MyD88-dependent and TLR4-TRIF-dependent pathways (IL-8, IL-6, SAA3, CCL20, MX1, IRF1, and ISG20). The between-culture differential expression of TLR4 was confirmed and extended by quantitative PCR analysis (QPCR) that revealed a 33-fold lower expression of TLR4 in the LR(array) fibroblast culture. After LPS treatment, the difference in TLR4 expression increased to almost 50-fold and was  associated with more than 8-fold lower expression of IL-8 and IL-6. No DNA sequence variations were identified in the proximal 1,300-bp promoter region of the TLR4 gene, and microarray analysis did not reveal a molecular explanation for the reduced TLR4 expression under either basal conditions or following exposure to LPS. The attenuated innate immune response of the LR(array) fibroblast culture to LPS may be caused by reduced TLR4 receptor expression. Also, the primary dermal fibroblast cells can be used to examine underlying causes for between-cow  variations in key immune response pathways. 
22196016	 RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA,  which encodes for a periplasmic protein known to be involved with Zn(II) import,  (2) yodA, which encodes for a periplasmic protein with unknown function, and (3)  ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding  protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a  ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis. 
22442305	 The BasS-BasR two-component system is known as an iron- and zinc-sensing transcription regulator in Escherichia coli, but so far only a few genes have been identified to be under the direct control of phosphorylated BasR. Using Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening, we have identified a total of at least 38 binding sites of phosphorylated BasR on the E. coli genome, and based on the BasR-binding sites, have predicted more than 20 novel targets of regulation. By DNase I footprint analysis for high-affinity BasR-binding sites, a direct repeat of a TTAAnnTT sequence was identified as the BasR box. Transcription regulation in vivo of the  target genes was confirmed after Northern blot analysis of target gene mRNAs from both wild-type E. coli and an otherwise isogenic basR deletion mutant. The BasR regulon can be classified into three groups of genes: group 1 includes the genes  for the formation and modification of membrane structure; group 2 includes genes  for modulation of membrane functions; and group 3 includes genes for stress-response cell functions, including csgD, the master regulator of biofilm formation. 
22344651	 The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based  RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance  genes floR, bla(CMY-2), aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking  antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that  it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators. 
22268572	 Plant development and function are underpinned by redox reactions that depend on  co-factors such as nicotinamide adenine dinucleotide (NAD). NAD has recently been shown to be involved in several signalling pathways that are associated with stress tolerance or defence responses. However, the mechanisms by which NAD influences plant gene regulation, metabolism and physiology still remain unclear. Here, we took advantage of Arabidopsis thaliana lines that overexpressed the nadC gene from E. coli, which encodes the NAD biosynthesis enzyme quinolinate phosphoribosyltransferase (QPT). Upon incubation with quinolinate, these lines accumulated NAD and were thus used as inducible systems to determine the consequences of an increased NAD content in leaves. Metabolic profiling showed clear changes in several metabolites such as aspartate-derived amino acids and NAD-derived nicotinic acid. Large-scale transcriptomic analyses indicated that NAD promoted the induction of various pathogen-related genes such as the salicylic acid (SA)-responsive defence marker PR1. Extensive comparison with transcriptomic databases further showed that gene expression under high NAD content was similar to that obtained under biotic stress, eliciting conditions or SA treatment. Upon inoculation with the avirulent strain of Pseudomonas syringae  pv. tomato Pst-AvrRpm1, the nadC lines showed enhanced resistance to bacteria infection and exhibited an ICS1-dependent build-up of both conjugated and free SA pools. We therefore concluded that higher NAD contents are beneficial for plant immunity by stimulating SA-dependent signalling and pathogen resistance. 
22267665	 Expression arrays were used to identify 4 putative oxidoreductases that were upregulated (>3-fold) by furfural (15 mM, 15 min). Plasmid expression of one (ucpA) increased furan tolerance in ethanologenic strain LY180 and wild-type strain W. Deleting ucpA decreased furfural tolerance. Although the mechanism remains unknown, the cryptic ucpA gene is now associated with a phenotype: furan  resistance. 
22336764	 In bacteria, adaptive response to external stimuli is often regulated by small RNAs (sRNAs). In Escherichia coli, the organism in which sRNAs have been best characterized so far, no function could be attributed to 40 out of 79 sRNAs. Here we decipher the function of RybA, one of these orphan sRNAs. RybA was discovered  in 2001 by Wassarman et al. using comparative genomics. This sRNA is conserved between E. coli, Salmonella typhimurium and Klebsiella pneumoniae. We determined  the expression pattern of RybA under different growth conditions and identified its exact 5' and 3' ends. Using microarray and Northern analysis we show that, under peroxide stress, the absence of RybA leads to an upregulation of key genes  of the TyrR regulon involved in the metabolism of aromatic compounds including the aromatic amino acids. Although containing an open reading frame, which might  have an independent function, RybA does not require translation for this activity and therefore acts at the RNA level. Furthermore we demonstrate that regulation requires the transcription regulator TyrR. The mechanism of activation of TyrR, probably the primary target of RybA, remains to be elucidated. The downregulation of aromatic amino acid biosynthesis might regulate the cellular concentration of  chorismate and its availability for other downstream products like ubiquinone or  enterobactin. While ubiquinone participates in the defense against oxidative stress in the cytoplasmic membrane, enterobactin is involved in iron import and is therefore detrimental under oxidative stress. 
22336761	 Small non-coding regulatory RNAs (sRNAs) have been studied in many bacterial pathogens during infection. However, few studies have focused on how intracellular pathogens modulate sRNA expression inside eukaryotic cells. Here, we monitored expression of all known sRNAs of Salmonella enterica serovar Typhimurium (S. Typhimurium) in bacteria located inside fibroblasts, a host cell  type in which this pathogen restrains growth. sRNA sequences known in S. Typhimurium and Escherichia coli were searched in the genome of S. Typhimurium virulent strain SL1344, the subject of this study. Expression of 84 distinct sRNAs was compared in extra- and intracellular bacteria. Non-proliferating intracellular bacteria upregulated six sRNAs, including IsrA, IsrG, IstR-2, RyhB-1, RyhB-2 and RseX while repressed the expression of the sRNAs DsrA, GlmZ, IsrH-1, IsrI, SraL, SroC, SsrS(6S) and RydC. Interestingly, IsrH-1 was previously reported as an sRNA induced by S. Typhimurium inside macrophages. Kinetic analyses unraveled changing expression patterns for some sRNAs along the infection. InvR and T44 expression dropped after an initial induction phase while IstR-2 was induced exclusively at late infection times (> 6 h). Studies focused on the Salmonella-specific sRNA RyhB-2 revealed that intracellular bacteria use this sRNA to regulate negatively YeaQ, a cis-encoded protein of unknown function. RyhB-2, together with RyhB-1, contributes to attenuate intracellular bacterial growth. To our knowledge, these data represent the first comprehensive study of S. Typhimurium sRNA expression in intracellular bacteria and provide the first insights into sRNAs that may direct pathogen adaptation to a non-proliferative state inside the host cell. 
22415002	 Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration and efficient separation and extraction based on ionic mobility. For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood. ITP achieves focusing and separation using an applied electric  field and two buffers within a fluidic channel system. For anionic analytes, the  leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively  low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus.  Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species. We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial  analyte concentration and/or the amount of time allotted for sample accumulation. We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids. 
22053080	 Bacterial Hfq is a protein that plays an important role in the regulation of genes in cooperation with sRNAs. Escherichia coli Hfq (EcHfq) has two or more sites that bind RNA(s) including U-rich and/or the poly(A) tail of mRNA. However, functional and structural information about Bacillus subtilis Hfq (BsHfq) including the RNA sequences that specifically bind to it remain unknown. Here, we describe RNA aptamers including fragment (AG)(3)A that are recognized by BsHfq and crystal structures of the BsHfq-(AG)(3)A complex at 2.2 Å resolution. Mutational and structural studies revealed that the RNA fragment binds to the distal site, one of the two binding sites on Hfq, and identified amino acid residues that are critical for sequence-specific interactions between BsHfq and (AG)(3)A. In particular, R32 appears to interact with G bases in (AG)(3)A. Poly(A) also binds to the distal site of EcHfq, but the overall RNA structure and protein-RNA interaction patterns engaged in the R32 residues of BsHfq-(AG)(3)A differ from those of EcHfq-poly(A). These findings provide novel insight into how the Hfq homologue recognizes RNA. 
22272603	 BACKGROUND: Spot 42 was discovered in Escherichia coli nearly 40 years ago as an  abundant, small and unstable RNA. Its biological role has remained obscure until  recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous  in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. RESULTS: We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named  VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. CONCLUSIONS: We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin. 
22232676	 RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic  to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq. 
22166202	 Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes life-threatening symptoms in humans worldwide. To rapidly and properly identify the pathogen and avoid its toxic effects, ligands which can directly and specifically bind to the virulent E. coli O157:H7 serotype should be identified.  In this study, a RNA aptamer-based ligand which can specifically distinguish the  pathogen E. coli O157:H7 from others was developed by a subtractive cell-SELEX method. To this end, an RNA library was first incubated with the E. coli K12 strain, and the RNAs binding to the strain were discarded. The precluded RNAs were then used for the selection of O157:H7-specific aptamers. After 6 rounds of  the subtractive cell-SELEX process, the selected aptamer was found to specifically bind to the O157:H7 serotype, but not to the K12 strain. This was evidenced by aptamer-immobilized ELISA, real-time PCR analysis, or an aptamer-linked precipitation experiment. Importantly, the isolated RNA aptamer that distinguishes between the virulent serotype and the nonpathogenic strain specifically bound to an O157:H7-specific lipopolysaccharide which includes the O antigen. This novel O157:H7-specific aptamer could be of potential application as a diagnostic ligand against the pathogen-related food borne illness. 
22064540	 Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the  determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed in Escherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain  and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates. 
22009253	 Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region  has been implicated in cellular processes--including polarity, migration, mitosis, and chromosomal instability (CIN)—that have been postulated as critical  for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but  leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T) animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus. Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression. 
22275871	 Gene expression is subject to random perturbations that lead to fluctuations in the rate of protein production. As a consequence, for any given protein, genetically identical organisms living in a constant environment will contain different amounts of that particular protein, resulting in different phenotypes.  This phenomenon is known as "phenotypic noise." In bacterial systems, previous studies have shown that, for specific genes, both transcriptional and translational processes affect phenotypic noise. Here, we focus on how the promoter regions of genes affect noise and ask whether levels of promoter-mediated noise are correlated with genes' functional attributes, using data for over 60% of all promoters in Escherichia coli. We find that essential genes and genes with a high degree of evolutionary conservation have promoters that confer low levels of noise. We also find that the level of noise cannot be attributed to the evolutionary time that different genes have spent in the genome of E. coli. In contrast to previous results in eukaryotes, we find no association between promoter-mediated noise and gene expression plasticity. These results are consistent with the hypothesis that, in bacteria, natural selection can act to reduce gene expression noise and that some of this noise is controlled through the sequence of the promoter region alone. 
23056556	 Our understanding of the Escherichia genus is heavily biased toward pathogenic or commensal isolates from human or animal hosts. Recent studies have recovered Escherichia isolates that persist, and even grow, outside these hosts. Although the environmental isolates are typically phylogenetically distinct, they are highly related to and phenotypically indistinguishable from their human counterparts, including for the coliform test. To gain insights into the genomic  diversity of Escherichia isolates from diverse habitats, including freshwater, soil, animal, and human sources, we carried out comparative DNA-DNA hybridizations using a multi-genome E. coli DNA microarray. The microarray was validated based on hybridizations with selected strains whose genome sequences were available and used to assess the frequency of microarray false positive and  negative signals. Our results showed that human fecal isolates share two sets of  genes (n>90) that are rarely found among environmental isolates, including genes  presumably important for evading host immune mechanisms (e.g., a multi-drug transporter for acids and antimicrobials) and adhering to epithelial cells (e.g., hemolysin E and fimbrial-like adhesin protein). These results imply that environmental isolates are characterized by decreased ability to colonize host cells relative to human isolates. Our study also provides gene markers that can distinguish human isolates from those of warm-blooded animal and environmental origins, and thus can be used to more reliably assess fecal contamination in natural ecosystems. 
22666463	 Previously, we have found that an insertional inactivation of aao(So), a gene encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of Streptococcus oligofermentans. Here, we found that aao(So) and mutT, a homolog of pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of either gene did not impair the growth of S. oligofermentans, but double deletion  of both aao(So) and mutT was lethal. Quantitative PCR showed that the transcript  abundance of mutT was reduced for 13-fold in the aao(So) insertional mutant, indicating that gene polarity derived from the inactivation of aao(So) attenuated the expression of mutT. Enzymatic assays were conducted to determine the biochemical functions of LAAO and MutT of S. oligofermentans. The results indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H(2)O(2) (min · mg protein)(-1)]; and MutT showed the pyrophosphohydrolase activity, which removed mutagens such as 8-oxo-dGTP. Like paraquat, aao(So) mutations increased the expression of SOD, and addition of aminoacetone (final concentration, 5 mM) decreased the mutant's growth by 11%, indicating that the aao(So) mutants are under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in  both the deletion and insertional gene mutants of aao(So). Electron spin resonance spectroscopy showed increased hydroxyl radicals in both types of aao(So) mutant. This demonstrated that inactivation of aao(So) caused the elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our  study indicates that the presence of both LAAO and MutT can prevent endogenous metabolites-generated ROS and mutagens. In this way, we were able to determine the role of the aao(So)-mutT operon in antioxidant defense in S. oligofermentans. 
22615800	 Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that  BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria. 
22615794	 Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence  from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells.  Further optimization of this method should allow rapid and scalable genome engineering in human cells. 
22438905	 Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces  oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and  require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of  the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells. 
22912581	 Removal of the reproductive system of many animals including fish, flies, nematodes, mice and humans can increase lifespan through mechanisms largely unknown. The abrogation of the germline in Caenorhabditis elegans increases longevity by 60% due to a signal emitted from the somatic gonad. Apart from increased longevity, germline-less C. elegans is also resistant to other environmental stressors such as feeding on bacterial pathogens. However, the evolutionary conservation of this pathogen resistance, its genetic basis and an understanding of genes involved in producing this extraordinary survival phenotype are currently unknown. To study these evolutionary aspects we used the  necromenic nematode Pristionchus pacificus, which is a genetic model system used  in comparison to C. elegans. By ablation of germline precursor cells and subsequent feeding on the pathogen Serratia marcescens we discovered that P. pacificus shows remarkable resistance to bacterial pathogens and that this response is evolutionarily conserved across the Genus Pristionchus. To gain a mechanistic understanding of the increased resistance to bacterial pathogens and  longevity in germline-ablated P. pacificus we used whole genome microarrays to profile the transcriptional response comparing germline ablated versus un-ablated animals when fed S. marcescens. We show that lipid metabolism, maintenance of the proteasome, insulin signaling and nuclear pore complexes are essential for germline deficient phenotypes with more than 3,300 genes being differentially expressed. In contrast, gene expression of germline-less P. pacificus on E. coli  (longevity) and S. marcescens (immunity) is very similar with only 244 genes differentially expressed indicating that longevity is due to abundant gene expression also involved in immunity. By testing existing mutants of Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved function of both genes in resistance to bacterial pathogens and longevity. This is the first study to show that the influence of the reproductive system on extending lifespan and innate immunity is conserved in evolution. 
21930876	 Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone  (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging  to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis. 
21980953	 A variety of bacterial cell surface structures and quorum signalling molecules play a role in biofilm development in Escherichia coli. However, here we show that an engineered reduced-genome E. coli mutant that lacks 17.6% of the parental E. coli genome, including the genes involved in the synthesis of various cell surface structures, such as type 1 fimbriae, curli, exopolysaccharide polymers and the autoinducer-2 signalling molecule, is able to develop mature biofilms. Using temporal gene expression profiling, we investigated phenotypic changes in reduced-genome biofilms in relation with the genes encoding the synthesis of different amino acids that were differentially expressed during biofilm formation. We identified and characterized entB, marR, dosC, mcbR and yahK genes, as involved in biofilm formation by the reduced-genome E. coli. Of these, for a first time, we demonstrated that overproduction of entB and yahK, which encode an enterobactin for iron transport and a hypothetical oxidoreductase protein, respectively, promoted biofilm development and maturation. Our results indicate that specific types of genes contribute to phenotypic changes in reduced-genome E. coli biofilms. In addition, this work demonstrates that the functions of biofilm-specific genes could be analysed through experiments using the reduced-genome E. coli. 
22058017	 The entrance of influenza virus into host cells is facilitated by the attachment  of the globular region of viral hemagglutinin to the sialic acid receptors on host cell surfaces. In this study, we have cloned the cDNA fragment encoding the  entire globular region (residues 101-257) of hemagglutinin of the H9N2 type avian influenza virus (A/ck/Korea/ms96/96). The protein segment (denoted as the H9 peptide), which was expressed and purified in E. coli, was used for the immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific DNA aptamers with high affinity to H9 peptide, we conducted the SELEX method; 19  aptamers were newly isolated. A random mixture of these aptamers showed an increased level of binding affinity to the H9 peptide. The sequence alignment analysis of these aptamers revealed that 6 aptamers have highly conserved consensus sequences. Among these, aptamer C7 showed the highest similarity to the consensus sequences. Therefore, based on the C7 aptamer, we synthesized a new modified aptamer designated as C7-35M. This new aptamer showed strong binding capability to the viral particles. Furthermore, it could prevent MDCK cells from  viral infection by strong binding to the viral particles. These results suggest that our aptamers can recognize the hemagglutinin protein of avian influenza virus and inhibit the binding of the virus to target receptors required for the penetration of host cells. 
21936575	 The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA)  and the gene for E. coli 16S (hui). Optimization was performed using a Box-Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors  tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a  perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is  indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays. 
21930787	 The transcription factor Sox2 is a key player in the maintenance of pluripotency  and "stemness." We have previously shown that Sox2 maintains self-renewal in the  osteoblast lineage while inhibiting differentiation (U. Basu-Roy et al., Cell Death Differ. 17:1345-1353, 2010; A. Mansukhani, D. Ambrosetti, G. Holmes, L. Cornivelli, and C. Basilico, J. Cell Biol. 168:1065-1076, 2005). Sox2 also interferes with Wnt signaling by binding β-catenin, a central mediator of the Wnt pathway. Here we show that these multiple functions of Sox2 are encoded in distinct domains. The self-renewal function of Sox2 is dependent on its transcriptional activity and requires both its DNA-binding and C-terminal activation regions, while only the third C-terminal transactivation (TA) region is required for binding β-catenin and interfering with Wnt-induced transcription. The results of gene expression analysis upon Sox2 deletion strongly support the notion that Sox2 maintains stemness. We show also that Sox2 suppresses differentiation by attenuating Wnt signaling by posttranscriptional and transcriptional mechanisms and that adenomatous polyposis coli (APC) and GSK3β, which are negative regulators of the Wnt pathway, are direct Sox2 targets in osteoblasts. Several genes, such as the FoxP1 and BMI-1 genes, that are associated with stemness are downregulated upon Sox2 inactivation. Constitutive expression of the Polycomb complex member BMI-1 can bypass the Sox2 requirement for self-renewal but does not affect differentiation. Our results establish a connection between Sox2 and BMI-1 in maintaining self-renewal and identify BMI-1  as a key mediator of Sox2 function. 
21880599	 Next-generation sequencing has great potential for application in bacterial transcriptomics. However, unlike eukaryotes, bacteria have no clear mechanism to  select mRNAs over rRNAs; therefore, rRNA removal is a critical step in sequencing-based transcriptomics. Duplex-specific nuclease (DSN) is an enzyme that, at high temperatures, degrades duplex DNA in preference to single-stranded  DNA. DSN treatment has been successfully used to normalize the relative transcript abundance in mRNA-enriched cDNA libraries from eukaryotic organisms. In this study, we demonstrate the utility of this method to remove rRNA from prokaryotic total RNA. We evaluated the efficacy of DSN to remove rRNA by comparing it with the conventional subtractive hybridization (Hyb) method. Illumina deep sequencing was performed to obtain transcriptomes from Escherichia  coli grown under four growth conditions. The results clearly showed that our DSN  treatment was more efficient at removing rRNA than the Hyb method was, while preserving the original relative abundance of mRNA species in bacterial cells. Therefore, we propose that, for bacterial mRNA-seq experiments, DSN treatment should be preferred to Hyb-based methods. 
22125496	 As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested  reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These  networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target. 
21839093	 Using a recombinant, T=1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on  the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with "free" CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem-loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem-loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem-loops displaying the sequence motif AxxA. The implication is that there are many stem-loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem-loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism  based on kinetically driven folding of the RNA. 
21559929	 Cra is a pleiotropic regulatory protein that controls carbon and energy flux in enteric bacteria. Recent studies have shown that Cra also regulates other cell processes and influences biofilm formation. The purpose of the present study was  to investigate the role of Cra in biofilm formation in Escherichia coli. Congo red-binding studies suggested that curli biosynthesis is impaired in cra mutants. Microarray analysis of wild-type and mutant E. coli cultivated in conditions promoting biofilm formation revealed that the curli biosynthesis genes, csgBAC and csgDEFG, are poorly expressed in the mutant, suggesting that transcription of genes required for curli production is regulated by Cra. Four putative Cra-binding sites were identified in the curli intergenic region, which were experimentally validated by performing electromobility shift assays. Site-directed mutagenesis of three Cra-binding sites in the promoter region of the csgDEFG operon suggests that Cra activates transcription of this operon upon  binding to operator regions both downstream and upstream of the transcription start site. Based on the Cra-binding sites identified in this and other studies,  the Cra consensus sequence is refined. 
21865549	 Lipid A (a hexaacylated 1,4' bisphosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. In this article, we define the role of TLR4  and MD-2 in LPS signaling by using genome-wide expression profiling in TLR4- and  MD-2-deficient macrophages after stimulation with peptidoglycan-free LPS and synthetic Escherichia coli lipid A. Of the 1396 genes significantly induced or repressed by any one of the treatments in the wild-type macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin and that both are required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. According to our murine TLR4/MD-2-activation model, the two phosphates on lipid A were predicted to interact extensively with  the two positively charged patches on mouse TLR4. When either positive patch was  abolished by mutagenesis into Ala, the responses to LPS and lipid A were nearly abrogated. However, the MyD88-dependent and -independent pathways were impaired to the same extent, indicating that the adjuvant activity of monophosphorylated lipid A most likely arises from its decreased potential to induce an active receptor complex and not more downstream signaling events. Hence, we concluded that ionic interactions between lipid A and TLR4 are essential for optimal LPS receptor activation. 
21883529	 LeuO, the regulator of leucine biosynthesis operon of Escherichia coli, is involved in the regulation of as yet unspecified genes affecting the stress response and pathogenesis expression. To get insights into the regulatory role(s) of LeuO, Genomic SELEX screening has been performed to identify the whole set of  its regulation targets. A total of 140 LeuO-binding sites were identified on the  E. coli genome, of which as many as 133 (95%) were found to contain the binding sites of H-NS, the universal silencer of stress-response genes, supporting the concept that LeuO plays an antagonistic role with anti-silencing activity. Western blot analysis indicated that H-NS predominates in growing phase; however, after prolonged culture for 1 week, H-NS decreased instead LeuO increased, supporting the anti-silencing role of LeuO. In concert with this model, a set of  stress-response genes including cryptic chaperone/usher-type fimbriae operons are under the control of antagonistic interplay between LeuO and H-NS. Confocal laser scanning microscopic observation in flow-chambers showed that the mutants lacking leuO and some fimbriae genes are defective in biofilm formation or form altered biofilm architecture. Taken together we propose that LeuO is a major player in antagonistic interplay against the universal silencer H-NS. 
21803424	 Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in pigs and humans. The duration and severity of diarrhoea can be controlled using zinc supplementation, typically pharmacological levels of zinc oxide in pigs. In this  study, IPEC J2 cells were used as an in vitro model of intestinal ETEC infection, with separate and simultaneous zinc treatment. Genomic analysis identified increased expression of a variety of innate immune response genes (NF-κB targets) in response to ETEC exposure, and several stress response genes in response to zinc exposure, provided as ZnO. Expression of genes involved in the innate immune response was reduced when cells were simultaneously exposed to ZnO, and it is suggested that ZnO treatment inhibits the induction of NF-κB in response to pathogens, possibly through up-regulated heat shock proteins. A similar response  in vivo with consequent down-regulation in the inflammatory response would reduce further pathogen invasion, maintain normal gut function and maintain growth. 
21742923	 We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOS(FA,Ac)) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1,  and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P.  ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOS(FA,Ac), a corn fiber-derived substrate enriched in O-acetyl and ferulic acid  esters, but exhibited negligible activity on sugar acetates. As expected, the P.  ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose. 
21768388	 The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de  novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σ(E) activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell  from the loss of viability experienced when σ(E) activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σ(E) is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σ(E)) with the opposite repressor function. 
21642439	 Drug efflux represents an important protection mechanism in bacteria to withstand antibiotics and environmental toxic substances. Efflux genes constitute 6-18% of  all transporters in bacterial genomes, yet the expression and functions of only a handful of them have been studied. Among the 20 efflux genes encoded in the Escherichia coli K-12 genome, only the AcrAB-TolC system is constitutively expressed. The expression, activities, and physiological functions of the remaining efflux genes are poorly understood. In this study we identified a dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic growth condition of E. coli, which is independent of antibiotic exposure. We found that expression of MdtEF is up-regulated more than 20-fold under anaerobic  conditions by the global transcription factor ArcA, resulting in increased efflux activity and enhanced drug tolerance in anaerobically grown E. coli. Cells lacking mdtEF display a significantly decreased survival rate under the condition of anaerobic respiration of nitrate. Deletion of the genes responsible for the biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal electron acceptor during the anaerobic respiration restores the decreased survival of ΔmdtEF cells. Moreover, ΔmdtEF cells are susceptible to indole nitrosative derivatives, a class of toxic byproducts formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions. Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated  during the anaerobic physiology of E. coli to protect the bacterium from nitrosative damage through expelling the nitrosyl indole derivatives out of the cells. 
21398637	 Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ(70) (the housekeeping σ subunit) and σ(S) (an alternative  σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ(70) (Eσ(70)) or with σ(S) (Eσ(S)) using the whole Escherichia coli genome as DNA template. We found that Eσ(70), in the absence of any additional transcription factor, preferentially transcribes genes  associated with fast growth (e.g. ribosomal operons). In contrast, Eσ(S) efficiently transcribes genes involved in stress responses, secondary metabolism  as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the -35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the -10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the -10/+1 region could improve promoter recognition by Eσ(S). 
21627466	 In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of  detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88. 
21385089	 A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine.  Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for  antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes  detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB),  β-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and  tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in  common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal  sample shared resistance genes at a significant level (χ², p < 0.0000001). These  data suggest that there may have been horizontal exchange of AR genes between these bacteria or there may be a common source of AR genes in the swine environment for E. coli and Salmonella. 
21422176	 We previously isolated a spontaneous mutant of Escherichia coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, that  has colonization traits superior to the wild-type parent strain (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). This intestine-adapted strain (E. coli MG1655*) grew faster on several different carbon sources than the wild type and was nonmotile due to deletion of the flhD gene. We now report the results of several high-throughput genomic analysis approaches to further characterize E. coli MG1655*. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655*. Microarray analysis revealed modest yet significant induction of catabolic gene systems across the genome in both E.  coli MG1655* and an isogenic flhD mutant constructed in the laboratory. Catabolome analysis with Biolog GN2 microplates revealed an enhanced ability of both E. coli MG1655* and the isogenic flhD mutant to oxidize a variety of carbon  sources. The results show that intestine-adapted E. coli MG1655* is more fit than the wild type for intestinal colonization, because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, resulting  in more efficient carbon source utilization and a higher intestinal population. Hence, mutations that enhance metabolic efficiency confer a colonization advantage. 
21569465	 BACKGROUND: Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the E. coli genome. RESULTS: Through the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the in vivo binding of AtoC to the predicted sites. This process verified twenty-two  additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences. CONCLUSIONS: This study, by tracing a number of putative AtoC binding sites, has  indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation. 
21491941	 The transport properties of single-strand DNA probe-modified self-propelling micromachines are exploited for "on-the-fly" hybridization and selective single-step isolation of target nucleic acids from "raw" microliter biological samples (serum, urine, crude E. coli lysate, saliva). The rapid movement of the guided modified microrockets induces fluid convection, which enhances the hybridization efficiency, thus enabling the rapid and selective isolation of nucleic acid targets from untreated samples. The integration of these autonomous  microrockets into a lab-on-chip device that provides both nucleic acid isolation  and downstream analysis could thus be attractive for diverse applications. 
21421764	 CsgD, the master regulator of biofilm formation, activates the synthesis of curli fimbriae and extracellular polysaccharides in Escherichia coli. To obtain insights into its regulatory role, we have identified a total of 20 novel regulation target genes on the E. coli genome by using chromatin immunoprecipitation (ChIP)-on-chip analysis with a high-density DNA microarray. By DNase I footprinting, the consensus CsgD-binding sequence predicted from a total of 18 target sites was found to include AAAAGNG(N(2))AAAWW. After a promoter-lacZ fusion assay, the CsgD targets were classified into two groups: group I genes, such as fliE and yhbT, are repressed by CsgD, while group II genes, including yccT and adrA, are activated by CsgD. The fliE and fliEFGH operons for flagellum formation are directly repressed by CsgD, while CsgD activates the adrA gene, which encodes an enzyme for synthesis of cyclic di-GMP,  a bacterial second messenger, which in turn inhibits flagellum production and rotation. Taking these findings together, we propose that the cell motility for planktonic growth is repressed by CsgD, thereby promoting the switch to biofilm formation. 
21378183	 Hydrogen peroxide (H(2)O(2)) is commonly formed in microbial habitats by either chemical oxidation processes or host defense responses. H(2)O(2) can penetrate membranes and damage key intracellular biomolecules, including DNA and iron-dependent enzymes. Bacteria defend themselves against this H(2)O(2) by inducing a regulon that engages multiple defensive strategies. A previous microarray study suggested that yaaA, an uncharacterized gene found in many bacteria, was induced by H(2)O(2) in Escherichia coli as part of its OxyR regulon. Here we confirm that yaaA is a key element of the stress response to H(2)O(2). In a catalase/peroxidase-deficient (Hpx(-)) background, yaaA deletion mutants grew poorly, filamented extensively, and lost substantial viability when  they were cultured in aerobic LB medium. The results from a thyA forward mutagenesis assay and the growth defect of the yaaA deletion in a recombination-deficient (recA56) background indicated that yaaA mutants accumulated high levels of DNA damage. The growth defect of yaaA mutants could be suppressed by either the addition of iron chelators or mutations that slowed iron import, indicating that the DNA damage was caused by the Fenton reaction. Spin-trapping experiments confirmed that Hpx(-) yaaA cells had a higher hydroxyl  radical (HO(•)) level. Electron paramagnetic resonance spectroscopy analysis showed that the proximate cause was an unusually high level of intracellular unincorporated iron. These results demonstrate that during periods of H(2)O(2) stress the induction of YaaA is a critical device to suppress intracellular iron  levels; it thereby attenuates the Fenton reaction and the DNA damage that would otherwise result. The molecular mechanism of YaaA action remains unknown. 
21380435	 Gram-negative bacterium Escherichia coli and the Gram-positive Deinococcus radiodurans fundamentally differ in their cell structures and gene regulations. We have previously reported that IrrE, a Deinococcus genus-specific global regulator, confers significantly enhanced tolerance to various abiotic stresses.  To better understand the global effects of IrrE on the regulatory networks, we carried out combined transcriptome and proteome analysis of E. coli expressing the IrrE protein. Our analysis showed that 216 (4.8%) of all E. coli genes were induced and 149 (3.3%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon source utilization, amino acid utilization, acid resistance, a hydrogenase and an oxidase. Also regulated were the EvgSA two-component system, the GadE, GadX and PurR master regulators, and 10 transcription factors (AppY, GadW, YhiF, AsnC, BetI, CynR, MhpR, PrpR, TdcA and KdgR). These results demonstrated that IrrE acts as global regulator and consequently improves abiotic stress tolerances in the heterologous host E. coli. The implication of our findings is discussed in relation to the evolutionary role of horizontal gene transfer in bacterial regulatory networks and environmental adaptation. 
21516083	 Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology. Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ~0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells. 
21324911	 RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased  citrate concentration. Transcriptome analysis reveals a PNPase-mediated response  to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency. 
21417213	 Rapid analysis of pathogenic bacteria is essential for food and water control to  preserve the public health. Therefore, we report on a chemiluminescence (CL) flow-through DNA microarray assay for the rapid and sensitive quantification of the pathogenic bacteria Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni in water. Using the stopped polymerase chain reaction (PCR)  strategy, the amount of amplified target DNA was strongly dependent on the applied cell concentration. The amplification was stopped at the logarithmic phase of the PCR to quantify the DNA products on the DNA microarray chip. The generation of single-stranded DNA sequences is essential for DNA hybridization assays on microarrays. Therefore, the DNA strands of the PCR products were separated by streptavidin-conjugated magnetic nanoparticles. This was achieved by introducing a reverse primer labeled with biotin together with a digoxigenin labeled forward primer for CL microarray imaging. A conjugate of an antidigoxigenin antibody and horseradish peroxidase recognized the digoxigenin-labeled antistrands bound to the probes on the microarray surface and catalyzed the reaction of luminol and hydrogen peroxide. The generated light emission was recorded by a sensitive charge-coupled device (CCD) camera. The quantification was conducted by a flow-through CL microarray readout system. The  DNA microarrays were based on an NHS-activated poly(ethylene glycol)-modified glass substrate. The DNA probes which have the same DNA sequence as the reverse primer were immobilized on this surface. The full assay was characterized by spiking experiments with heat-inactivated bacteria in water. The total assay time was 3.5 h, and the detection limits determined on CL microarrays were for E. coli O157:H7, S. enterica , and C. jejuni 136, 500, and 1 cell/mL, respectively. The results of the DNA microarray assay were comparable to the SYBR green-based assays analyzed with a real-time PCR device. The advantage of the new microarray  analysis method is seen in the ability of a high multiplex degree on DNA microarrays, the high specificity of DNA hybridization on DNA microarrays, and the possibility to get quantitative results on an automated CL flow-through microarray analysis system. 
21149258	 Tiling microarrays have proven to be a valuable tool for gaining insights into the transcriptomes of microbial organisms grown under various nutritional or stress conditions. Here, we describe the use of such an array, constructed at the level of 20 nt resolution for the Escherichia coli MG1655 genome, to observe genome-wide changes in the steady-state RNA levels in mutants defective in either RNase E or RNase III. The array data were validated by comparison to previously published results for a variety of specific transcripts as well as independent northern analysis of additional mRNAs and sRNAs. In the absence of RNase E, 60% of the annotated coding sequences showed either increases or decreases in their steady-state levels. In contrast, only 12% of the coding sequences were affected  in the absence of RNase III. Unexpectedly, many coding sequences showed decreased abundance in the RNase E mutant, while more than half of the annotated sRNAs showed changes in abundance. Furthermore, the steady-state levels of many transcripts showed overlapping effects of both ribonucleases. Data are also presented demonstrating how the arrays were used to identify potential new genes, RNase III cleavage sites and the direct or indirect control of specific biological pathways. 
21435272	 BACKGROUND: Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress. RESULTS: The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the  complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS: We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis  targets for future global phenotype engineering. 
20679207	 Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a β-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the β-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during  growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans β-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles. 
21149585	 The regulated expression of virulence genes is critical for successful infection  by an intestinal pathogen. Bacteria rely on sensing environmental signals to find preferable niches and reach the infectious state. Orally ingested enterohemorrhagic Escherichia coli (EHEC) travels through the gastrointestinal tract and encounters a variety of environmental factors, some of which act as triggering signals for the induction of virulence genes. Butyrate, one of the main short-chain fatty acids (SCFAs), is such a signal, enhancing the expression  of genes for intimate attachment and type III secretion. We further explored the  role of SCFAs and found a positive effect of SCFAs on flagellar expression. Although EHEC did not produce flagella when grown in Dulbecco's modified Eagle's  medium (DMEM), a tissue culture medium that enhances virulence gene expression, the addition of SCFAs to the medium induced the production of flagella, and the EHEC bacteria became motile. Among SCFAs, butyrate simultaneously activates both  virulence and flagellar genes. Flagella did not affect initial adherence, and they were not expressed in adherent bacteria during microcolony formation. SCFAs  activated flagellar genes via two regulatory steps. Butyrate activated the flhDC  regulatory genes through leucine-responsive regulatory protein (Lrp), which is also a regulator of virulence genes. However, butyrate, acetate, and propionate also activated downstream genes independently of flhDC activation. Consequently,  when encountering increased concentrations of SCFAs, which are abundant in acetate, in the intestine, EHEC first activates flagellar production and motility, followed by genes involved in adherence and type III secretion, which leads to efficient adherence in a preferable niche. 
20676725	 Previous results have demonstrated that the silencing of adjacent genes encoding  NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ Microbiol 75:4315-4323, 2009]. This gene silencing is now reported to result from the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream  gene. YqhC shares homology with transcriptional regulators belonging to the AraC/XylS family and was shown to act as a positive regulator of the adjacent operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and by a direct comparison of furfural resistance and NADPH-dependent furfural reductase activity. Closely related bacteria contain yqhC, yqhD, and dkgA orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also present in more distantly related Gram-negative bacteria. Disruption of yqhC offers a useful approach to increase furfural tolerance in bacteria. 
21115656	 Cra (catabolite repressor activator) is a global regulator of the genes for carbon metabolism in Escherichia coli. To gain insights into the regulatory roles of Cra, attempts were made to identify the whole set of regulation targets using  an improved genomic SELEX (systematic evolution of ligands by exponential enrichment) system. Surprisingly, a total of 164 binding sites were identified for Cra, 144 (88%) of which were newly identified. The majority of known targets  were included in the SELEX chip pattern. The promoters examined by the lacZ reporter assay in vivo were all regulated by Cra. These two lines of evidence indicate that a total of as many as 178 promoters are under the control of Cra. The majority of Cra targets are the genes coding for the enzymes involved in central carbon metabolism, covering all the genes for the enzymes involved in glycolysis and metabolism downstream of glycolysis, including the tricarboxylic acid (TCA) cycle and aerobic respiration. Taken together, we propose that Cra plays a key role in balancing the levels of the enzymes for carbon metabolism. 
21299643	 Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to UV light or chemicals but is likely to encounter reactive oxygen species during infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack  an SOS system, although NG1427 shows amino acid sequence similarity to the SOS response regulator LexA from Escherichia coli. Similar to LexA and other S24 peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis requires the active and cleavage site residues conserved between LexA and NG1427. NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific, putative integral membrane protein; and recN, a DNA repair gene known to be required for oxidative damage survival. Full NG1427 regulon de-repression requires RecA following methyl methanesulphonate or mitomycin C treatment, but is largely RecA-independent following hydrogen peroxide treatment. NG1427 binds specifically to the operator regions of the genes it controls, and DNA binding is abolished by oxidation of the single cysteine residue encoded in NG1427. We propose that NG1427 is inactivated independently of RecA by oxidation. 
21299642	 Bacteria comprehensively reorganize their global gene expression when faced with  starvation. The alarmone ppGpp facilitates this massive response by co-ordinating the downregulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response. Such a large reorientation requires the activities of multiple regulators, yet the regulatory network downstream of ppGpp remains poorly defined. Transcription profiling during isoleucine depletion, which leads to gradual starvation (over > 100 min), allowed us to identify genes that required ppGpp, Lrp and RpoS for their induction and to deduce the regulon response times. Although the Lrp and RpoS regulons required ppGpp for their activation, they were not induced simultaneously. The data suggest that metabolic genes, i.e. those of the Lrp regulon, require only a low level of ppGpp for their induction. In contrast, the RpoS regulon was induced only when high levels of ppGpp accumulated. We tested several predictions of a model that explains how bacteria allocate transcriptional resources between metabolism and stress response by discretely tuning two regulatory circuits to different levels of ppGpp. The emergent regulatory structure insures that stress  survival circuits are only triggered if homeostatic metabolic networks fail to compensate for environmental deficiencies. 
20649646	 Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and bacterial indole derivatives are unstable in the microbial community because they are quickly degraded by diverse bacterial oxygenases. Hence, this work sought to  identify novel, non-toxic, stable and potent indole derivatives from plant sources for inhibiting the biofilm formation of E. coli O157:H7 and P. aeruginosa. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit the biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN more effectively inhibited biofilms than indole for the two pathogenic bacteria. Additionally, IAN decreased the production of virulence factors including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), pyocyanin and pyoverdine in P. aeruginosa. DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, whereas IAN induced indole-related genes and prophage genes in E. coli O157:H7. It appeared that IAN  inhibited the biofilm formation of E. coli by reducing curli formation and inducing indole production. Also, corroborating phenotypic results of P. aeruginosa, whole-transcriptomic data showed that IAN repressed virulence-related genes and motility-related genes, while IAN induced several small molecule transport genes. Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa. Additionally, indole-3-carboxyaldehyde was another natural biofilm inhibitor for both E. coli and P. aeruginosa. 
21324192	 BACKGROUND: The alarmone (p)ppGpp mediates a global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavorable conditions. Synthesis of (p)ppGpp is, in most bacteria, controlled by RelA/SpoT (Rsh) proteins. The role of (p)ppGpp has been characterized primarily in Escherichia coli and several Gram-positive bacteria. Here, we report the first in-depth analysis of the (p)ppGpp-regulon in an α-proteobacterium using a high-resolution tiling array to better understand the pleiotropic stress phenotype of a relA/rsh mutant. RESULTS: We compared gene expression of the Rhizobium etli wild type and rsh (previously rel) mutant during exponential and stationary phase, identifying numerous (p)ppGpp targets, including small non-coding RNAs. The majority of the 834 (p)ppGpp-dependent genes were detected during stationary phase. Unexpectedly, 223 genes were expressed (p)ppGpp-dependently during early exponential phase, indicating the hitherto unrecognized importance of (p)ppGpp during active growth. Furthermore, we identified two (p)ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, namely extracytoplasmic function sigma factor EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA. CONCLUSIONS: The regulatory role of (p)ppGpp in R. etli stress adaptation is far-reaching in redirecting gene expression during all growth phases. Genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. This work is an important step toward a full understanding of the regulatory network underlying stress responses in α-proteobacteria. 
21931480	 BACKGROUND: The focus of this study is on the antibacterial properties of silver  nanoparticles embedded within a zeolite membrane (AgNP-ZM). METHODS AND RESULTS: These membranes were effective in killing Escherichia coli and were bacteriostatic against methicillin-resistant Staphylococcus aureus. E. coli suspended in Luria Bertani (LB) broth and isolated from physical contact with the membrane were also killed. Elemental analysis indicated slow release of  Ag(+) from the AgNP-ZM into the LB broth. The E. coli killing efficiency of AgNP-ZM was found to decrease with repeated use, and this was correlated with decreased release of silver ions with each use of the support. Gene expression microarrays revealed upregulation of several antioxidant genes as well as genes coding for metal transport, metal reduction, and ATPase pumps in response to silver ions released from AgNP-ZM. Gene expression of iron transporters was reduced, and increased expression of ferrochelatase was observed. In addition, upregulation of multiple antibiotic resistance genes was demonstrated. The expression levels of multicopper oxidase, glutaredoxin, and thioredoxin decreased with each support use, reflecting the lower amounts of Ag(+) released from the membrane. The antibacterial mechanism of AgNP-ZM is proposed to be related to the exhaustion of antioxidant capacity. CONCLUSION: These results indicate that AgNP-ZM provide a novel matrix for gradual release of Ag(+). 
20829106	 To understand the responses of Escherichia coli to high succinate stress and to determine the roles of upregulated genes in high succinate tolerance, a continuous culture of wild-type E. coli W3110 was performed for 268 days in a gradually increasing concentration of succinate. Growth of the final adapted strain, designated DST160, proceeded growth rate of 0.20 h(-1) without a lag phase in medium containing 0.592 M succinate, while the wild-type strain showed 0.02 h(-1) in 38 h. The growth rates of DST160 in media containing either 0.61 M  NaCl, 0.61 M KCl, or at pH 4.5 were 25% higher, 18% lower, and 57% higher than those of wild-type, respectively, implying DST160 acquired salt tolerance and pH  shock tolerance as well as succinate tolerance. DNA microarray and real-time PCR  results indicated that genes controlling active transport and biosynthesis of osmoprotectants were upregulated in DST160 compared to W3110. When ygjE, encoding a putative tartrate/succinate antiporter, and betA, encoding betaine biosynthesis, were expressed in a wild-type E. coli as represent genes for active transport and osmoprotectant synthesis, respectively, greater growth rates were achieved under 0.592 M succinate stress conditions (seven times higher due to ygjE expression and six times higher due to betA expression) than wild-type. The  potential to design a metabolic engineering for microbial succinate production is suggested based on the transcriptional regulation of the long-term adapted DST160. 
21535697	 E. coli O157:H7 is an important foodborne pathogen, and ground beef is a common vehicle of infection. DNA microarrays have been used for transcriptomic studies of E. coli O157:H7 using laboratory media; however, analysis of gene expression in complex matrices such as food are lacking. This study compared gene expression profiles of E. coli O157:H7 Sakai strain in raw ground beef extract (GBE) and tryptic soy broth (TSB). Total RNA was isolated from GBE and TSB after 2 h of incubation with E. coli O157:H7. Following reverse transcription (RT) of the RNA, labeled cDNA was hybridized to microarrays representing 9608 open reading frames  (Operon; Genome Array-Ready Oligo Set) corresponding to 4 genomes of E. coli strains and 3 plasmids. There were 74 up-regulated (genes involved in protein and polysaccharide biosynthesis, transcription factors, membrane transport proteins,  and acid shock proteins) and 54 down-regulated (encoding proteins for energy metabolism, biosynthesis of cofactors, transporters of small molecules, and transcription factors and enzymes responsible for protein degradation) genes in E. coli O157:H7 grown in GBE compared to TSB, respectively. Furthermore, compared to incubation in TSB, E. coli O157:H7 incubated in GBE for 2 h showed significantly increased survival when exposed to synthetic gastric fluid, pH 1.5. This study demonstrated that microarray analyses can be performed using complex food matrices, and gene expression of E. coli O157:H7 differs in TSB compared to  GBE. The information will be useful for identification of genes that can be employed as potential targets for interventions to control E. coli O157:H7. 
21601690	 The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions vary depending upon the organism and the nature of the DNA-binding proteins under study. Here, we describe a customized ChIP protocol to examine the genome-wide distribution of a mobile DNA-binding enzyme, Escherichia coli RNA Polymerase (RNAP) as well as the factors that dynamically associate with RNAP during different stages of transcription. We describe new data analysis methods for determining the association of a broadly distributed DNA-binding complex. Further, we describe our approach of combining small molecules and antibiotics that perturb specific cellular events with ChIP and genomic platforms to dissect  mechanisms of gene regulation in vivo. The chemical genomic methods can be leveraged to map natural and cryptic promoters and transcription units, annotate  genomes, and reveal coupling between different processes in regulation of genes.  This approach provides the framework for engineering gene networks and controlling biological output in a desired manner. 
21815091	 Cellular processes are carried out through a series of molecular interactions. Various experimental approaches can be used to investigate these functional relationships on a large-scale. Recently, the power of investigating biological systems from the perspective of genetic (gene-gene or epistatic) interactions has been evidenced by the ability to elucidate novel functional relationships. Examples of functionally related genes include genes that buffer each other's function or impinge on the same biological process. Genetic interactions have traditionally been investigated in bacteria by combining pairs of mutations (e.g., gene deletions) and assessing deviation of the phenotype of each double mutant from an expected neutral (or no interaction) phenotype. Fitness is a particularly convenient phenotype to measure: when the double mutant grows faster or slower than expected, the two mutated genes are said to show alleviating or aggravating interactions, respectively. The most commonly used neutral model assumes that the fitness of the double mutant is equal to the product of individual single mutant fitness. A striking genetic interaction is exemplified by the loss of two nonessential genes that buffer each other in performing an essential biological function: deleting only one of these genes produces no detectable fitness defect; however, loss of both genes simultaneously results in  systems failure, leading to synthetic sickness or lethality. Systematic large-scale genetic interaction screens have been used to generate functional maps for model eukaryotic organisms, such as yeast, to describe the functional organization of gene products into pathways and protein complexes within a cell.  They also reveal the modular arrangement and cross talk of pathways and complexes within broader functional neighborhoods (Dixon et al., Annu Rev Genet 43:601-625, 2009). Here, we present a high-throughput quantitative Escherichia coli Synthetic Genetic Array (eSGA) screening procedure, which we developed to systematically infer genetic interactions by scoring growth defects among large numbers of double mutants in a classic Gram-negative bacterium. The eSGA method exploits the rapid colony growth, ease of genetic manipulation, and natural efficient genetic  exchange via conjugation of laboratory E. coli strains. Replica pinning is used to grow and mate arrayed sets of single gene mutant strains and to select double  mutants en masse. Strain fitness, which is used as the eSGA readout, is quantified by the digital imaging of the plates and subsequent measuring and comparing single and double mutant colony sizes. While eSGA can be used to screen select mutants to probe the functions of individual genes, using eSGA more broadly to collect genetic interaction data for many combinations of genes can help reconstruct a functional interaction network to reveal novel links and components of biological pathways as well as unexpected connections between pathways. A variety of bacterial systems can be investigated, wherein the genes impinge on a essential biological process (e.g., cell wall assembly, ribosome biogenesis, chromosome replication) that are of interest from the perspective of  drug development (Babu et al., Mol Biosyst 12:1439-1455, 2009). We also show how  genetic interactions generated by high-throughput eSGA screens can be validated by manual small-scale genetic crosses and by genetic complementation and gene rescue experiments. 
21857910	 Transcriptional networks consist of multiple regulatory layers corresponding to the activity of global regulators, specialized repressors and activators as well  as proteins and enzymes shaping the DNA template. Such intrinsic complexity makes uncovering connections difficult and it calls for corresponding methodologies, which are adapted to the available data. Here we present a new computational method that predicts interactions between transcription factors and target genes  using compendia of microarray gene expression data and documented interactions between genes and transcription factors. The proposed method, called Kernel Embedding of Regulatory Networks (KEREN), is based on the concept of gene-regulon association, and captures hidden geometric patterns of the network via manifold embedding. We applied KEREN to reconstruct transcription regulatory interactions  on a genome-wide scale in the model bacteria Escherichia coli (E. coli). Application of the method not only yielded accurate predictions of verifiable interactions, which outperformed on certain metrics comparable methodologies, but also demonstrated the utility of a geometric approach in the analysis of high-dimensional biological data. We also described possible applications of kernel embedding techniques to other function and network discovery algorithms. 
21673794	 CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets,  reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons. 
21203457	 BACKGROUND: Despite comprehensive investigation, the Escherichia coli SOS response system is not yet fully understood. We have applied custom designed whole genome tiling arrays to measure UV invoked transcriptional changes in E. coli. This study provides a more complete insight into the transcriptome and the  UV irradiation response of this microorganism. RESULTS: We detected a number of novel differentially expressed transcripts in addition to the expected SOS response genes (such as sulA, recN, uvrA, lexA, umuC and umuD) in the UV treated cells. Several of the differentially expressed transcripts might play important roles in regulation of the cellular response to  UV damage. We have predicted 23 novel small peptides from our set of detected non-gene transcripts. Further, three of the predicted peptides were cloned into protein expression vectors to test the biological activity. All three constructs  expressed the predicted peptides, in which two of them were highly toxic to the cell. Additionally, a remarkably high overlap with previously in-silico predicted non-coding RNAs (ncRNAs) was detected. Generally we detected a far higher transcriptional activity than the annotation suggests, and these findings correspond with previous transcription mappings from E. coli and other organisms. CONCLUSIONS: Here we demonstrate that the E. coli transcriptome consists of far more transcripts than the present annotation suggests, of which many transcripts  seem important to the bacterial stress response. Sequence alignment of promoter regions suggest novel regulatory consensus sequences for some of the upregulated  genes. Finally, several of the novel transcripts identified in this study encode  putative small peptides, which are biologically active. 
20848492	 BACKGROUND: A custom phylogenetic microarray composed of small subunit ribosomal  RNA probes, representing ≈500 bacterial species from the human and animal gut, was developed and evaluated for analysis of gut microbial diversity using fecal samples from healthy subjects and Crohn's disease (CD) patients. METHODS: Oligonucleotide probes (≈40 mer) used on the microarray were selected from published articles or designed with the "GoArray" microarray probe design program using selected bacterial 16S rRNA sequences. Fecal 16S rDNA from individual samples of six healthy subjects and six CD patients were used as template to generate fluorescently labeled cRNA that was hybridized to the microarray. Differences revealed by the microarray in relative abundance of microbial populations between healthy and diseased patients were verified using quantitative real-time polymerase chain reaction (PCR) with species-specific primer sets. RESULTS: The microarray analyses showed that Eubacterium rectale, Bacteroides fragilis group, B. vulgatus, Ruminococcus albus, R. callidus, R. bromii, and Faecalibacterium prausnitzii were 5-10-fold more abundant in the healthy subjects than in the CD patients, while Enterococcus sp., Clostridium difficile, Escherichia coli, Shigella flexneri, and Listeria sp. were more abundant in the CD group. CONCLUSIONS: The microarray detected differences in abundance of bacterial populations within the phylum Firmicutes that had been reported previously for the same samples based on phylogenetic analysis of metagenomic clone libraries. In addition, the microarray showed that Enterococcus sp. was in higher abundance  in the CD patients. This microarray should be another useful tool to examine the  diversity and abundance of human intestinal microbiota. 
20846334	 AIMS: To determine how stress response and virulence gene expression of stationary phase (SP) Escherichia coli O157:H7 are affected by nutrient levels. METHODS AND RESULTS: A targeted microarray (n=125 genes) was used to determine the impact of nutrient deprivation [15 min in 3-(N-Morpholino)propanesulfonic acid buffer] on SP E. coli O157:H7. In total, 24 genes were significantly affected (>1·5-fold; P <0·05) with 17 induced and seven attenuated. Additionally, 11 genes belonging to significantly affected stress response regulons were significantly induced (P<0·05), though <1·5-fold. Induced genes included global and specific stress response regulators, the mar operon, iron acquisition and virulence genes. In contrast, transcript for major porins and replicative genes were repressed. Comparison of the nutrient deprived transcriptome to that derived from nutrient replenished cells revealed a disparate transcriptome, with 44 genes expressed at significantly elevated levels in nutrient replenished cells, including all queried global and specific stress response regulators and key virulence genes. Genes expressed at elevated levels in nutrient deprived cells were related to σ(S) . The microarray data were validated by qRT-PCR. CONCLUSIONS:   SP E. coli O157:H7 were affected by nutrient deprivation, with both starvation-related and unrelated networks induced, thereby demonstrating how the E. coli O157:H7 stress response transcriptome is fine-tuned to environmental  conditions. Further, by comparison of starved cells to cells provided with fresh  nutrients, it is clear starved E. coli O157:H7 undergo massive physiological reprogramming dominated initially by stress response induction to adapt to a nutrient rich environment. SIGNIFICANCE AND IMPACT OF THE STUDY:   This study demonstrated how σ(S) -induced SP E. coli O157:H7 remain highly sensitive and adaptable to environmental conditions. Further, by examining how starved cells respond to nutrient-rich conditions, we show preliminary adaptation to a nutrient rich environment is dominated by the induction of diverse stress response networks. Combined, this provides E. coli O157:H7 stress physiology-based knowledge that can be used to design more effective food safety interventions. 
20688154	 Transmission of antimicrobial drug resistance from resistant bacteria to non-resistant strains is an important public health issue. In this study, we have examined the possibility of multiple resistance gene transfer between Escherichia coli and Salmonella in the natural setting. Bacteria isolated from calves concurrently shedding E. coli and Salmonella showed similar antimicrobial drug resistance patterns as measured by a broth dilution method. However, microarray analysis of the antibiotic resistance at the gene level revealed several differences in resistance gene profile. Resistance profiles of E. coli isolated from different farms were closer than the profile of E. coli and Salmonella isolated from the same farm. This shows that the chance of multiple resistance gene transfers between these species is unlikely. 
21108805	 BACKGROUND: Gene expression regulation is still poorly documented in bacteria with highly reduced genomes. Understanding the evolution and mechanisms underlying the regulation of gene transcription in Buchnera aphidicola, the primary endosymbiont of aphids, is expected both to enhance our understanding of  this nutritionally based association and to provide an intriguing case-study of the evolution of gene expression regulation in a reduced bacterial genome. RESULTS: A Bayesian predictor was defined to infer the B. aphidicola transcription units, which were further validated using transcriptomic data and RT-PCR experiments. The characteristics of B. aphidicola predicted transcription  units (TUs) were analyzed in order to evaluate the impact of operon map organization on the regulation of gene transcription.On average, B. aphidicola TUs contain more genes than those of E. coli. The global layout of B. aphidicola  operon map was mainly shaped by the big reduction and the rearrangements events,  which occurred at the early stage of the symbiosis. Our analysis suggests that this operon map may evolve further only by small reorganizations around the frontiers of B. aphidicola TUs, through promoter and/or terminator sequence modifications and/or by pseudogenization events. We also found that the need for  specific transcription regulation exerts some pressure on gene conservation, but  not on gene assembling in the operon map in Buchnera. Our analysis of the TUs spacing pointed out that a selection pressure is maintained on the length of the  intergenic regions between divergent adjacent gene pairs. CONCLUSIONS: B. aphidicola can seemingly only evolve towards a more polycistronic operon map. This implies that gene transcription regulation is probably subject to weak selection pressure in Buchnera conserving operons composed of genes with  unrelated functions. 
20809562	 We report here a new small molecule fluorogen and RNA aptamer pair for RNA labeling. The small-molecule fluorogen is designed on the basis of fluorescently  quenched sulforhodamine dye. The SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure and fluorescence screening in E. coli have been applied to discover the aptamer that can specifically activate the fluorogen with micromolar binding affinity. The systematic mutation and truncation study on the  aptamer structure determined the minimum binding domain of the aptamer. A series  of rationally modified fluorogen analogues have been made to probe the interacting groups of fluorogen with the aptamer. These results led to the design of a much improved fluorogen ASR 7 that displayed a 33-fold increase in the binding affinity for the selected aptamer in comparison to the original ASR 1 and an 88-fold increase in the fluorescence emission after the aptamer binding. This  study demonstrates the value of combining in vitro SELEX and E. coli fluorescence screening with rational modifications in discovering and optimizing new fluorogen-RNA aptamer labeling pairs. 
21072209	 BACKGROUND: Escherichia coli AlkB is a 2-oxoglutarate- and iron-dependent dioxygenase that reverses alkylated DNA damage by oxidative demethylation. Mouse  AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of mammalian dioxygenases. METHODS AND FINDINGS: In the present study we show non-Mendelian inheritance of the Alkbh1 targeted allele in mice. Both Alkbh1(-/-) and heterozygous Alkbh1(+/-) offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is considerably skewed against female offspring, with one female born for every three to four males. Most mechanisms that cause segregation distortion, act in the male gametes and affect male fertility. The skewing of the sexes appears to be of paternal origin, and might be set in the pachythene stage of meiosis during spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. In testes, apoptotic spermatids were revealed in 5-10% of the tubules in Alkbh1(-/-) adults. The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5 during embryonic development. This is consistent with the incompletely penetrant  phenotypes observed, particularly recurrent unilateral eye defects and craniofacial malformations. CONCLUSIONS: Genetic and phenotypic assessment suggests that Alkbh1 mediates gene regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio  distribution and embryonic development in mice. 
20929247	 The utility and performance of label-free, oligonucleotide probes for reagentless detection of dilute target analytes was examined using a voltammetric transduction principle in an array format. Multistep, solid-state fabrication yielded preproduction arrays of 16 individually addressable, 30 μm diameter microelectrodes in a 30 mm × 6.5 mm × 0.5 mm dipstick disposable device. The specificity of 16 nucleotide (nt) 2'-O-methylribonucleic acid and 22 nt DNA backbone probes bound through Mg(2+)-phosphonate bridges to polypyrrole films on  the microelectrodes were studied using microbial target RNAs of various lengths.  Probe-specific interactions with Escherichia coli O157 H7 23S rRNA (2907 nt) and  Candida albicans 18S rRNA (1788 nt) were detected at 65 and 58 fmol/mL, respectively, in volumes as low as 0.5 mL. Specificity studies showed that, for a given probe, "nontarget" transcripts can contribute to changes in the voltammetric detection signal, though with responses that never exceed 70% of the detection signal acquired for specifically designed complementary targets. These  results statistically validate the use of the voltammetric microelectrode array for obtaining a "yes-no" answer on complementary specific binding. The study also identifies challenges and pitfalls for the selection strategies of oligonucleotide probes. 
20805330	 The Haemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in Escherichia coli. CpxR, the cytoplasmic response regulator, was shown previously  to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs  also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response. 
20727995	 Much of our knowledge on heredity, development, physiology and the underlying cellular and molecular processes is derived from the studies of model, or reference, organisms. Despite the great variety of life, a common base of shared  principles could be extracted by studying a few life forms, selected based on their amenability to experimental studies. Very briefly, the origins of a few model organisms are described, including E. coli, yeast, C. elegans, Drosophila,  Xenopus, zebrafish, mouse, maize and Arabidopsis. These model organisms were chosen because of their importance and wide use, which made them systems of choice for genome-wide studies. Many of their genomes were between the first to be fully sequenced, opening unprecedented opportunities for large-scale transcriptomics and proteomics studies. 
20707865	 Single-gene mutants with extended lifespan have been described in several model organisms. We performed a genome-wide screen for long-lived mutants in Escherichia coli, which revealed strains lacking tricarboxylic acid (TCA)-cycle-related genes that exhibit longer stationary-phase survival and increased resistance to heat stress compared to wild-type. Extended lifespan in the sdhA mutant, lacking subunit A of succinate dehydrogenase, is associated with the reduced production of superoxide and increased stress resistance. On the other hand, the longer lifespan of the lipoic acid synthase mutant (lipA) is associated with reduced oxygen consumption and requires the acetate-producing enzyme pyruvate oxidase, as well as acetyl-CoA synthetase, the enzyme that converts extracellular acetate to acetyl-CoA. The hypoxia-inducible transcription factor ArcA, acting independently of acetate metabolism, is also required for maximum lifespan extension in the lipA and lpdA mutants, indicating that these mutations promote entry into a mode normally associated with a low-oxygen environment. Because analogous changes from respiration to fermentation have been observed in long-lived Saccharomyces cerevisiae and Caenorhabditis elegans strains, such metabolic alterations may represent an evolutionarily conserved strategy to extend lifespan. 
20825684	 BACKGROUND: Transcriptional regulatory network inference (TRNI) from large compendia of DNA microarrays has become a fundamental approach for discovering transcription factor (TF)-gene interactions at the genome-wide level. In correlation-based TRNI, network edges can in principle be evaluated using standard statistical tests. However, while such tests nominally assume independent microarray experiments, we expect dependency between the experiments  in microarray compendia, due to both project-specific factors (e.g., microarray preparation, environmental effects) in the multi-project compendium setting and effective dependency induced by gene-gene correlations. Herein, we characterize the nature of dependency in an Escherichia coli microarray compendium and explore its consequences on the problem of determining which and how many arrays to use in correlation-based TRNI. RESULTS: We present evidence of substantial effective dependency among microarrays in this compendium, and characterize that dependency with respect to  experimental condition factors. We then introduce a measure neff of the effective number of experiments in a compendium, and find that corresponding to the dependency observed in this particular compendium there is a huge reduction in effective sample size i.e., neff = 14.7 versus n = 376. Furthermore, we found that the neff of select subsets of experiments actually exceeded neff of the full compendium, suggesting that the adage 'less is more' applies here. Consistent with this latter result, we observed improved performance in TRNI using subsets of the data compared to results using the full compendium. We identified experimental condition factors that trend with changes in TRNI performance and neff , including growth phase and media type. Finally, using the set of known E.  coli genetic regulatory interactions from RegulonDB, we demonstrated that false discovery rates (FDR) derived from neff -adjusted p-values were well-matched to FDR based on the RegulonDB truth set. CONCLUSIONS: These results support utilization of neff as a potent descriptor of  microarray compendia. In addition, they highlight a straightforward correlation-based method for TRNI with demonstrated meaningful statistical testing for significant edges, readily applicable to compendia from any species,  even when a truth set is not available. This work facilitates a more refined approach to construction and utilization of mRNA expression compendia in TRNI. 
20559621	 Biofilms are communities of bacteria whose formation on surfaces requires a large portion of the bacteria's transcriptional network. To identify environmental conditions and transcriptional regulators that contribute to sensing these conditions, we used a high-throughput approach to monitor biofilm biomass produced by an isogenic set of Escherichia coli K-12 strains grown under combinations of environmental conditions. Of the environmental combinations, growth in tryptic soy broth at 37 degrees C supported the most biofilm production. To analyze the complex relationships between the diverse cell-surface organelles, transcriptional regulators, and metabolic enzymes represented by the  tested mutant set, we used a novel vector-item pattern-mining algorithm. The algorithm related biofilm amounts to the functional annotations of each mutated protein. The pattern with the best statistical significance was the gene ontology 'pyruvate catabolic process,' which is associated with enzymes of acetate metabolism. Phenotype microarray experiments illustrated that carbon sources that are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are particularly supportive of biofilm formation. Scanning electron microscopy revealed structural differences between mutants that lack acetate metabolism enzymes and their parent and confirmed the quantitative differences. We conclude  that acetate metabolism functions as a metabolic sensor, transmitting changes in  environmental conditions to biofilm biomass and structure. 
20491932	 The vast majority of experimental data have been accumulated on the transcription regulation of individual genes within a single model prokaryote, Escherichia coli, which form the well-established on-off switch model of transcription by DNA-binding regulatory proteins. After the development of modern high-throughput  experimental systems such as microarray analysis of whole genome transcription and the Genomic SELEX search for the whole set of regulation targets by transcription factors, a number of E. coli promoters are now recognized to be under the control of multiple transcription factors, as in the case of eukaryotes. The number of regulation targets of a single transcription factor has also been found to be more than hitherto recognized, ranging up to hundreds of promoters, genes or operons for several global regulators. The multifactor promoters and the multitarget transcription factors can be assembled into complex networks of transcription regulation, forming hierarchical networks. 
20692677	 Escherichia coli growth in biofilms and growth at a suboptimal temperature of 20  °C have been shown to decrease sensitivity to monochloramine (Berry, D., C. Xi, L. Raskin. 2009. Environ. Sci. Technol. 43, 884-889). In order to better understand why growth conditions affect sensitivity to monochloramine, a comparative transcriptomic approach was used to identify common patterns of differentially-expressed genes under these growth conditions and during monochloramine exposure. This approach revealed a set of differentially-expressed genes shared under multiple conditions (planktonic growth at 20 °C, biofilm growth, and exposure of planktonic cells to monochloramine), with nine genes shared under all three conditions. Functional gene categories enriched in the shared gene sets included: general metabolic inhibition, redox and oxidoreductase response, cell envelope integrity response, control of iron and sulfur transport  metabolism and several genes of unknown function. Single gene deletion mutant analyses verified that loss of 15 of the 24 genes up-regulated during monochloramine exposure as well as during other tested conditions increased E. coli sensitivity to monochloramine up to two fold. Constitutive expression of down-regulated genes in single gene mutants yielded mixed results, indicating that the expression of some down-regulated genes actually decreases sensitivity to monochloramine. These results contribute to the understanding of the bacterial response to disinfectants by characterizing the overlap between growth condition  associated stress responses and monochloramine-associated stress responses. This  characterization highlights the bacterial responses responsible for decreased sensitivity to monochloramine under different growth conditions. 
20865122	 Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain's evolution in different deliberately colonized patients  and the surprising insight that hosts appear to personalize their microflora. By  first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization. 
20562308	 AphB is a LysR-type activator that initiates the expression of the virulence cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we carried out a microarray analysis and show here that AphB influences the expression of a number of genes that are not associated with the VPI. One gene strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional activator responsible for activating the expression of lysine decarboxylase, which plays an important role in survival at low pH. Other genes activated by AphB encode a Na(+)/H(+) antiporter, a carbonic anhydrase, a member of the ClC family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB influences each of these genes directly by recognizing a conserved binding site within their promoters, as determined by gel mobility shift assays. Transcriptional lacZ fusions indicate that AphB activates the expression of these genes under aerobic conditions in response to low pH and also under anaerobic conditions at neutral pH. Further experiments show that the regulation of cadC by AphB in response to low pH and anaerobiosis is mirrored in the heterologous organism Escherichia coli, is independent of the global regulators Fnr and ArcAB, and depends upon the region of the promoter that contains the AphB binding site.  These results raise the possibility that the activity of AphB is influenced by the pH and oxygen tension of the environment. 
20639866	 A fundamental goal in biotechnology and biology is the development of approaches  to better understand the genetic basis of traits. Here we report a versatile method, trackable multiplex recombineering (TRMR), whereby thousands of specific  genetic modifications are created and evaluated simultaneously. To demonstrate TRMR, in a single day we modified the expression of >95% of the genes in Escherichia coli by inserting synthetic DNA cassettes and molecular barcodes upstream of each gene. Barcode sequences and microarrays were then used to quantify population dynamics. Within a week we mapped thousands of genes that affect E. coli growth in various media (rich, minimal and cellulosic hydrolysate) and in the presence of several growth inhibitors (beta-glucoside, D-fucose, valine and methylglyoxal). This approach can be applied to a broad range of traits to identify targets for future genome-engineering endeavors. 
20427277	 Peroxynitrite is formed in macrophages by the diffusion-limited reaction of superoxide and nitric oxide. This highly reactive species is thought to contribute to bacterial killing by interaction with diverse targets and nitration of protein tyrosines. This work presents for the first time a comprehensive analysis of transcriptional responses to peroxynitrite under tightly controlled chemostat growth conditions. Up-regulation of the cysteine biosynthesis pathway and an increase in S-nitrosothiol levels suggest S-nitrosylation to be a consequence of peroxynitrite exposure. Genes involved in the assembly/repair of iron-sulfur clusters also show enhanced transcription. Unexpectedly, arginine biosynthesis gene transcription levels were also elevated after treatment with peroxynitrite. Analysis of the negative regulator for these genes, ArgR, showed that post-translational nitration of tyrosine residues within this protein is responsible for its degradation in vitro. Further up-regulation was seen in oxidative stress response genes, including katG and ahpCF. However, genes known to be up-regulated by nitric oxide and nitrosating agents (e.g. hmp and norVW) were unaffected. Probabilistic modeling of the transcriptomic data identified five altered transcription factors in response to peroxynitrite exposure, including OxyR and ArgR. Hydrogen peroxide can be present as a contaminant in commercially available peroxynitrite preparations. Transcriptomic analysis of cells treated with hydrogen peroxide alone also revealed up-regulation of oxidative stress response genes but not of many other genes that are up-regulated by peroxynitrite. Thus, the cellular responses to peroxynitrite and hydrogen peroxide are distinct. 
20362014	 A microarray was developed to simultaneously screen Escherichia coli and Salmonella enterica for multiple genetic traits. The final array included 203 60-mer oligonucleotide probes, including 117 for resistance genes, 16 for virulence genes, 25 for replicon markers, and 45 other markers. Validity of the array was tested by assessing inter-laboratory agreement among four collaborating groups using a blinded study design. Internal validation indicated that the assay was reliable (area under the receiver-operator characteristic curve=0.97). Inter-laboratory agreement, however, was poor when estimated using the intraclass correlation coefficient, which ranged from 0.27 (95% confidence interval 0.24, 0.29) to 0.29 (0.23, 0.34). These findings suggest that extensive testing and procedure standardization will be needed before bacterial genotyping arrays can be readily shared between laboratories. 
20299454	 Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed  NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of  NADH and ATP, respectively. We used a global interaction network, comprising of protein interactions, transcriptional regulation, and metabolic networks, to integrate data from transcription profiles, metabolic fluxes, and the metabolite  levels. We identified high-scoring networks for the two strains. The results revealed a smaller, but denser network for perturbations of ATP level, compared with that of NADH level. The action of many global transcription factors such as  ArcA, Fnr, CRP, and IHF commonly involved both NADH and ATP, whereas others responded to either ATP or NADH. Overexpressing NADH oxidase invokes response in  widespread aspects of metabolism involving the redox cofactors (NADH and NADPH),  whereas ATPase has a more focused response to restore ATP level by enhancing proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH played a key role in restoring redox homeostasis through the concerted activity of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled network of regulation that illustrates the overlapping and distinct aspects of metabolism controlled by NADH and ATP. Our study contributes to the general understanding of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli. 
20368404	 Bovine-origin Escherichia coli isolates were tested for resistance phenotypes using a disk diffusion assay and for resistance genotypes using a DNA microarray. An isolate with gentamicin and amikacin resistance but with no corresponding genes detected yielded a 1,056-bp DNA sequence with the closest homologues for its inferred protein sequence among a family of 16S rRNA methyltransferase enzymes. These enzymes confer high-level aminoglycoside resistance and have only  recently been described in Gram-negative bacteria. 
20453079	 The development of codon bias indices (CBIs) remains an active field of research  due to their myriad applications in computational biology. Recently, the relative codon usage bias (RCBS) was introduced as a novel CBI able to estimate codon bias without using a reference set. The results of this new index when applied to Escherichia coli and Saccharomyces cerevisiae led the authors of the original publications to conclude that natural selection favours higher expression and enhanced codon usage optimization in short genes. Here, we show that this conclusion was flawed and based on the systematic oversight of an intrinsic bias  for short sequences in the RCBS index and of biases in the small data sets used for validation in E. coli. Furthermore, we reveal that how the RCBS can be corrected to produce useful results and how its underlying principle, which we here term relative codon adaptation (RCA), can be made into a powerful reference-set-based index that directly takes into account the genomic base composition. Finally, we show that RCA outperforms the codon adaptation index (CAI) as a predictor of gene expression when operating on the CAI reference set and that this improvement is significantly larger when analysing genomes with high mutational bias. 
20356716	 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. 
20410062	 OBJECTIVES: To gain further insight into the defence mechanisms against triclosan in a mutant derived from an Escherichia coli strain carrying the triclosan-resistant target enzyme, FabI(G93V). METHODS: An E. coli imp4231 FabI(G93V) strain was constructed by replacing intact fabI with a linear DNA cassette, fabI(G93V)-CmR, that contains a single mutation, GGT to GTT, at codon 93 of fabI(G93V) and a chloramphenicol resistance gene (CmR) as a marker for the mutant allele by a Red-mediated recombination system. Using this E. coli imp4231 FabI(G93V) strain, nitrosoguanidine (NTG) mutagenesis was performed to generate E. coli IFNs [imp4231 FabI(G93V) treated with NTG] displaying higher MICs of triclosan than its parent strain. The genes overexpressed in E. coli IFN4 were identified by DNA microarray analysis. RESULTS: An E. coli imp4231 FabI(G93V) strain displays approximately 400-fold increased MICs of triclosan (MIC approximately 8 mg/L) compared with the parent strain (MIC approximately 0.02 mg/L). Furthermore, E. coli IFN4 has the highest MIC of triclosan (MIC approximately 80 mg/L). DNA microarray analysis of E. coli  IFN4 shows that many genes involved in the biosynthesis of membrane proteins, including transporters, reductases/dehydrogenases and stress response regulators, were highly expressed in the mutant. CONCLUSIONS: These results strongly indicate that E. coli IFN cells might protect themselves from triclosan by activating various defence mechanisms, such as (i) changing efflux activities; (ii) capturing the triclosan; and (iii) increasing the expression of important regulators or metabolic enzymes. 
19919618	 AIMS: To complement our proteome study, whole-transcriptome analyses were utilized here to identify proteins related to degrading cis-1,2-dichloroethylene  (cis-DCE). METHODS AND RESULTS: Metabolically engineered Escherichia coli strains were utilized expressing an evolved toluene ortho-monooxygenase along with either (i)  glutathione S-transferase and altered gamma-glutamylcysteine synthetase or (ii) a rationally engineered epoxide hydrolase. cis-DCE degradation induced 30 known stress genes and 32 uncharacterized genes. Because of the reactive cis-DCE epoxides formed, we hypothesized that some of these uncharacterized genes may be  related to a variety of stresses. Using isogenic mutants, IbpB, YchH, YdeI, YeaR, YgiW, YoaG and YodD were related to hydrogen peroxide, cadmium and acid stress. Additional whole-transcriptome studies with hydrogen peroxide stress using the most hydrogen peroxide-sensitive mutants, ygiW and ychH, identified that FliS, GalS, HcaR, MglA, SufE, SufS, Tap, TnaB, YhcN and YjaA are also involved in the stress response of E. coli to hydrogen peroxide, cadmium and acid, as well as are involved in biofilm formation. CONCLUSION: Seventeen proteins are involved in the stress network for this organism, and YhcN and YchH were shown to be important for the degradation of cis-DCE. SIGNIFICANCE AND IMPACT OF THE STUDY: Six previously uncharacterized proteins (YchH, YdeI, YgiW, YhcN, YjaA and YodD) were shown to be stress proteins. 
20348540	 An unexpectedly high number of regulatory RNAs have been recently discovered that fine-tune the function of genes at all levels of expression. We employed Genomic  SELEX, a method to identify protein-binding RNAs encoded in the genome, to search for further regulatory RNAs in Escherichia coli. We used the global regulator protein Hfq as bait, because it can interact with a large number of RNAs, promoting their interaction. The enriched SELEX pool was subjected to deep sequencing, and 8865 sequences were mapped to the E. coli genome. These short sequences represent genomic Hfq-aptamers and are part of potential regulatory elements within RNA molecules. The motif 5'-AAYAAYAA-3' was enriched in the selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed  to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the antisense strand of protein coding genes than on the sense strand. They were enriched opposite to translation start sites or opposite to intervening sequences between ORFs in operons. These results expand the repertoire of Hfq targets and also suggest that Hfq might regulate the expression of a large number of genes via interaction with cis-antisense RNAs. 
20156994	 A systematic search was performed for DNA-binding sequences of YgiP, an uncharacterized transcription factor of Escherichia coli, by using the Genomic SELEX. A total of 688 YgiP-binding loci were identified after genome-wide profiling of SELEX fragments with a high-density microarray (SELEX-chip). Gel shift and DNase-I footprinting assays indicated that YgiP binds to multiple sites along DNA probes with a consensus GTTNATT sequence. Atomic force microscope observation indicated that at low concentrations, YgiP associates at various sites on DNA probes, but at high concentrations, YgiP covers the entire DNA surface supposedly through protein-protein contact. The intracellular concentration of YgiP is very low in growing E. coli cells under aerobic conditions, but increases more than 100-fold to the level as high as the major nucleoid proteins under anaerobic conditions. An E. coli mutant lacking ygiP showed retarded growth under anaerobic conditions. High abundance and large number of binding sites together indicate that YgiP is a nucleoid-associated protein with both architectural and regulatory roles as the nucleoid proteins Fis and IHF. We then propose that YgiP is a novel nucleoid protein of E. coli under anaerobiosis and propose to rename it Dan (DNA-binding protein under anaerobic conditions). 
20227352	 (c) 2010 Elsevier B.V. All rights reserved. 
20452492	 OBJECTIVE: The purpose of this study was to test the hypothesis that periodontopathic bacteria exert potent proinflammatory effects in human decidua. STUDY DESIGN: The immunostimulatory effects of Gram-positive and negative periodontopathic bacteria and their lipopolysaccharides were tested in human decidual cell cultures in comparison with Escherichia coli. Cytokine production was measured by enzyme-linked immunosorbent assay; inflammatory gene expression was measured by oligonucleotide arrays and quantitative real time-polymerase chain reaction. RESULTS: All bacteria that were tested elicited an inflammatory response, although concentration-dependence and efficacy varied considerably with organism  and culture. Lipopolysaccharides were more potent stimuli than intact bacterial cells, although bacteria exerted greater effects at high concentrations. Of 112 genes on the arrays, 18 genes were stimulated significantly by one or more lipopolysaccharide preparation. CONCLUSION: The ability of periodontopathic bacteria to stimulate a decidual inflammatory response is highly variable and partly dependent on the presence and structure of constituent lipopolysaccharides. This adds to our understanding of the causal association between periodontal disease and preterm birth. 
20364295	 A rapid detection method for Enterohemorrhagic Escherichia coli (EHEC), which has the virulent stx2 gene, was developed using a two-step, ultra-rapid real-time (URRT) PCR. URRT PCR was designed to detect the stx2 gene using a microchip-based, real-time PCR system, GenSpector TMC-1000, which only has a 6 microl total reaction volume with an extremely short denaturation step and combined annealing/extension step (1 and 3 s, respectively) for each cycle. Specific primers for the stx2 gene were designed to amplify a 100 bp region known for genetic stability among the various EHEC strains. Using the URRT PCR method,  stx2 gene could be detected in 7 min 8 s including melting point (Tm) analysis. The detection limit for the stx2 gene for URRT-PCR was estimated to be 3 c.f.u./PCR with the amplification product having a consistent Tm of 85.2 +/- 0.4  degrees C. This method was tested for the various applications relevant to the different EHEC strains and was useful for the rapid detection of stx2-carrying EHEC strains. 
20105222	 Previously we identified that the Escherichia coli protein MqsR (YgiU) functions  as a toxin and that it is involved in the regulation of motility by quorum sensing signal autoinducer-2 (AI-2). Furthermore, MqsR is directly associated with biofilm development and is linked to the development of persister cells. Here we show that MqsR and MqsA (YgiT) are a toxin/antitoxin (TA) pair, which, in significant difference to other TA pairs, regulates additional loci besides its own. We have recently identified that MqsR functions as an RNase. However, using  three sets of whole-transcriptome studies and two nickel-enrichment DNA binding microarrays coupled with cell survival studies in which MqsR was overproduced in  isogenic mutants, we identified eight genes (cspD, clpX, clpP, lon, yfjZ, relB, relE and hokA) that are involved in a mode of MqsR toxicity in addition to its RNase activity. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) showed that (i) the MqsR/MqsA complex (and MqsA alone) represses the toxin gene cspD, (ii) MqsR overproduction induces cspD, (iii) stress induces cspD, and (iv) stress fails to induce cspD when MqsR/MqsA are overproduced or when mqsRA is deleted. Electrophoretic mobility shift assays show that the MqsA/MqsR complex binds the promoter of cspD. In addition, proteases Lon and ClpXP are necessary for MqsR toxicity. Together, these results indicate the MqsR/MqsA complex represses cspD which may be derepressed by titrating MqsA with  MqsR or by degrading MqsA via stress conditions through proteases Lon and ClpXP.  Hence, we demonstrate that the MqsR/MqsA TA system controls cell physiology via its own toxicity as well as through its regulation of another toxin, CspD. 
20237100	 Oligonucleotide chips targeting the bacterial internal transcribed spacer region  (ITS) of the 16S-23S rRNA gene, which contains genus- and species-specific regions, were developed and evaluated. Forty-three sequences were designed consisting of 1 universal, 3 Gram stain-specific, 9 genus-specific, and 30 species-specific probes. The specificity of the probes was confirmed using bacterial type strains including 54 of 52 species belonging to 18 genera. The performance of the probes was evaluated using 825 consecutive samples that were positive by blood culture in broth medium. Among the 825 clinical specimens, 708  (85.8%) were identified correctly by the oligonucleotide chip. Most (536 isolates, or 75.7%) were identified as staphylococci, Escherichia coli, or Klebsiella pneumoniae. Thirty-seven isolates (4.5%) did not bind to the corresponding specific probes. Most of these also were staphylococci, E. coli, or K. pneumoniae and accounted for 6.3% of total number of the species. Sixty-two specimens (7.5%) did not bind the genus- or species-specific probes because of lack of corresponding specific probes. Among them, Acinetobacter baumannii was the single most frequent isolate (26/62). The oligonucleotide chip was highly specific and sensitive in detecting the causative agents of bacteremia directly from positive blood cultures. 
19998280	 (c) 2009 Wiley Periodicals, Inc. 
20075074	 Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post-transcriptional regulation of many genes. A number of these highly conserved ribo-regulators are stringently regulated at the level of transcription and are part of major regulons that deal  with the immediate response to various stress conditions, indicating that every major transcription factor may control the expression of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named FnrS, possesses signatures of  base-pairing RNAs, and we show by employing global proteomic and transcriptomic profiling that the expression of multiple genes is negatively regulated by the sRNA. Intriguingly, many of these genes encode enzymes with "aerobic" functions or enzymes linked to oxidative stress. Furthermore, in previous work most of the  potential target genes have been shown to be repressed by FNR through an undetermined mechanism. Collectively, our results provide insight into the mechanism by which FNR negatively regulates genes such as sodA, sodB, cydDC, and  metE, thereby demonstrating that adaptation to anaerobic growth involves the action of a small regulatory RNA. 
20066496	 Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium. 
20222947	 BACKGROUND: 6S RNA from E. coli is known to bind to RNA polymerase interfering with transcription initiation. Because 6S RNA concentrations are maximal at stationary phase and binding occurs preferentially to the holoenzyme associated with sigma(70) (Esigma(70)) it is believed that 6S RNA supports adjustment to stationary phase transcription. Previous studies have also suggested that inhibition is specific for sigma(70)-dependent promoters characterized by a weak  -35 recognition motif or extended -10 promoters. There are many exceptions to this precept, showing that other types of promoters, including stationary phase-specific (sigma(38)-dependent) promoters are inhibited. RESULTS: To solve this apparent ambiguity and to better understand the role of 6S RNA in stationary phase transition we have performed a genome-wide transcriptional analysis of wild-type and 6S RNA deficient cells growing to mid-log or early stationary phase. We found 245 genes at the exponential growth phase and 273 genes at the early stationary phase to be > or = 1.5-fold differentially expressed. Up- and down-regulated genes include many transcriptional regulators, stress-related proteins, transporters and several enzymes involved in purine metabolism. As the most striking result during stationary phase, however, we obtained in the 6S RNA deficient strain a concerted expression reduction of genes constituting the translational apparatus. In accordance, primer extension analysis showed that transcription of ribosomal RNAs, representing the key molecules for ribosome biogenesis, is also significantly reduced under the same conditions. Consistent with this finding biochemical analysis of the 6S RNA deficient strain indicates that the lack of 6S RNA is apparently compensated by an increase of the basal ppGpp concentration, known to affect growth adaptation and ribosome biogenesis. CONCLUSIONS: The analysis demonstrated that the effect of 6S RNA on transcription is not strictly confined to sigma(70)-dependent promoters. Moreover, the results  indicate that 6S RNA is embedded in stationary phase adaptation, which is governed by the capacity of the translational machinery. 
20008776	 Elevated levels of fluoroquinolone resistance are frequently found among Escherichia coli clinical isolates. This study investigated the antibiotic resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli clinical isolate, PS5, to two selection steps with increasing concentrations of norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by Western blotting that NorE5 has a multidrug resistance phenotype resulting from the overexpression of the antibiotic resistance efflux pump AcrAB-TolC. Microarray and gene fusion analyses revealed significantly increased expression in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS activity is attributable to a frameshift mutation that truncates SoxR, rendering  it a constitutive transcriptional activator of soxS. Furthermore, microarray and  reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter,  showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox sequence is in the backward or class I orientation within the promoter, and its disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus, chromosomal mutations in parC and soxR are responsible for the increased antibiotic resistance of NorE5. 
20016996	 Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora paradoxa (Glaucophyta). The primers were designed according to the conserved regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is characterized by a region with no transmembrane alpha-helices (80 aa), followed by a region with 7alpha-helices (219 aa) and a shorter 35-aa C-terminal region. The DNA sequence of the N-terminal region was expressed in E. coli and the recombinant purified peptide was used as antigen in hens to obtain polyclonal antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked labeling of the muroplast (aka cyanelle) membrane. 
19942657	 Alternative sigma factor 54 (RpoN) is an important regulator of stress resistance and virulence genes in many bacterial species. In this study, we report on the gene expression alterations that follow rpoN inactivation in Escherichia coli O157 : H7 strain Sakai (Sakai rpoN : : kan), and the influence of RpoN on the acid resistance phenotype. Microarray gene expression profiling revealed the differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This included the growth-phase-dependent upregulation of genes required for glutamate-dependent acid resistance (GDAR) ( gadA, gadB, gadC and gadE), and the  downregulation of locus of enterocyte effacement (LEE) genes, which encode a type III secretion system. Upregulation of gad genes in SakairpoN : : kan during exponential growth correlated with increased GDAR and survival in a model stomach system. Complementation of SakairpoN : : kan with a cloned version of rpoN restored acid susceptibility. Genes involved in GDAR regulation, including rpoS (sigma factor 38) and gadE (acid-responsive regulator), were shown to be required for the survival of SakairpoN : : kan by the GDAR mechanism. This study describes the contribution of rpoN to acid resistance and GDAR gene regulation, and reveals RpoN to be an important regulator of stress resistance and virulence genes in E.  coli O157 : H7. 
20008510	 We report that greA expression is driven by two strong, overlapping P1 and P2 promoters. The P1 promoter is sigma(70)-dependent and P2 is sigma(E)-dependent. Two-thirds of transcripts terminate within the leader region and the remaining third comprises greA mRNA. Termination efficiency seems to be unaffected by growth phase. Two collections of small 40-50 (initiating from P2) and 50-60 nt (from P1) RNA chains, termed GraL, are demonstrable in vivo and in vitro. We document that GraL arrays arise from an intrinsic terminator with an 11 bp stem followed by an AU(7)GCU(2) sequence. Atypical chain termination occurs at multiple sites; the 3'-ends differ by 1 nt over a range of 10 nt. Transcripts observed are shown to be insensitive to Gre factors and physically released from  RNAP-DNA complexes. The abundance of individual chains within each cluster displays a characteristic pattern, which can be differentially altered by oligonucleotide probes. Multiple termination sites are particularly sensitive to  changes at the bottom of the stem. Evolutionarily conserved GraL stem structures  and fitness assays suggest a biological function for the RNA clusters themselves. Although GraL overexpression induces >/=3-fold transcriptional changes of over 100 genes, a direct target remains elusive. 
19966274	 High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system coupled to chromatin purification for genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino-acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We found that the fusion BioTag::H3.3 was efficiently biotinylated in vivo. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. We found that embryonic chromatin is differentially extracted with increasing salt concentrations. Interestingly, chromatin that remains insoluble after washing in 600 mM salt is enriched at 5' and 3' ends, suggesting the presence of large protein complexes that render chromatin insoluble at transcriptional initiation and termination sites. We also found that H3.3 landscapes from these salt fractions display consistent features that correlate with gene activity: the most highly expressed genes contain the most H3.3. This versatile two-component approach has the potential of facilitating genome-wide chromatin dynamics and regulatory site identification in C. elegans. 
20348429	 Saponins, an important group of bioactive plant natural products, are glycosides  of triterpenoid or steroidal aglycones (sapogenins). Saponins possess many biological activities, including conferring potential health benefits for humans. However, most of the steps specific for the biosynthesis of triterpene saponins remain uncharacterized at the molecular level. Here, we use comprehensive gene expression clustering analysis to identify candidate genes involved in the elaboration, hydroxylation, and glycosylation of the triterpene skeleton in the model legume Medicago truncatula. Four candidate uridine diphosphate glycosyltransferases were expressed in Escherichia coli, one of which (UGT73F3) showed specificity for multiple sapogenins and was confirmed to glucosylate hederagenin at the C28 position. Genetic loss-of-function studies in M. truncatula confirmed the in vivo function of UGT73F3 in saponin biosynthesis. This report provides a basis for future studies to define genetically the roles of multiple cytochromes P450 and glycosyltransferases in triterpene saponin biosynthesis in Medicago. 
20161784	 BACKGROUND: SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate SELEX and assess the extent of these  effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of  SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library,  but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection. CONCLUSIONS/SIGNIFICANCE: Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We  conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers. 
19880649	 Rapid and specific detection of Shiga toxin-producing Escherichia coli (STEC) strains with a high level of virulence for humans has become a priority for public health authorities. This study reports on the development of a low-density macroarray for simultaneously testing the genes stx1, stx2, eae, and ehxA and six different nle genes issued from genomic islands OI-122 (ent, nleB, and nleE) and  OI-71 (nleF, nleH1-2, and nleA). Various strains of E. coli isolated from the environment, food, animals, and healthy children have been compared with clinical isolates of various seropathotypes. The eae gene was detected in all enteropathogenic E. coli (EPEC) strains as well as in enterohemorrhagic E. coli (EHEC) strains, except in EHEC O91:H21 and EHEC O113:H21. The gene ehxA was more  prevalent in EHEC (90%) than in STEC (42.66%) strains, in which it was unequally  distributed. The nle genes were detected only in some EPEC and EHEC strains but with various distributions, showing that nle genes are strain and/or serotype specific, probably reflecting adaptation of the strains to different hosts or environmental niches. One characteristic nle gene distribution in EHEC O157:[H7], O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O5:H-, O55:H7, O123:H11,  O172:H25, and O165:H25 was ent/espL2, nleB, nleE, nleF, nleH1-2, nleA. (Brackets  indicate genotyping of the flic or rfb genes.) A second nle pattern (ent/espL2, nleB, nleE, nleH1-2) was characteristic of EHEC O103:H2, O145:[H28], O45:H2, and  O15:H2. The presence of eae, ent/espL2, nleB, nleE, and nleH1-2 genes is a clear  signature of STEC strains with high virulence for humans. 
20212332	 A method for identifying and quantifying proteins with relatively low-cost orthogonal acceleration time-of- flight mass spectrometry (oa-ToF-MS) was tested. Escherichia coli (E. coli) K12 MG1655 cell lysate was separated by 1D gel-electrophoresis; fractions were digested and separated fast and reproducibly  by ultra-performance liquid chromatography (UPLC). Peptides were identified using oa-ToF-MS to measure exact masses of parent ions and the fragment ions generated  by up-front collision-induced dissociation. Fragmentation of all compounds was achieved by rapidly cycling between high- and low values of energy applied to ions. More than 100 proteins from E. coli K12 proteome were identified and relatively quantified. Results were found to correlate with transcriptome data determined by DNA microarrays. 
19734312	 More than 80 small regulatory RNAs (sRNAs) and 60 proteins of 16 to 50 amino acids (small proteins) are encoded in the Escherichia coli genome. The vast majority of the corresponding genes have no known function. We screened 125 DNA bar-coded mutants to identify novel cell envelope stress and acute acid shock phenotypes associated with deletions of genes coding for sRNAs and small proteins. Nine deletion mutants (ssrA, micA, ybaM, ryeF, yqcG, sroH, ybhT, yobF,  and glmY) were sensitive to cell envelope stress and two were resistant (rybB and blr). Deletion mutants of genes coding for four small proteins (yqgB, mgrB, yobF, and yceO) were sensitive to acute acid stress. We confirmed each of these phenotypes in one-on-one competition assays against otherwise-wild-type lacZ mutant cells. A more detailed investigation of the SsrA phenotype suggests that ribosome release is critical for resistance to cell envelope stress. The bar-coded deletion collection we generated can be screened for sensitivity or resistance to virtually any stress condition. 
19861135	 Specific growth rate dependent gene expression changes of Escherichia coli K12 MG1655 were studied by microarray and real-time PCR analyses. The bacteria were cultivated on glucose limited minimal medium using the accelerostat method (A-stat) where starting from steady state conditions (chemostat culture) dilution rate is constantly increased. At specific growth rate (mu) 0.47h(-1), E. coli had focused its metabolism to glucose utilization by down-regulation of alternative substrate transporters expression compared to mu=0.3h(-1). It was found that acetic acid accumulation began at mu=0.34+/-0.01h(-1) and two acetate synthesis pathways - phosphotransacetylase-acetate kinase (pta-ackA) and pyruvate oxidase (poxB) - contributed to the synthesis at the beginning of overflow metabolism, i.e. onset of acetate excretion. On the other hand, poxB, pta and ackA expression patterns suggest that pyruvate oxidase may be the only enzyme synthesizing acetate at mu=0.47h(-1). Loss of glucose and acetate co-utilization represented by down-regulation of acs-yjcH-actP operon between specific growth rates 0.3-0.42h(-1) and acetic acid accumulation from mu=0.34+/-0.01h(-1) allows one to surmise that the acetate utilization operon expression might play an important role in overflow metabolism. 
19962194	 A 70-mer-oligonucleotide-based microarray (1152 features) that emphasizes stress  and immune responses factors was constructed to study transcriptomic responses of the snail Biomphalaria glabrata to different immune challenges. In addition to sequences with relevant putative ID and Gene Ontology (GO) annotation, the array  features non-immune factors and unknown B. glabrata ESTs for functional gene discovery. The transcription profiles of B. glabrata (3 biological replicates, each a pool of 5 snails) were recorded at 12h post-wounding, exposure to Gram negative or Gram positive bacteria (Escherichia coli and Micrococcus luteus, respectively), or infection with compatible trematode parasites (Schistosoma mansoni or Echinostoma paraensei, 20 miracidia/snail), relative to controls, using universal reference RNA. The data were subjected to Significance Analysis for Microarrays (SAM), with a false positive rate (FPR) <or=10%. Wounding yielded a modest differential expression profile (27 up/21 down) with affected features mostly dissimilar from other treatments. Partially overlapping, yet distinct expression profiles were recorded from snails challenged with E. coli (83 up/20 down) or M. luteus (120 up/42 down), mostly showing up-regulation of defense and  stress-related features. Significantly altered expression of selected immune features indicates that B. glabrata detects and responds differently to compatible trematodes. Echinostoma paraensei infection was associated mostly with down-regulation of many (immune-) transcripts (42 up/68 down), whereas S. mansoni exposure yielded a preponderance of up-regulated features (140 up/23 down), with  only few known immune genes affected. These observations may reflect the divergent strategies developed by trematodes during their evolution as specialized pathogens of snails to negate host defense responses. Clearly, the immune defenses of B. glabrata distinguish and respond differently to various immune challenges. 
20018699	 The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA transcript abundance with 24-h periodicity under continuous light conditions. The mechanism underlying these oscillations remains elusive--neither cis nor trans-factors controlling circadian gene expression phase have been identified. Here, we show that the topological status of the chromosome is highly correlated  with circadian gene expression state. We also demonstrate that DNA sequence characteristics of genes that appear monotonically activated and monotonically repressed by chromosomal relaxation during the circadian cycle are similar to those of supercoiling-responsive genes in Escherichia coli. Furthermore, perturbation of superhelical status within the physiological range elicits global changes in gene expression similar to those that occur during the normal circadian cycle. 
19801409	 In Pseudomonas aeruginosa, cyclic AMP (cAMP) signaling regulates the transcription of hundreds of genes encoding diverse virulence factors, including  the type II secretion system (T2SS) and type III secretion system (T3SS) and their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent  transcriptional regulator that is homologous to the Escherichia coli catabolite repressor protein, is thought to be the major cAMP-binding protein that regulates these important virulence determinants. Using a bioinformatic approach, we have identified a gene (PA4704) encoding an additional putative cAMP-binding protein in P. aeruginosa PAO1, which we herein refer to as CbpA, for cAMP-binding protein A. Structural modeling predicts that CbpA is composed of a C-terminal cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. We show  that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using in vitro trypsin protection assays, we demonstrate that CbpA undergoes a conformational change upon cAMP binding. Reporter gene assays and electrophoresis mobility shift assays defined the cbpA promoter and a Vfr-binding site that are necessary for Vfr-dependent transcription. Although CbpA is highly regulated by Vfr, deletion of cbpA did not affect known Vfr-dependent functions, including the T2SS, the T3SS, flagellum- or TFP-dependent motility, virulence in a mouse model  of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green fluorescent protein was found to be localized to the flagellated old cell pole in a cAMP-dependent manner. These results suggest that polar localization of CbpA may be important for its function. 
19763343	 Molecular interactions define the functional organization of the cell. Epistatic  (genetic, or gene-gene) interactions, one of the most informative and commonly encountered forms of functional relationships, are increasingly being used to map process architecture in model eukaryotic organisms. In particular, 'systems-level' screens in yeast and worm aimed at elucidating genetic interaction networks have led to the generation of models describing the global modular organization of gene products and protein complexes within a cell. However, comparable data for prokaryotic organisms have not been available. Given its ease of growth and genetic manipulation, the Gram-negative bacterium Escherichia coli appears to be an ideal model system for performing comprehensive genome-scale examinations of genetic redundancy in bacteria. In this review, we highlight emerging experimental and computational techniques that have been developed recently to examine functional relationships and redundancy in E. coli  at a systems-level, and their potential application to prokaryotes in general. Additionally, we have scanned PubMed abstracts and full-text published articles to manually curate a list of approximately 200 previously reported synthetic sick or lethal genetic interactions in E. coli derived from small-scale experimental studies. 
19889087	 Non-coding small RNAs (sRNAs) play a major role in post-transcriptional regulation of gene expression. Of the 80 sRNAs that have been identified in E. coli, one-third bind to the RNA chaperone Hfq. Hfq both stabilizes these sRNAs in vivo and stimulates pairing to targets in vitro. A novel Hfq-dependent RNA, called here MgrR, was identified by its ability to bind Hfq. Expression of MgrR requires the PhoQ/PhoP two-component system; the PhoP response regulator is active under low Mg2+ concentrations and is an important virulence regulator in Salmonella; mgrR is also found in Salmonella species. Negatively regulated targets of MgrR identified using microarrays include eptB, involved in lipopolysaccharide (LPS) modification, and ygdQ, encoding a hypothetical protein. Cell sensitivity to the antimicrobial polymyxin B is affected by LPS modifications, and cells carrying an mgrR deletion were approximately 10 times more resistant than wild-type cells to polymyxin B. Thus, lower Mg2+ concentrations, sensed by PhoQ/PhoP, lead to expression of MgrR, changing LPS. sRNAs have previously been shown to regulate many outer membrane proteins. This work demonstrates that LPS, a major contributor of bacterial interactions with mammalian cells, is also subject to regulation by sRNAs. 
19801397	 Genetic analysis of interspecific populations derived from crosses between the wild tomato species Solanum habrochaites f. sp. glabratum, which synthesizes and  accumulates insecticidal methylketones (MK), mostly 2-undecanone and 2-tridecanone, in glandular trichomes, and cultivated tomato (Solanum lycopersicum), which does not, demonstrated that several genetic loci contribute  to MK metabolism in the wild species. A strong correlation was found between the  shape of the glandular trichomes and their MK content, and significant associations were seen between allelic states of three genes and the amount of MK produced by the plant. Two genes belong to the fatty acid biosynthetic pathway, and the third is the previously identified Methylketone Synthase1 (MKS1) that mediates conversion to MK of beta-ketoacyl intermediates. Comparative transcriptome analysis of the glandular trichomes of F2 progeny grouped into low- and high-MK-containing plants identified several additional genes whose transcripts were either more or less abundant in the high-MK bulk. In particular, a wild species-specific transcript for a gene that we named MKS2, encoding a protein with some similarity to a well-characterized bacterial thioesterase, was  approximately 300-fold more highly expressed in F2 plants with high MK content than in those with low MK content. Genetic analysis in the segregating population showed that MKS2's significant contribution to MK accumulation is mediated by an  epistatic relationship with MKS1. Furthermore, heterologous expression of MKS2 in Escherichia coli resulted in the production of methylketones in this host. 
19877125	 Colicin D is a plasmid-encoded antibacterial protein that specifically cleaves the anticodon loops of four Escherichia coli tRNA(Arg) species. Here, we report that the catalytic domain of colicin D, which is expressed in Saccharomyces cerevisiae, impairs cell growth by cleaving specific tRNAs. DNA microarray analysis revealed that mating-related genes were upregulated, while genes involved in a range of metabolic processes were downregulated, thereby impairing  cell growth. The pheromone-signalling pathway was activated only in alpha cells by tRNA cleavage, which was not observed in 'a' cells or diploid cells. On the basis of these results and on the recent identification of two killer toxins that cleave specific tRNAs, the relationship between tRNA depletion and the resultant  cellular response is discussed. 
19917103	 BACKGROUND: The aging process of bacteria in stationary phase is halted if cells  are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during  lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells). RESULTS: A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell  functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old  cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old  and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells.Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were  up-regulated in both young and old cells. Numerous genes encoding transporters for carbohydrates and organic alcohols and acids were down-regulated in old cells only. CONCLUSION: Network analysis revealed very different transcriptional activities during the lag period in old and young cells. Rejuvenation seems to take place during exponential growth by replicative dilution of old cellular components. 
19749070	 The impact of feed supplementation with bambermycin, monensin, narasin, virginiamycin, chlortetracycline, penicillin, salinomycin, and bacitracin on the  distribution of Escherichia coli pathotypes in broiler chickens was investigated  using an E. coli virulence DNA microarray. Among 256 E. coli isolates examined, 59 (23%) were classified as potentially extraintestinal pathogenic E. coli (ExPEC), while 197 (77%) were considered commensal. Except for chlortetracycline  treatment, the pathotype distribution was not significantly different among treatments (P > 0.05). Within the 59 ExPEC isolates, 44 (75%) were determined to  be potentially avian pathogenic E. coli (APEC), with the remaining 15 (25%) considered potentially "other" ExPEC isolates. The distribution within phylogenetic groups showed that 52 (88%) of the ExPEC isolates belonged to groups B2 and D, with the majority of APEC isolates classified as group D and most commensal isolates (170, 86%) as group A or B1. Indirect assessment of the presence of the virulence plasmid pAPEC-O2-ColV showed a strong association of the plasmid with APEC isolates. Among the 256 isolates, 224 (88%) possessed at least one antimicrobial resistance gene, with nearly half (107, 42%) showing multiple resistance genes. The majority of resistance genes were distributed among commensal isolates. Considering that the simultaneous detection of antimicrobial resistance tet(A), sulI, and bla(TEM) genes and the integron class  I indicated a potential presence of the resistance pAPEC-O2-R plasmid, the results revealed that 35 (14%) of the isolates, all commensals, possessed this multigene resistance plasmid. The virulence plasmid was never found in combination with the antimicrobial resistance plasmid. The presence of the ColV plasmid or the combination of iss and tsh genes in the majority of APEC isolates  supports the notion that when found together, the plasmid, iss, and tsh serve as  good markers for APEC. These data indicate that different resistant E. coli pathotypes can be found in broiler chickens and that the distribution of such pathotypes and certain virulence determinants could be modulated by antimicrobial agent feed supplementation. 
19759044	 OBJECTIVES: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-TolC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. METHODS: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. RESULTS: Despite important differences in the triclosan-dependent transcriptomes  of the two species, increased expression of efflux pump component genes was seen  in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in  Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An  unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. CONCLUSIONS: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents. 
19625371	 ANG is a plasma protein with angiogenic and ribonucleolytic activity implicated in tumor growth, heart failure, wound healing, asthma, and the composition of the adult gut microflora. Human mast cells (HuMC) are similarly associated with modulation of vascular permeability, angiogenic processes, wound healing, and asthma. We hypothesized that HuMC express and secrete ANG in response to divergent stimuli. ANG expression was evaluated in the LAD2 HMC, the HMC-1, and CD34+-derived HuMC, following exposure to live Escherichia coli, TLR ligands, or  neuropeptides and following FcepsilonRI aggregation. Expression and production of ANG were determined by microarray analysis, qRT-PCR, confocal microscopy, and ELISA. Microarray analysis showed that ANG is up-regulated by LAD2 cells exposed  to live E. coli. qRT-PCR analysis revealed that LAD2, HMC-1, and HuMC constitutively expressed ANG mRNA and that it was up-regulated by exposure to E.  coli. Activation of HuMC by FcepsilonRI aggregation resulted in release of small  amounts of ANG (<100 pg/mL), whereas compound 48/80, NGF, LPS, PGN, and flagellin activated HuMC to secrete >160 pg/mL ANG. These observations demonstrate that HuMC store and secrete ANG to a variety of stimuli and suggest that MC-derived ANG is available in the subsequent inflammatory response. 
19684063	 Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in the presence of low concentrations of oxygen. Microarray analysis revealed alteration in gene expression in the presence of 6 % oxygen. During the exponential growth phase, 96 genes were upregulated and 79 genes were downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative stress protection were upregulated, including alkyl hydroperoxide reductase (ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant changes in gene expression of proteins that mediate oxidative metabolism, such as cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The  expression of genes encoding formate uptake transporter (PG0209) and formate tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that formate metabolism plays a major role under aerobic conditions. The concomitant reduction of expression of a gene encoding the lactate transporter PG1340 suggests decreased utilization of this nutrient. The concentrations of both formate and lactate were assessed in culture supernatants and cells, and they were in agreement with the results obtained at the transcriptional level. Also, genes encoding gingipain protease secretion/maturation regulator (porR) and protease transporter (porT) had reduced expression in the presence of oxygen, which also correlated with reduced protease activities under aerobic conditions.  In addition, metal transport was affected, and while iron-uptake genes such as the genes encoding the haemin uptake locus (hmu) were downregulated, expression of manganese transporter genes, such as feoB2, was elevated in the presence of oxygen. Finally, genes encoding putative regulatory proteins such as extracellular function (ECF) sigma factors as well as small proteins had elevated expression levels in the presence of oxygen. As P. gingivalis is distantly related to the well-studied model organism Escherichia coli, results from our work may provide further understanding of oxygen metabolism and protection in other related bacteria belonging to the phylum Bacteroidetes. 
19571306	 Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures  using high-performance liquid chromatography coupled to photodiode and mass spectrometry detection revealed the accumulation of the aurone hispidol (6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one) as a major response to yeast elicitor. Parallel, large-scale transcriptome profiling indicated that three peroxidases, MtPRX1, MtPRX2, and MtPRX3, were coordinately induced with the accumulation of hispidol. MtPRX1 and MtPRX2 exhibited aurone synthase activity based upon in vitro substrate specificity and product profiles  of recombinant proteins expressed in Escherichia coli. Hispidol possessed significant antifungal activity relative to other M. truncatula phenylpropanoids  tested but has not been reported in this species before and was not found in differentiated roots in which high levels of the peroxidase transcripts accumulated. We propose that hispidol is formed in cell cultures by metabolic spillover when the pool of its precursor, isoliquiritigenin, builds up as a result of an imbalance between the upstream and downstream segments of the phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism. The results illustrate that integration of metabolomics and transcriptomics in genetically reprogrammed plant cell cultures is a powerful approach for the discovery of novel bioactive secondary metabolites and the mechanisms underlying  their generation. 
19893610	 Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic. We sought to elucidate genetic mechanisms whereby a polymorphism evolved in Escherichia coli under glucose limitation and persisted because of cross-feeding among multiple adaptive clones. Apart from a 29 kb deletion in the dominant clone, no large-scale genomic changes distinguished evolved clones from their common ancestor. Using transcriptional profiling on co-evolved clones cultured separately under glucose-limitation we identified 180 genes significantly altered in expression relative to the common ancestor grown under similar conditions. Ninety of these were similarly expressed in all clones, and many of the genes affected (e.g., mglBAC, mglD, and lamB) are  in operons coordinately regulated by CRP and/or rpoS. While the remaining significant expression differences were clone-specific, 93% were exhibited by the majority clone, many of which are controlled by global regulators, CRP and CpxR.  When transcriptional profiling was performed on adaptive clones cultured together, many expression differences that distinguished the majority clone cultured in isolation were absent, suggesting that CpxR may be activated by overflow metabolites removed by cross-feeding strains in co-culture. Relative to  their common ancestor, shared expression differences among adaptive clones were partly attributable to early-arising shared mutations in the trans-acting global  regulator, rpoS, and the cis-acting regulator, mglO. Gene expression differences  that distinguished clones may in part be explained by mutations in trans-acting regulators malT and glpK, and in cis-acting sequences of acs. In the founder, a cis-regulatory mutation in acs (acetyl CoA synthetase) and a structural mutation  in glpR (glycerol-3-phosphate repressor) likely favored evolution of specialists  that thrive on overflow metabolites. Later-arising mutations that led to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system. Taken together, these findings underscore the importance of regulatory change, founder genotype, and the biotic  environment in the adaptive evolution of microbes. 
19626364	 Genetic engineering of lactic acid bacteria (LAB) requires a reliable gene expression system. Especially, a stable promoter is an important genetic element  to induce gene expression in such a system. We report on a novel tuf promoter (Ptuf) of Lactococcus lactis subsp. lactis IL1403 that was screened and selected  through analysis of previously published microarray data. Ptuf activity was examined and compared with three other known lactococcal promoters (PdnaJ, PpfkA, and Pusp45) using different bacteria as expression hosts. Each promoter was, respectively, fused to the promoterless and modified bmpB gene as a reporter, and we estimated promoter activity through BmpB expression. All promoters were active in IL1403, and Ptuf activity was strongest among them. The activity of each promoter differed by host bacteria (Lactobacillus plantarum Lb25, Lactobacillus reuteri ATCC23272, and Escherichia coli Top10F'). Ptuf had the highest activity in IL1403 when growth reached late log phase. The activity of each promoter correlated with the expression of each cognate gene in the microarray data (R (2) = 0.7186, P = 0.06968). This study revealed that novel food-grade promoters such  as IL1403 Ptuf can be selected from microarray data for food-grade microorganisms and Ptuf can be used to develop a reliable gene expression system in L. lactis. 
19758836	 Following the pioneering screens for small regulatory RNAs (sRNAs) in Escherichia coli in 2001, sRNAs are now being identified in almost every branch of the eubacterial kingdom. Experimental strategies have become increasingly important for sRNA discovery, thanks to increased availability of tiling arrays and fast progress in the development of high-throughput cDNA sequencing (RNA-Seq). The new technologies also facilitate genome-wide discovery of potential target mRNAs by sRNA pulse-expression coupled to transcriptomics, and immunoprecipitation with RNA-binding proteins such as Hfq. Moreover, the staggering rate of new sRNAs demands mechanistic analysis of target regulation. We will also review the available toolbox for wet lab-based research, including in vivo and in vitro reporter systems, genetic methods and biochemical co-purification of sRNA interaction partners. 
19633077	 The asc operon of Escherichia coli is one of the cryptic genetic systems for beta-D-galactoside utilization as a carbon source. The ascFB genes for beta-D-galactoside transport and catabolism are repressed by the AscG regulator.  After genomic SELEX screening, AscG was found to recognize and bind the consensus palindromic sequence TGAAACC-GGTTTCA. AscG binding was detected at two sites upstream of the ascFB promoter and at three sites upstream of the prpBC operon for propionate catabolism. In an ascG-disrupted mutant, transcription of ascFB was enhanced, in agreement with the repressor model of AscG. This repression was  indicated to be due to interference of binding of cyclic AMP-CRP to the CRP box,  which overlaps with the AscG-binding site 1, as well as binding of RNA polymerase to the promoter. Under conditions of steady-state E. coli growth in a rich medium, the intracellular level of AscG stayed constant at a level supposedly leading to tight repression of the ascFB operon. The level of prpR, encoding the  activator of prpBCDE, was also increased in the absence of AscG, indicating the involvement of AscG in repression of prpR. Taken together, these data suggest a metabolic link through interplay between the asc and prp operons. 
19703108	 Hfq is a conserved RNA-binding protein that regulates diverse cellular processes  through post-transcriptional control of gene expression, often by functioning as  a chaperone for regulatory sRNAs. Here, we explored the role of Hfq in enterohaemorrhagic Escherichia coli (EHEC), a group of non-invasive intestinal pathogens. EHEC virulence is dependent on a Type III secretion system encoded in  the LEE pathogenicity island. The abundance of transcripts for all 41 LEE genes and more than half of confirmed non-LEE-encoded T3 effectors were elevated in an  EHEC hfq deletion mutant. Thus, Hfq promotes co-ordinated expression of the LEE-encoded T3S apparatus and both LEE- and non-LEE-encoded effectors. Increased  transcript levels led to the formation of functional secretion complexes capable  of secreting high quantities of effectors into the supernatant. The increase in LEE-derived transcripts and proteins was dependent on Ler, the LEE-encoded transcriptional activator, and the ler transcript appears to be a direct target of Hfq-mediated negative regulation. Finally, we found that Hfq contributes to the negative regulation of T3SSs in several other pathogens, suggesting that Hfq, potentially along with species-specific sRNAs, underlies a common means to prevent unfettered expression of T3SSs. 
19728878	 BACKGROUND: The Ada-dependent adaptive response system in Escherichia coli is important for increasing resistance to alkylation damage. However, the global transcriptional and translational changes during this response have not been reported. Here we present time-dependent global gene and protein expression profiles following treatment with methyl methanesulfonate (MMS) in E. coli W3110  and its ada mutant strains. RESULTS: Transcriptome profiling showed that 1138 and 2177 genes were differentially expressed in response to MMS treatment in the wild-type and mutant strains, respectively. A total of 81 protein spots representing 76 nonredundant proteins differentially expressed were identified using 2-DE and LC-MS/MS. In the wild-type strain, many genes were differentially expressed upon long-exposure to  MMS, due to both adaptive responses and stationary phase responses. In the ada mutant strain, the genes involved in DNA replication, recombination, modification and repair were up-regulated 0.5 h after MMS treatment, indicating its connection to the SOS and other DNA repair systems. Interestingly, expression of the genes involved in flagellar biosynthesis, chemotaxis, and two-component regulatory systems related to drug or antibiotic resistance, was found to be controlled by Ada. CONCLUSION: These results show in detail the regulatory components and pathways controlling adaptive response and how the related genes including the Ada regulon are expressed with this response. 
19592586	 Pseudomonas aeruginosa exhibits swarming motility on semisolid surfaces (0.5 to 0.7% agar). Swarming is a more than just a form of locomotion and represents a complex adaptation resulting in changes in virulence gene expression and antibiotic resistance. In this study, we used a comprehensive P. aeruginosa PA14  transposon mutant library to investigate how the complex swarming adaptation process is regulated. A total of 233 P. aeruginosa PA14 transposon mutants were verified to have alterations in swarming motility. The swarming-associated genes  functioned not only in flagellar or type IV pilus biosynthesis but also in processes as diverse as transport, secretion, and metabolism. Thirty-three swarming-deficient and two hyperswarming mutants had transposon insertions in transcriptional regulator genes, including genes encoding two-component sensors and response regulators; 27 of these insertions were newly identified. Of the 25  regulatory mutants whose swarming motility was highly impaired (79 to 97%), only  1 (a PA1458 mutant) had a major defect in swimming, suggesting that this regulator might influence flagellar synthesis or function. Twitching motility, which requires type IV pili, was strongly affected in only two regulatory mutants (pilH and PA2571 mutants) and was moderately affected in three other mutants (algR, ntrB, and nosR mutants). Microarray analyses were performed to compare the gene expression profile of a swarming-deficient PA3587 mutant to that of the wild-type PA14 strain under swarming conditions. PA3587 showed 63% homology to metR, which encodes a regulator of methionine biosynthesis in Escherichia coli. The observed dysregulation in the metR mutant of nine different genes required for swarming motility provided a possible explanation for the swarming-deficient  phenotype of this mutant. 
19652016	 The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively,  these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at these sites and clearance of (32)P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were  abnormally low in CD macrophages. Coupled with reduced secretion, these findings  indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway. 
19706172	 BACKGROUND: The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials of this genome should increase our understanding of their impact on M. leprae physiology. RESULTS: Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome.  Transcribed pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like  structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene  promoters. Mechanisms for translational "silencing" of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno (SD) sequences. Transcribed pseudogenes also contained multiple "in-frame" stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong  SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. CONCLUSION: Approximately half of M. leprae's transcriptome consists of inactive  gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large  number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational "silencing" mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts. 
19703276	 BACKGROUND: Glucose is the preferred carbon and energy source for Bacillus subtilis and Escherichia coli. A complex regulatory network coordinates gene expression, transport and enzymatic activities, in response to the presence of this sugar. We present a comparison of the cellular response to glucose in these  two model organisms, using an approach combining global transcriptome and regulatory network analyses. RESULTS: Transcriptome data from strains grown in Luria-Bertani medium (LB) or LB+glucose (LB+G) were analyzed, in order to identify differentially transcribed  genes in B. subtilis. We detected 503 genes in B. subtilis that change their relative transcript levels in the presence of glucose. A similar previous study identified 380 genes in E. coli, which respond to glucose. Catabolic repression was detected in the case of transport and metabolic interconversion activities for both bacteria in LB+G. We detected an increased capacity for de novo synthesis of nucleotides, amino acids and proteins. A comparison between orthologous genes revealed that global regulatory functions such as transcription, translation, replication and genes relating to the central carbon  metabolism, presented similar changes in their levels of expression. An analysis  of the regulatory network of a subset of genes in both organisms revealed that the set of regulatory proteins responsible for similar physiological responses observed in the transcriptome analysis are not orthologous. An example of this observation is that of transcription factors mediating catabolic repression for most of the genes that displayed reduced transcript levels in the case of both organisms. In terms of topological functional units in both these bacteria, we found interconnected modules that cluster together genes relating to heat shock,  respiratory functions, carbon and peroxide metabolism. Interestingly, B. subtilis functions not found in E. coli, such as sporulation and competence were shown to  be interconnected, forming modules subject to catabolic repression at the level of transcription. CONCLUSION: Our results demonstrate that the response to glucose is partially conserved in model organisms E. coli and B. subtilis, including genes encoding basic functions such as transcription, translation, replication and genes involved in the central carbon metabolism. 
19682387	 BACKGROUND: It has long been recognized that Klebsiella pneumoniae can grow anaerobically on citrate. Genes responsible for citrate fermentation of K. pneumoniae were known to be located in a 13-kb gene cluster on the chromosome. By whole genome comparison of the available K. pneumoniae sequences (MGH 78578, 342, and NTUH-K2044), however, we discovered that the fermentation gene cluster was present in MGH 78578 and 342, but absent in NTUH-K2044. In the present study, the previously unknown genome diversity of citrate fermentation among K. pneumoniae clinical isolates was investigated. RESULTS: Using a genomic microarray containing probe sequences from multiple K. pneumoniae strains, we investigated genetic diversity among K. pneumoniae clinical isolates and found that a genomic region containing the citrate fermentation genes was not universally present in all strains. We confirmed by PCR analysis that the gene cluster was detectable in about half of the strains tested. To demonstrate the metabolic function of the genomic region, anaerobic growth of K. pneumoniae in artificial urine medium (AUM) was examined for ten strains with different clinical histories and genomic backgrounds, and the citrate fermentation potential was found correlated with the genomic region. PCR  detection of the genomic region yielded high positive rates among a variety of clinical isolates collected from urine, blood, wound infection, and pneumonia. Conserved genetic organizations in the vicinity of the citrate fermentation gene  clusters among K. pneumoniae, Salmonella enterica, and Escherichia coli suggest that the 13-kb genomic region were not independently acquired. CONCLUSION: Not all, but nearly half of the K. pneumoniae clinical isolates carry the genes responsible for anaerobic growth on citrate. Genomic variation of citrate fermentation genes in K. pneumoniae may contribute to metabolic diversity and adaptation to variable nutrient conditions in different environments. 
19481094	 Escherichia coli regulates cytosolic free Ca(2+) in the micromolar range through  influx and efflux. Herein, we show for the first time that ATP is essential for Ca(2+) efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca(2+) (41 elevated, 69 depressed). Of these, 3 transport proteins and  4 membrane proteins were identified as potential Ca(2+) transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca(2+) relative to time zero. Based on the microarray results and other predicted possible Ca(2+) transporters, the level of cytosolic free Ca(2+)  was measured in selected mutants from the Keio knockout collection using intracellular aequorin. In this way, we identified a knockout of atpD, coding for a component of the F(o)F(1) ATPase, as defective in Ca(2+) efflux. Seven other putative Ca(2+) transport proteins exhibited normal Ca(2+) handling. The defect in the DeltaatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the DeltaatpD knockout cells to that of the wild type and restored Ca(2+) efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the DeltaatpD cells. Under these conditions, a similar defect in Ca(2+) efflux in wild type was observed in DeltaatpD cells. Ten millimolar concentration of Ca(2+) resulted in a 30% elevation in ATP in wild type and was accompanied by a 10% reduction in generation time under these conditions. Knockouts of pitB, a potential Ca(2+) transporter, atoA, the beta subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca(2+) efflux. We therefore propose that ATP is most likely to regulate Ca(2+) efflux in E. coli through an ATPase. 
19288441	 Although importance of the rpoS gene on biofilm formation by Escherichia coli has been suggested, there has not been any report showing where the rpoS is expressed during biofilm formation process. Since physiological state of the cells in the biofilms is considerably heterogeneous, the expression of the rpoS gene must be heterogeneous. In this study, in situ spatial expression of the rpoS gene during  biofilm formation was investigated with an rpoS-gfp transcriptional fusion mutant strain. A ribosomal binding site and a gene encoding a green fluorescent protein  were introduced into the downstream of the rpoS gene, which enabled us to observe the in situ spatial expression of the rpoS gene during biofilm formation processes without any disturbance of the rpoS expression. In the early stages of  the biofilm formation process, the rpoS gene was expressed in the most of the cells. On the other hand, the rpoS expression was observed only at the outside of the biofilms during the late stages of the biofilm formation process. The in situ spatial expression of the rpoS gene in the biofilm was verified by quantifying the expression levels of the rpoS at the outside and the inside of the biofilms with the real time RT-PCR. In addition, global gene expression analysis was performed with DNA microarray to investigate physiological difference between the outside and the inside of the biofilms. This heterogeneous rpoS expression profile suggested that the cells at the outside of the biofilm need to express the rpoS to shift the physiological state to the stationary growth mode such as induction of various stress responses and suppression of the motility. 
19502395	 Escherichia coli is not considered naturally competent, yet it has homologues of  the genes that most competent bacteria use for DNA uptake and processing. In Haemophilus influenzae and Vibrio cholerae, these genes are regulated by the Sxy  and cyclic AMP receptor (CRP) proteins. We used microarrays to find out whether similar regulation occurs in E. coli. Expression of sxy strongly induced 63 transcriptional units, 34 of which required CRP for transcriptional activation and had promoter sites resembling the Sxy- and CRP-dependent CRP-S motif previously characterized in H. influenzae. As previously reported, sxy expression also induced the sigma-H regulon. Flagellar operons were downregulated by sxy expression, although motility remained unaffected. The CRP-S regulon included all of E. coli's known competence gene homologues, so we investigated Sxy's effect on competence-associated phenotypes. A sxy knockout reduced both "natural" plasmid transformation and competitive fitness in long-term culture. In addition, expression of plasmid-borne sxy led to production of type IV pilin, the main subunit of the DNA uptake machinery of most bacteria. Although H. influenzae Sxy  only weakly activated the E. coli Sxy regulon, induction was dramatically improved when it was coexpressed with its cognate CRP, suggesting that intimate interactions between Sxy and CRP are required for transcriptional activation at CRP-S sites. 
19482050	 An oligonucleotide array based on PCR method containing a combination of probes for taxonomic markers and species-specific virulence genes was developed for the  simultaneous identification of 14 Lactic Acid Bacteria (LAB) and food-borne pathogenic bacteria in raw milk. The hypervariable regions V3 and V6 of 16S, together with a variable region of 23S rRNA genes and several genes specific for  virulence factors were selected. Universal primers and multiplex PCR were used for the rapid differentiation of bacterial species and low concentration of specific pathogenic and spoilage bacteria were detected in milk. The dominant species such as Streptococcus thermophilus, Enterococcus faecalis, Lactococcus lactis, andLeuconostoc lactis were identified by indirect-labelling reactions based upon incorporation of amino-allyl dUTPs. The results regarding food-borne pathogens detection showed highly specific hybridisation patterns with the genomic DNA from Campylobacter jejuni, Escherichia coli, Salmonella thyphimurium, Listeria monocytogenes and Yersinia enterocolitica. A clear differentiation of dominant species present in a complex microbial community such as raw milk was achieved by the application of short oligonucleotide probes which discriminate sequences differing by few nucleotides. 
19621081	 BACKGROUND: Amino acid absorption in the form of di- and tripeptides is mediated  by the intestinal proton-coupled peptide transporter PEPT-1 (formally OPT-2) in Caenorhabditits elegans. Transporter-deficient animals (pept-1(lg601)) show impaired growth, slowed postembryonal development and major changes in amino acid status. PRINCIPAL FINDINGS: Here we demonstrate that abolished intestinal peptide transport also leads to major metabolic alterations that culminate in a two fold  increase in total body fat content. Feeding of C. elegans with [U-(13)C]-labelled E. coli revealed a decreased de novo synthesis of long-chain fatty acids in pept-1(lg601) and reduced levels of polyunsaturated fatty acids. mRNA profiling revealed increased transcript levels of enzymes/transporters needed for peroxisomal beta-oxidation and decreased levels for those required for fatty acid synthesis, elongation and desaturation. As a prime and most fundamental process that may account for the increased fat content in pept-1(lg601) we identified a highly accelerated absorption of free fatty acids from the bacterial food in the  intestine. CONCLUSIONS: The influx of free fatty acids into intestinal epithelial cells is strongly dependent on alterations in intracellular pH which is regulated by the interplay of PEPT-1 and the sodium-proton exchanger NHX-2. We here provide evidence for a central mechanism by which the PEPT-1/NHX-2 system strongly influences the in vivo fat content of C. elegans. Loss of PEPT-1 decreases intestinal proton influx leading to a higher uptake of free fatty acids with fat  accumulation whereas loss of NHX-2 causes intracellular acidification by the PEPT-1 mediated proton/dipeptide symport with an almost abolished uptake of fatty acids and a lean phenotype. 
19496624	 The transactivating responsive (TAR) element is a RNA hairpin located in the 5' untranslated region of HIV-1 mRNA. It is essential for full-length transcription  of the retroviral genome and therefore for HIV-1 replication. Hairpin aptamers that generate highly stable and specific complexes with TAR were previously identified, thus decreasing the level of TAR-dependent expression in cultured cells [Kolb, G., et al. (2006) RNA Biol. 3, 150-156]. We performed genomic SELEX  against TAR using a human RNA library to identify human transcripts that might interact with the retroviral genome through loop-loop interactions and potentially contribute to the regulation of TAR-mediated processes. We identified a genomic aptamer termed a1 that folds as a hairpin with an apical loop complementary to five nucleotides of the TAR hexanucleotide loop. Surface plasmon resonance experiments performed on a truncated or mutated version of the a1 aptamer, in the presence of the Rop protein of Escherichia coli, indicate the formation of a highly stable a1-TAR kissing complex. The 5' ACCCAG loop of a1 constitutes a new motif of interaction with the TAR loop. 
19575794	 BACKGROUND: Evolutionary analyses of enterohemorrhagic Escherichia coli (EHEC) have identified two distantly related clonal groups: EHEC 1, including serotype O157:H7 and its inferred ancestor O55:H7; and EHEC 2, comprised of several serogroups (O26, O111, O118, etc.). These two clonal groups differ in their virulence and global distribution. Although several fully annotated genomic sequences exist for strains of serotype O157:H7, much less is known about the genomic composition of EHEC 2. In this study, we analyzed a set of 24 clinical EHEC 2 strains representing serotypes O26:H11, O111:H8/H11, O118:H16, O153:H11 and O15:H11 from humans and animals by comparative genomic hybridization (CGH) on an oligoarray based on the O157:H7 Sakai genome. RESULTS: Backbone genes, defined as genes shared by Sakai and K-12, were highly conserved in EHEC 2. The proportion of Sakai phage genes in EHEC 2 was substantially greater than that of Sakai-specific bacterial (non-phage) genes. This proportion was inverted in O55:H7, reiterating that a subset of Sakai bacterial genes is specific to EHEC 1. Split decomposition analysis of gene content revealed that O111:H8 was more genetically uniform and distinct from other EHEC 2 strains, with respect to the Sakai O157:H7 gene distribution. Serotype O26:H11 was the most heterogeneous EHEC 2 subpopulation, comprised of strains with the highest as well as the lowest levels of Sakai gene content conservation. Of the 979 parsimoniously informative genes, 15% were found to be compatible and their distribution in EHEC 2 clustered O111:H8 and O118:H16 strains by serotype. CGH data suggested divergence of the LEE island from the LEE1 to the LEE4 operon, and also between animal and human isolates irrespective  of serotype. No correlation was found between gene contents and geographic locations of EHEC 2 strains. CONCLUSION: The gene content variation of phage-related genes in EHEC 2 strains supports the hypothesis that extensive modular shuffling of mobile DNA elements has occurred among EHEC strains. These results suggest that EHEC 2 is a multiform pathogenic clonal complex, characterized by substantial intra-serotype genetic variation. The heterogeneous distribution of mobile elements has impacted the diversification of O26:H11 more than other EHEC 2 serotypes. 
19377097	 Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein  possessed a single, high affinity metal-binding site that can accommodate Zn(2+)  or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+)  finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene  encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture  vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. 
19429622	 LeuO, a LysR family transcription factor, exists in a wide variety of bacteria of the family Enterobacteriaceae and is involved in the regulation of as yet unidentified genes affecting the stress response and pathogenesis expression. Using genomic screening by systematic evolution of ligands by exponential enrichment (SELEX) in vitro, a total of 106 DNA sequences were isolated from 12 different regions of the Escherichia coli genome. All of the SELEX fragments formed complexes in vitro with purified LeuO. After Northern blot analysis of the putative target genes located downstream of the respective LeuO-binding sequence, a total of nine genes were found to be activated by LeuO, while three genes were  repressed by LeuO. The LeuO target gene collection included several multidrug resistance genes. A phenotype microarray assay was conducted to identify the gene(s) responsible for drug resistance and the drug species that are under the control of the LeuO target gene(s). The results described herein indicate that the yjcRQP operon, one of the LeuO targets, is involved in sensitivity control against sulfa drugs. We propose to rename the yjcRQP genes the sdsRQP genes (sulfa drug sensitivity determinant). 
19395492	 Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella  oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation  of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as  DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP. 
19468044	 The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was  studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs  and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal  DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the  b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship. 
19346340	 Escherichia coli O157:H7 has caused serious outbreaks of food-borne illness via transmission in a variety of food vehicles, including unpasteurized apple juice,  dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns before and after exposure to model apple juice. Transcriptomes of mid-exponential- and stationary-phase cells were evaluated after 10 min in model  apple juice (pH 3.5) using microarrays probing 4,886 open reading frames. A total of 331 genes were significantly induced upon exposure of cells to model apple juice, including genes involved in the acid, osmotic, and oxidative stress responses as well as the envelope stress response. Acid and osmotic stress response genes, including asr, osmC, osmB, and osmY, were significantly induced in response to model apple juice. Multiple envelope stress responses were activated as evidenced by increased expression of CpxR and Rcs phosphorelay-controlled genes. Genes controlled by CpxR (cpxP, degP, and htpX) were significantly induced 2- to 15-fold upon exposure to apple juice. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 genes induced in model apple juice, 104 are O157-specific genes, including those encoding type three secretion effectors (espJ, espB, espM2, espL3, and espZ). Elucidating the response of O157:H7 to acidic foods provides insight into how this pathogen is able to survive in food matrices and how exposure to foods influences subsequent transmission and virulence. 
19767843	 The global gene expression and biomolecular composition in an Escherichia coli model strain exposed to 10 adverse conditions (sodium chloride, ethanol, glycerol, hydrochloric and acetic acid, sodium hydroxide, heat (46 degrees C), and cold (15 degrees C), as well as ethidium bromide and the disinfectant benzalkonium chloride) were determined using DNA microarrays and Fourier transform infrared (FT-IR) spectroscopy. In total, approximately 40% of all investigated genes (1682/4279 genes) significantly changed expression, compared with a nonstressed control. There were, however, only 3 genes (ygaW (unknown function), rmf (encoding a ribosomal modification factor), and ghrA (encoding a glyoxylate/hydroxypyruvate reductase)) that significantly changed expression under all conditions (not including benzalkonium chloride). The FT-IR analysis showed an increase in unsaturated fatty acids during ethanol and cold exposure, and a decrease during acid and heat exposure. Cold conditions induced changes in  the carbohydrate composition of the cell, possibly related to the upregulation of outer membrane genes (glgAP and rcsA). Although some covariance was observed between the 2 data sets, principle component analysis and regression analyses revealed that the gene expression and the biomolecular responses are not well correlated in stressed populations of E. coli, underlining the importance of multiple strategies to begin to understand the effect on the whole cell. 
19473256	 The response to elevated temperature [heat shock (HS)] is highly conserved. The transcriptome of Escherichia coli K-12 has been studied under a variety of conditions while such studies involving E. coli O157:H7 are only now being conducted. To better understand the impact of HS on E. coli O157:H7, global transcript levels of strain EDL933 cells shifted from 37 to 50 degrees C for 15 min were compared with cells held at 37 degrees C by microarray. Using a mixed model analysis, 193 genes were found to be differentially transcribed at P<0.0042 with a q value <0.1. The 111 downregulated genes include the curli pili-associated genes csgABCDEFG, maltose transport-associated proteins malEFK, and NADH dehydrogenase subunit encoding nuoCEHIJN. The 82 genes upregulated include the HS-induced genes rpoH, dnaK, dnaJ, groEL, groES, and grpE along with  two LEE-encoded genes: hypothetical gene Z5121 and sepZ. Twenty-three additional  genes located in O-islands were found to be differentially expressed. Quantitative real-time PCR (qRT-PCR) was performed to validate the microarray results. Also, samples subjected to a 30-42 degrees C shift were examined by qRT-PCR to confirm differential transcription of selected genes. These results indicate that this pathogen may regulate its virulence factors in response to temperature changes. 
19329643	 The LEE pathogenicity island has been acquired on multiple occasions within the different lineages of enteropathogenic and enterohemorrhagic Escherichia coli. In each lineage, LEE expression is regulated by complex networks of pathways, including core pathways shared by all lineages and lineage-specific pathways. Within the O157:H7 lineage of enterohemorrhagic E. coli, strain-to-strain variation in LEE expression has been observed, implying that expression patterns  can diversify even within highly related subpopulations. Using comparative genomics of E. coli O157:H7 subpopulations, we have identified one source of strain-level variation affecting LEE expression. The variation occurs in prophage-dense regions of the genome that lie immediately adjacent to the late regions of the pch prophage carrying pchA, pchB, pchC, and a newly identified pch gene, pchX. Genomic segments extending from the holin S region to the pchA, pchB, pchC, and pchX genes of their respective prophage are highly conserved but are nonetheless embedded within adjacent genomic segments that are extraordinarily variable, termed pch adjacent genomic regions (pch AGR). Despite the remarkable degree of variation, the pattern of variation in pch AGR is highly correlated with the distribution of phylogenetic markers on the backbone of the genome. Quantitative analysis of transcription from the LEE1 promoter further revealed that variation in the pch AGR has substantial effects on absolute levels and patterns of LEE1 transcription. Variation in the pch AGR therefore serves as a mechanism to diversify LEE expression patterns, and the lineage-specific pattern  of pch AGR variation could ultimately influence ecological or virulence characteristics of subpopulations within each lineage. 
19492094	 BACKGROUND: Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA).  The mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nDNA. Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. To investigate how the cellular transcriptome is modified in response to the absence of mtDNA, we used Affymetrix HG-U133A GeneChip arrays to study the gene expression profile of two human cell lines, 143BTK(-) and A549, which had been entirely depleted of mtDNA (rho(o) cells), and compared it with that of corresponding undepleted parental cells (rho(+) cells). RESULTS: Our data indicate that absence of mtDNA is associated with: i) a down-regulation of cell cycle control genes and a reduction of cell replication rate, ii) a down-regulation of nuclear-encoded subunits of complex III of the respiratory chain and iii) a down-regulation of a gene described as the human homolog of ELAC2 of E. coli, which encodes a protein that we show to also target  to the mitochondrial compartment. CONCLUSIONS: Our results indicate a strong correlation between mitochondrial biogenesis and cell cycle control and suggest that some proteins could have a double role: for instance in controlling both cell cycle progression and mitochondrial functions. In addition, the finding that ELAC2 and maybe other transcripts that are located into mitochondria, are down-regulated in rho(o) cells, make them good candidates for human disorders associated with defective replication and expression of mtDNA. 
19303281	 One of the major limitations in the development of ultrasensitive electrochemical biosensors based on one-dimensional nanostructures is the difficulty involved with reliably fabricating nanoelectrode arrays (NEAs). In this work, we describe  a simple, robust and scalable wafer-scale fabrication method to produce multiplexed biosensors. Each sensor chip consists of nine individually addressable arrays that uses electron beam patterned vertically aligned carbon nanofibers (VACNFs) as the sensing element. To ensure nanoelectrode behavior with higher sensitivity, VACNFs were precisely grown on 100 nm Ni dots with 1 microm spacing on each micro pad. Pretreatments by the combination of soaking in 1.0 M HNO(3) and electrochemical etching in 1.0M NaOH dramatically improved the electrode performance, indicated by the decrease of redox peak separation in cyclic voltammogram (DeltaE(p)) to approximately 100 mV and an approximately 200% increase in steady-state currents. The electrochemical detection of the hybridization of DNA targets from E. coli O157:H7 onto oligonucleotide probes were successfully demonstrated. The 9 arrays within the chip were divided into three groups with triplicate sensors for positive control, negative control and specific hybridization. The proposed method has the potential to be scaled up to  NxN arrays with N up to 10, which is ideal for detecting a myriad of organisms. In addition, such sensors can be used as a generic platform for many electroanalysis applications. 
19273673	 Simocyclinone D8 (SD8) exhibits antibiotic activity against gram-positive bacteria but not against gram-negative bacteria. The molecular basis of the cytotoxicity of SD8 is not fully understood, although SD8 has been shown to inhibit the supercoiling activity of Escherichia coli gyrase. To understand the mechanism of SD8, we have employed biochemical assays to directly measure the sensitivities of E. coli and Staphylococcus aureus type II topoisomerases to SD8  and microarray analysis to monitor the cellular responses to SD8 treatment. SD8 is a potent inhibitor of either E. coli or S. aureus gyrase. In contrast, SD8 exhibits only a moderate inhibitory effect on S. aureus topoisomerase IV, and E.  coli topoisomerase IV is virtually insensitive to SD8. The antimicrobial effect of SD8 against E. coli has become evident in the absence of the AcrB multidrug efflux pump. As expected, SD8 treatment exhibits the signature responses to the loss of supercoiling activity in E. coli: upregulation of gyrase genes and downregulation of the topoisomerase I gene. Unlike quinolone treatment, however,  SD8 treatment does not induce the SOS response. These results suggest that DNA gyrase is the target of SD8 in both gram-positive and gram-negative bacteria and  that the lack of the antibacterial effect against gram-negative bacteria is due,  in part, to the activity of the AcrB efflux pump. 
19449976	 Gene regulatory networks capture interactions between genes and other cell substances, resulting in various models for the fundamental biological process of transcription and translation. The expression levels of the genes are typically measured as mRNA concentration in micro-array experiments. In a so-called genetic perturbation experiment, small perturbations are applied to equilibrium states and the resulting changes in expression activity are measured. One of the most important problems in systems biology is to use these data to identify the interaction pattern between genes in a regulatory network, especially in a large  scale network. The authors develop a novel algorithm for identifying the smallest genetic network that explains genetic perturbation experimental data. By construction, our identification algorithm is able to incorporate and respect a priori knowledge known about the network structure. A priori biological knowledge is typically qualitative, encoding whether one gene affects another gene or not,  or whether the effect is positive or negative. The method is based on a convex programming relaxation of the combinatorially hard problem of L(0) minimisation.  The authors apply the proposed method to the identification of a subnetwork of the SOS pathway in Escherichia coli, the segmentation polarity network in Drosophila melanogaster, and an artificial network for measuring the performance  of the method. 
19251846	 The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the  global transcriptional patterns of gene expression in strains deficient in ppGpp  (ppGpp(0)) and/or DksA (DeltadksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksA-deficient strains, respectively, increasing to 13% of all genes in the ppGpp(0) DeltadksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli. 
19220470	 It has been widely reported that pH mediates cadmium toxicity to bacteria. We used a tripartite approach to investigate mechanisms by which pH affects cadmium  toxicity that included analyses of: (1) growth kinetics, (2) global gene expression, and (3) cadmium speciation. Cadmium extended the lag phase at pH 7, but not at pH 5. DNA microarray analysis revealed that stress response genes including hdeA, otsA, and yjbJ were more highly expressed at pH 5 than at pH 7 after only 5 min of exposure to cadmium, suggesting that acidic pH more rapidly induced genes that confer cadmium resistance. In addition, genes involved in transport and many hypothetical genes were more highly expressed at pH 5 than at  pH 7 in the presence of cadmium. Concentrations of two cadmium species, including one previously implicated in the mechanism by which pH mediates cadmium toxicity  (CdOH+), increased with pH. Our data demonstrate that transcriptional responses of Escherichia coli to cadmium are substantially affected by pH and suggest that  several stress response, transport, and hypothetical genes play roles in the mechanism by which pH mediates cadmium toxicity. 
19218383	 The human gastric pathogen Helicobacter pylori has many virulence factors involved in pathogenesis, but the mechanisms regulating these virulence factors are not yet fully understood. In this study, we cloned HP1248, which is similar in sequence to Escherichia coli vacB, which was previously shown to be associated with the expression of virulence in Shigella and enteroinvasive E. coli. E. coli  vacB encodes RNase R. RNase R is involved in the posttranscriptional regulation of mRNA stability. By global transcriptional microarray profiling of an H. pylori HP1248 deletion mutant, we defined six virulence-related genes which were posttranscriptionally downregulated by HP1248, including the motility-related genes HP1192 and flaB, the chemotaxis-related gene cheY, and the apoptosis-inducing genes HP0175, cagA, and gtt. In this study, recombinant HP1248 protein expressed in E. coli showed 3'-to-5' exoribonuclease activity. Motility and apoptosis induction were increased in the H. pylori HP1248 deletion mutant. We also showed that HP1192 is associated with H. pylori motility, possibly through HP1248 regulation. Further, we suggested and studied the possible mechanisms of this specific regulation of virulent genes by HP1248. In addition,  the expression level of HP1248 mRNA changed dramatically in response to a variety of altered environmental conditions, including pH and temperature. Hence, HP1248  in H. pylori seems to play a role in environmental sensing and in regulation of virulent phenotypes, such as motility and host apoptosis induction. 
19151137	 We performed chromatin immunoprecipitation-microarray analysis to investigate differences in function between StpA and H-NS in Escherichia coli cells. StpA binding regions essentially overlap those of H-NS in wild-type cells, while they  are reduced to one-third in the hns mutant. The H-NS binding profile was unaffected by stpA inactivation. 
19193861	 Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how it signals was not clear. Here, we provide evidence that T6P functions as an inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the SNF1-related group of protein kinases. T6P, but not other sugars and sugar phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling extracts strongly (50%) at low concentrations (1-20 microM). Inhibition was noncompetitive with respect to ATP. In immunoprecipitation studies using antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were physically separable, with T6P inhibition of SnRK1 dependent on an intermediary factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues analyzed except those of mature leaves, which did not contain the intermediary factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression  was determined in seedlings expressing Escherichia coli otsA encoding T6P synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1 target genes showed opposite regulation, consistent with the regulation of SnRK1  by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes  involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron transport, which are normally down-regulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were down-regulated by T6P. These experiments provide strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in growing tissues. 
19162465	 STD (sexually transmitted disease, Gonorrhoea) sensor based on nucleic acid probe (from Opa, a multi-copy gene of Neisseria gonorrhoeae) functionalized nanostructured-polyaniline coated onto indium-tin-oxide-coated glass plate has been fabricated using avidin-biotin as cross-linking agent. This DNA functionalized electrode can specifically detect upto 0.5 x 10(-15)M of complementary target within 60s of hybridization time at 25 degrees C by differential pulse voltammetry (DPV) using methylene blue as electro-active DNA hybridization indicator. This highly sensitive and specific nucleic acid functionalized nanostructured-polyaniline electrode can distinguish presence of N. gonorrhoeae from Neisseria meningitidis and Escherichia coli culture and spiked samples from the urethral swabs of the patients. 
19028086	 We present the rapid and sensitive detection of amplified DNA on a giant magneto-resistance sensor using superparamagnetic particles as a detection label. The one-step assay is performed on an integrated and miniaturized detection platform suitable for application into point-of-care devices. A double-tagged PCR amplification product of the LamB gene of the Escherichia coli bacterium was used to investigate binding kinetics of the assay. We applied magnetic actuation to concentrate the target-particle complexes at the sensor surface and to remove unbound particles from the sensor surface. We achieved biological dose-response curves detecting 4-250pM amplicon concentrations in a one-step format in total assay times of less than 3min. Using various tag-antibody combinations specific for one of the individual genes, multi-analyte detection is shown of several antibiotic resistance genes of the food pathogen Salmonella. 
19107467	 A novel strategy was developed for the specific immobilization of DNA probes on poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD)  of PHB depolymerase as an active binding motif. To demonstrate whether this method can be used for the detection of clinical pathogens, the pathogen-specific biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for four model pathogens, while the target DNAs were prepared by PCR using universal  primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was immobilized on the PHB-coated slide by microspotting. This DNA-protein complex microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the specific pathogens could be diagnosed in the presence of other microorganisms. Thus, the DNA-protein complex microarray platform technology employing PHB and the SBD reported here can be widely used for the detection of DNA-DNA and DNA-biomolecule interactions without synthetic or chemical modification of biomolecules or solid surface. 
19348629	 Thanks to the availability of high-throughput omics data, bioinformatics approaches are able to hypothesize thus-far undocumented genetic interactions. However, due to the amount of noise in these data, inferences based on a single data source are often unreliable. A popular approach to overcome this problem is  to integrate different data sources. In this study, we describe DISTILLER, a novel framework for data integration that simultaneously analyzes microarray and  motif information to find modules that consist of genes that are co-expressed in  a subset of conditions, and their corresponding regulators. By applying our method on publicly available data, we evaluated the condition-specific transcriptional network of Escherichia coli. DISTILLER confirmed 62% of 736 interactions described in RegulonDB, and 278 novel interactions were predicted. 
19168658	 SdiA is a homolog of quorum-sensing regulators that detects N-acylhomoserine lactone (AHL) signals from other bacteria. Escherichia coli uses SdiA to reduce its biofilm formation in the presence of both AHLs and its own signal indole. Here we reconfigured SdiA (240 amino acids) to control biofilm formation using protein engineering. Four SdiA variants were obtained with altered biofilm formation, including truncation variants SdiA1E11 (F7L, F59L, Y70C, M94K, and K153X) and SdiA14C3 (W9R, P49T, N87T, frameshift at N96, and L123X), which reduced biofilm formation by 5- to 20-fold compared to wild-type SdiA in the presence of endogenous indole. Whole-transcriptome profiling revealed that wild-type SdiA reduced biofilm formation by repressing genes related to indole synthesis and curli synthesis compared to when no SdiA was expressed, while variant SdiA1E11 induced genes related to indole synthesis in comparison to wild-type SdiA. These results suggested altered indole metabolism, and corroborating the DNA microarray results in regard to indole synthesis, variant SdiA1E11 produced ninefold more indole, which led to reduced swimming motility and cell density. Also, wild-type SdiA decreased curli production and tnaA transcription, while SdiA1E11 increased tnaA transcription (tnaA encodes tryptophanase, which forms indole) compared to wild-type SdiA. Hence, wild-type SdiA decreased biofilm formation by reducing curli production and motility, and SdiA1E11 reduced biofilm formation via indole. Furthermore, an AHL-sensitive variant (SdiA2D10, having four mutations at E31G, Y42F, R116H, and L165Q) increased biofilm formation sevenfold in the presence of N-octanoyl-DL-homoserine lactone and N-(3-oxododecatanoyl)-L-homoserine lactone. Therefore, SdiA can be evolved to increase or decrease biofilm formation, and biofilm formation may be controlled by altering sensors rather than signals. 
19074378	 The uncharacterized protein family UPF0042 of the Swiss-Prot database is predicted to be a member of the conserved group of bacterium-specific P-loop-containing proteins. Here we show that two of its members, YvcJ from Bacillus subtilis and YhbJ, its homologue from Escherichia coli, indeed bind and  hydrolyze nucleotides. The cellular function of yvcJ was then addressed. In contrast to results recently obtained for E. coli, which indicated that yhbJ mutants strongly overproduced glucosamine-6-phosphate synthase (GlmS), comparison of the wild type with the yvcJ mutant of B. subtilis showed that GlmS expression  was quite similar in the two strains. However, in mutants defective in yvcJ, the  transformation efficiency and the fraction of cells that expressed competence were reduced. Furthermore, our data show that YvcJ positively controls the expression of late competence genes. The overexpression of comK or comS compensates for the decrease in competence of the yvcJ mutant. Our results show that even if YvcJ and YhbJ belong to the same family of P-loop-containing proteins, the deletion of corresponding genes has different consequences in B. subtilis and in E. coli. 
19246752	 We recently reported that carbon monoxide (CO) has bactericidal activity. To understand its mode of action we analysed the gene expression changes occurring when Escherichia coli, grown aerobically and anaerobically, is treated with the CO-releasing molecule CORM-2 (tricarbonyldichlororuthenium(II) dimer). Microarray analysis shows that the E. coli CORM-2 response is multifaceted, with a high number of differentially regulated genes spread through several functional categories, namely genes involved in inorganic ion transport and metabolism, regulators, and genes implicated in post-translational modification, such as chaperones. CORM-2 has a higher impact in E. coli cells grown anaerobically, as judged by the repression of genes belonging to eight functional classes which are not seen in the response of aerobically CORM-2-treated cells. The biological relevance of the variations caused by CORM-2 was substantiated by studying the CORM-2 sensitivity of selected E. coli mutants. The results show that the deletion of redox-sensing regulators SoxS and OxyR increased the sensitivity to CORM-2 and suggest that while SoxS plays an important role in protection against  CORM-2 under both growth conditions, OxyR seems to participate only in the aerobic CORM-2 response. Under anaerobic conditions, we found that the heat-shock proteins IbpA and IbpB contribute to CORM-2 defence since the deletion of these genes increases the sensitivity of the strain. The induction of several met genes and the hypersensitivity to CORM-2 of the DeltametR, DeltametI and DeltametN mutant strains suggest that CO has effects on the methionine metabolism of E. coli. CORM-2 also affects the transcription of several E. coli biofilm-related genes and increases biofilm formation in E. coli. In particular, the absence of tqsA or bhsA increases the resistance of E. coli to CORM-2, and deletion of tsqA  leads to a strain that has lost its capacity to form biofilm upon treatment with  CORM-2. In spite of the relatively stable nature of the CO molecule, our results  show that CO is able to trigger a significant alteration in the transcriptome of  E. coli which necessarily has effects in several key metabolic pathways. 
18823050	 We propose that transcript levels for some genes are affected by the bacterial cell division cycle and this may be an important factor to consider when designing synthetic bacterial genomes. To test this hypothesis, transcript levels of 58 genes in Escherichia coli B/r A were determined at five times during the cell division cycle. A two-step ANOVA technique was used to analyze data from custom oligonucleotide microarrays containing genes involved in important cellular processes including central metabolism, macromolecular synthesis, and transport and secretion. Consistent with results previously found in Caulobacter, approximately 17% of the transcript levels were cell cycle dependent. Cell cycle  regulation can be divided into two classes: genes displaying increased transcript concentrations following gene replication and genes displaying an increased transcript concentration prior to replication initiation. Transcripts levels for  hns, uspA, and zwf were affected by the cell division cycle, but did not fit well into either class. These results indicate that transcription of a significant fraction of the genome is affected by replication cycle progression. The results  also show that both physical gene position and the physiological function of a gene affect when it is transcribed. In addition to the simple association with replication fork progression, other phenomena must be occurring to account for some of our observations. In conclusion, gene position, with regard to the C period, and gene function are important factors to incorporate into design criteria for synthetic bacterial genomes. 
19091747	 Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO)(3)Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially  expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation. 
19040357	 Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33x10(-9) m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However,  selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By  transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with  various modifications, as well as dissecting signal transduction pathways. 
19028889	 Hypermutator Pseudomonas aeruginosa strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in P. aeruginosa, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new P. aeruginosa antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described P. aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD, mutT, ung, and mutY, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the P. aeruginosa PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of pfpI resulted in a dramatic increase in the H(2)O(2)-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced  protection against the basal level of reactive oxygen species. Finally, pfpI mutants are more sensitive to different types of stress and are affected in biofilm formation. 
19011022	 DNA microarrays demonstrate that H-NS controls 69% of the temperature regulated genes in Escherichia coli K-12. H-NS is shown to be a common regulator of multiple iron and other nutrient acquisition systems preferentially expressed at  37 degrees C and of general stress response, biofilm formation, and cold shock genes highly expressed at 23 degrees C. 
19144914	 Whereas ribosomal proteins (r-proteins) are known primarily as components of the  translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the  abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases  along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay  may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments. 
19022649	 To meet the requirement of World Health Organization for zero tolerance of E. coli cell in 100mL drinking water, a new procedure based on photodeposition of nano-Ag at TiO(2)-coated piezoelectric quartz crystal (PQC) electrode was developed to fabricate a highly sensitive PQC/DNA biosensor. Enhancement of 3.3 times for binding of complementary DNA has been shown and attributed to the following effects arising from the nano-Ag coating. First, a large increase in the active surface area and packing density of neutravidin enhances the maximum neutravidin load to 1.8 times of a normal electrode. Second, the functional activity of neutravidin is enhanced by chemical interaction with nano-Ag to give  rise to an increase in the binding ratio between neutravidin and biotinylated DNA probe from 1.00:1.76 to 1.00:3.01. Third, the stronger binding leads to a higher  stability of the biotinylated DNA probes bound and increase in hybridization with the complementary DNA. Under the optimized conditions for flow analysis with online PCR product denaturing and hybridization, a detection limit of eight E. coli cells are obtained which require sampling at least 800mL water to detect a single E. coli cell in 100mL water. 
18809310	 High-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of nucleic acids identifying specific bacterial pathogens  is reported. The biosensor consists of a high-performance SPR imaging sensor with polarization contrast and internal referencing (refractive index resolution 2 x 10(-7) RIU) and an array of DNA probes microspotted on the surface of the SPR sensor. It is demonstrated that short sequences of nucleic acids (20-23 bases) characteristic for bacterial pathogens such as Brucella abortus, Escherichia coli, and Staphylococcus aureus can be detected at 100 pM levels. Detection of specific DNA or RNA sequences can be performed in less than 15 min by the reported SPR sensor. 
19538023	 Antioxidant activity of isorhamnetin 3-O neohesperidoside (I3ON), isolated from the leaves of Acacia salicina, was determined by the ability of this compound to  inhibit lipid peroxidation and to protect against hydroxyl radical-induced DNA damage in pKS plasmid DNA and Escherichia coli cultures. Antigenotoxic activity was assessed by using the comet assay. The IC(50) value of the inhibitory activity toward lipid peroxidation by I3ON is 0.6 mM. This compound was also able to protect against hydroxyl radical-induced DNA damage in pKS plasmid DNA. Moreover, this compound induced an inhibitory activity toward H2O2-induced genotoxicity. The protective effect exhibited by this molecule was also determined by analysis of gene expression as a response to an oxidative stress, using a cDNA microarray. Transcription of several genes related to the antioxidant system (HMOX2 and TXNL) and to the DNA repair pathway (XPC, POLD1, POLD2, PCNA, DDIT3, APEX, and LIG4) were upregulated after incubation with I3ON.  Taken together, these observations provide evidence that the I3ON, isolated from  the leaves of A. salicina, is able to protect cells against oxidative stress. 
18793315	 Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using Escherichia coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above approximately 1.0 Os kg(-1) causes a dose-dependent K(+) leak from the cell, resulting in a substantial decrease in cytosolic K(+) content and a concurrent accumulation of Na(+) in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K(+) uptake. Ion flux data are consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K(+) uptake  to efflux. Microarray experiments reveal that about 40% of upregulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic and non-ionic osmotica. The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting  the importance of voltage-gated K(+) transporters for bacterial adaptation to hyperosmotic stress. 
18952787	 In Escherichia coli, the gcvB gene encodes a nontranslated RNA (referred to as GcvB) that regulates OppA and DppA, two periplasmic binding proteins for the oligopeptide and dipeptide transport systems. An additional regulatory target of  GcvB, sstT, was found by microarray analysis of RNA isolated from a wild-type strain and a gcvB deletion strain grown to mid-log phase in Luria-Bertani broth.  The SstT protein functions to transport L-serine and L-threonine by sodium transport into the cell. Reverse transcription-PCR and translational fusions confirmed that GcvB negatively regulates sstT mRNA levels in cells grown in Luria-Bertani broth. A series of transcriptional fusions identified a region of sstT mRNA upstream of the ribosome binding site needed for negative regulation by GcvB. Analysis of the GcvB RNA identified a sequence complementary to this region of the sstT mRNA. The region of GcvB complementary to sstT mRNA is the same region of GcvB identified to regulate the dppA and oppA mRNAs. Mutations predicted to disrupt base pairing between sstT mRNA and GcvB were made in gcvB, which resulted in the identification of a small region of GcvB necessary for negative regulation of sstT-lacZ. Additionally, the RNA chaperone protein Hfq was found to be necessary for GcvB to negatively regulate sstT-lacZ in Luria-Bertani  broth and glucose minimal medium supplemented with glycine. The sstT mRNA is the  first target found to be regulated by GcvB in glucose minimal medium supplemented with glycine. 
19074655	 Shigella is the cause of shigellosis or bacillary dysentery, the occurrence of which is estimated to be 165 million cases per year worldwide, resulting in 1.1 million deaths. Rapid and reliable assays for detecting and identifying Shigella  in food, environmental and clinical samples are therefore necessary. Shigella species are traditionally identified by their O antigens. This study developed a  DNA microarray targeting O-serotype-specific genes to detect all 34 distinct O-antigen forms of Shigella, including Shigella boydii types 1-18, Shigella dysenteriae types 1-13, Shigella flexneri types 1-5 and 6, and Shigella sonnei. A total of 282 strains were used to test the specificity of the microarray, including 186 Shigella and Escherichia coli representative strains, 86 Shigella clinical isolates and ten strains of other bacterial species that are commonly isolated from food or clinical stool specimens. The oligonucleotide probes were printed on the microarray in concentrations from 1 to 100 muM, and 10 muM proved  to be the optimal probe concentration. The detection sensitivity for each serotype was 50 ng genomic DNA or 1 c.f.u. in 25 g milk powder sample following a 6 h enrichment in broth. The microarray is specific, sensitive and reproducible,  and, to our knowledge, is the first report of a microarray for serotyping all O-antigen forms of Shigella. 
19159110	 A prototype handheld, compact, rapid thermocycler was developed for multiplex analysis of nucleic acids in an inexpensive, portable configuration. Instead of the commonly used Peltier heating/cooling element, electric thin-film resistive heater and a miniature fan enable rapid heating and cooling of glass capillaries  leading to a simple, low-cost Thin-Film Resistive Thermocycler (TFRT). Computer-based pulse width modulation control yields heating rates of 6-7 K/s and cooling rates of 5 K/s. The four capillaries are closely coupled to the heater, resulting in low power consumption. The energy required by a nominal PCR cycle (20 s at each temperature) was found to be 57+/-2 J yielding an average power of  approximately 1.0 W (not including the computer and the control system). Thus the device can be powered by a standard 9 V alkaline battery (or other 9 V power supply). The prototype TFRT was demonstrated (in a benchtop configuration) for detection of three important food pathogens (E. coli ETEC, Shigella dysenteriae,  and Salmonella enterica). PCR amplicons were analyzed by gel electrophoresis. The 35 cycle PCR protocol using a single channel was completed in less then 18 min. Simple and efficient heating/cooling, low cost, rapid amplification, and low power consumption make the device suitable for portable DNA amplification applications including clinical point of care diagnostics and field use. 
19118351	 In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria-Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA  expression in Luria-Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in  the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified  a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified  a region complementary to this region of cycA mRNA. However, mutations predicted  to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed. 
18843507	 RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli. The RpoS regulon expression has been well characterized in rich media that  support fast growth and high growth yields. In contrast, though RpoS levels are high in minimal media, how RpoS functions under such conditions has not been clearly resolved. In this study, we compared the global transcriptional profiles  of wild type and an rpoS mutant of E. coli grown in glucose minimal media using microarray analyses. The expression of over 200 genes was altered by loss of RpoS in exponential and stationary phases, with only 48 genes common to both conditions. The nature of the RpoS-controlled regulon in minimal media was substantially different from that expressed in rich media. Specifically, the expression of many genes encoding regulatory factors (e.g., hfq, csrA, and rpoE)  and genes in metabolic pathways (e.g., lysA, lysC, and hisD) were regulated by RpoS in minimal media. In early exponential phase, protein levels of RpoS in minimal media were much higher than that in Luria-Bertani media, which may at least partly account for the observed difference in the expression of RpoS-controlled genes. Expression of genes required for flagellar function and chemotaxis was elevated in the rpoS mutant. Western blot analyses show that the flagella sigma factor FliA was expressed much higher in rpoS mutants than in WT in all phase of growth. Consistent with this, the motility of rpoS mutants was enhanced relative to WT. In conclusion, RpoS and its controlled regulators form a complex regulatory network that mediates the expression of a large regulon in minimal media. 
19194530	 BACKGROUND: The Escherichia coli heterodimeric HU protein is a small DNA-bending  protein associated with the bacterial nucleoid. It can introduce negative supercoils into closed circular DNA in the presence of topoisomerase I. Cells lacking HU grow very poorly and display many phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the transcription profile of every Escherichia coli gene in the absence of one or both HU subunits. This genome-wide in silico transcriptomic approach, performed in parallel with in vivo genetic experimentation, defined the HU regulon. This large regulon, which comprises 8% of the genome, is composed of four biologically relevant gene classes whose regulation responds to anaerobiosis, acid stress, high osmolarity, and SOS induction. CONCLUSIONS/SIGNIFICANCE: The regulation a large number of genes encoding enzymes involved in energy metabolism and catabolism pathways by HU explains the highly pleiotropic phenotype of HU-deficient cells. The uniform chromosomal distribution of the many operons regulated by HU strongly suggests that the transcriptional and nucleoid architectural functions of HU constitute two aspects of a unique protein-DNA interaction mechanism. 
18782592	 The genomic DNA-DNA hybridization (DDH) method has been widely used as a practical method for the determination of phylogenetic relationships between closely related biological strains. Traditional DDH methods have serious limitations including low reproducibility, a high background and a time-consuming procedure. The DDH method using a genome-probing microarray (GPM) has been recently developed to complement conventional methods and could be used to overcome the limitations that are typically encountered. It is necessary to compare the GPM-based DDH method to the conventional methods before using the GPM for the estimation of genomic similarities since all of the previous scientific data have been entirely dependent on conventional DDH methods. In order to address this issue we compared the DDH values obtained using the GPM, microplate  and nylon membrane methods to multi-locus sequence typing (MLST) data for 9 Salmonella genomes and an Escherichia coli type strain. The results showed that the genome similarity values and the degrees of standard deviation obtained using the GPM method were lower than those obtained with the microplate and nylon membrane methods. The dendrogram from the cluster analysis of GPM DDH values was  consistent with the phylogenetic tree obtained from the multi-locus sequence typing (MLST) data but was not similar to those obtained using the microplate and nylon membrane methods. Although the signal intensity had to be maximal when the  targets were hybridized to their own probe, the methods using membranes and microplates frequently produced higher signals in the heterologous hybridizations than those obtained in the homologous hybridizations. Only the GPM method produced the highest signal intensity in homologous hybridizations. These results show that the GPM method can be used to obtain results that are more accurate than those generated by the other methods tested. 
18710431	 The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely  related bacteria, although the number of repeats varies. All five Sib RNAs in E.  coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion  strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18-  to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in  the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression  of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell. 
18819010	 Cistus creticus subsp. creticus is a plant of intrinsic scientific interest due to the distinctive pharmaceutical properties of its resin. Labdane-type diterpenes, the main constituents of the resin, exhibit considerable antibacterial and cytotoxic activities. In this study chemical analysis of isolated trichomes from different developmental stages revealed that young leaves of 1-2 cm length displayed the highest content of labdane-type diterpenes (80 mg/g fresh weight) whereas trichomes from older leaves (2-3 or 3-4 cm) exhibited  gradual decreased concentrations. A cDNA library was constructed enriched in transcripts from trichomes isolated from young leaves, which are characterized by high levels of labdane-type diterpenes. Functional annotation of 2,022 expressed  sequence tags (ESTs) from the trichome cDNA library based on homology to A. thaliana genes suggested that 8% of the putative identified sequences were secondary metabolism-related and involved primarily in flavonoid and terpenoid biosynthesis. A significant proportion of the ESTs (38%) displayed no significant similarity to any other DNA deposited in databases, indicating a yet unknown function. Custom DNA microarrays constructed with 1,248 individual clones from the cDNA library facilitated transcriptome comparisons between trichomes and trichome-free tissues. In addition, gene expression studies in various Cistus tissues and organs for one of the genes highlighted as the most differentially expressed by the microarray experiments revealed a putative sesquiterpene synthase with a trichome-specific expression pattern. Full length cDNA isolation  and heterologous expression in E. coli followed by biochemical analysis, led to the characterization of the produced protein as germacrene B synthase. 
19038054	 BACKGROUND: Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain chi7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. RESULTS: Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain chi7122. CONCLUSION: Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence. 
18774127	 Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2') from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by  resistance-causing enzymes have reduced affinities for binding their therapeutic  target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2'). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2') is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes. 
18648770	 To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra  mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double  genes knockout mutant to block this pathway, where the acetate overflowed due to  the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate  production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. 
18701636	 Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of  all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are  principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli sigma(70) (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes. 
18765720	 Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as  a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members  of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other  genes of C. rodentium whose expression required activation by RegA were required  for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence  of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required  for intestinal colonization. 
18710869	 Motility and adherence are two integral aspects of bacterial pathogenesis. Adherence, often mediated by fimbriae, permits bacteria to attach to host cells and establish infection, whereas flagellum-driven motility allows bacteria to disseminate to sites more advantageous for colonization. Both fimbriae and flagella have been proven important for virulence of uropathogenic Escherichia coli (UPEC). Reciprocal regulation is one mechanism by which bacteria may reconcile the contradictory actions of adherence and motility. PapX, a P fimbrial gene product of UPEC strain CFT073, is a functional homolog of MrpJ of Proteus mirabilis; ectopic expression of papX in P. mirabilis reduces motility. To define the connection between P fimbria expression and motility in UPEC, the role of papX in the regulation of motility of strain CFT073 was examined. Overexpression  of papX decreased motility of CFT073, which correlated with both a significant reduction in flagellin protein synthesized and flagella assembled on the cell surface. Conversely, an increase in motility and flagellin production was seen in an isogenic papX deletion mutant of CFT073. Microarray and quantitative reverse transcription-PCR analysis indicated that repression of motility of CFT073 by PapX appears to occur at the transcriptional level; expression of many motility-associated genes, including flhDC, the master regulator of motility, is  decreased when papX is overexpressed. Transcription of motility genes is increased in the papX mutant compared to wild type. Electrophoretic mobility gel  shift analysis revealed that PapX binds to the flhD promoter. We conclude that synthesis of P fimbriae regulates flagellum synthesis to repress motility via PapX. 
18805977	 The causative agent of Legionnaires' disease, Legionella pneumophila, is a natural parasite of environmental protozoa and employs a biphasic life style to switch between a replicative and a transmissive (virulent) phase. L. pneumophila  harbors the lqs (Legionella quorum sensing) cluster, which includes genes encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response regulator LqsR, and a homologue of HdeD, which is involved in acid resistance in  Escherichia coli. LqsR promotes host-cell interactions as an element of the stationary-phase virulence regulatory network. Here, we characterize L. pneumophila mutant strains lacking all four genes of the lqs cluster or only the  hdeD gene. While an hdeD mutant strain did not have overt physiological or virulence phenotypes, an lqs mutant showed an aberrant morphology in stationary growth phase and was defective for intracellular growth, efficient phagocytosis,  and cytotoxicity against host cells. Cytotoxicity was restored upon reintroduction of the lqs genes into the chromosome of an lqs mutant strain. The  deletion of the lqs cluster caused more-severe phenotypes than deletion of only lqsR, suggesting a synergistic effect of the other lqs genes. A transcriptome analysis indicated that in the stationary phase more than 380 genes were differentially regulated in the lqs mutant and wild-type L. pneumophila. Genes involved in protein production, metabolism, and bioenergetics were upregulated in the lqs mutant, whereas genes encoding virulence factors, such as effectors secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome  analysis revealed that a set of Icm/Dot substrates is not produced in the absence of the lqs gene cluster, which confirms the findings from DNA microarray assays and mirrors the virulence phenotype of the lqs mutant strain. 
18790864	 It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm  formation by generating the cell-to-cell mating F pili between pairs of F(+) cells (approximately two to four pili per cell) and by stimulating the formation  of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in  curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the  RpoS and HN-S global regulators). These data provided new insights into the role  of a natural F plasmid during the development of E. coli biofilms. 
18761688	 Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction  of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We  suggest that DinB targets Pol III, thereby acting as a brake on replication fork  progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication  to safeguard genome stability. 
19043542	 Response of cells to changing environmental conditions is governed by the dynamics of intricate biomolecular interactions. It may be reasonable to assume,  proteins being the dominant macromolecules that carry out routine cellular functions, that understanding the dynamics of protein:protein interactions might  yield useful insights into the cellular responses. The large-scale protein interaction data sets are, however, unable to capture the changes in the profile  of protein:protein interactions. In order to understand how these interactions change dynamically, we have constructed conditional protein linkages for Escherichia coli by integrating functional linkages and gene expression information. As a case study, we have chosen to analyze UV exposure in wild-type  and SOS deficient E. coli at 20 minutes post irradiation. The conditional networks exhibit similar topological properties. Although the global topological  properties of the networks are similar, many subtle local changes are observed, which are suggestive of the cellular response to the perturbations. Some such changes correspond to differences in the path lengths among the nodes of carbohydrate metabolism correlating with its loss in efficiency in the UV treated cells. Similarly, expression of hubs under unique conditions reflects the importance of these genes. Various centrality measures applied to the networks indicate increased importance for replication, repair, and other stress proteins  for the cells under UV treatment, as anticipated. We thus propose a novel approach for studying an organism at the systems level by integrating genome-wide functional linkages and the gene expression data. 
18762220	 Despite the extensive use of efficacious pertussis vaccines, Bordetella pertussis infections are still among the main causes for childhood morbidity and mortality. Severe pertussis occurs mostly in very young children, often too young to be sufficiently protected by current vaccines, which require several administrations in regimens that vary between countries. Since natural infection with B. pertussis is able to induce protection, we have developed the live attenuated B.  pertussis vaccine strain BPZE1 that protects mice upon a single intranasal administration. This strain was obtained by genetically inactivating pertussis toxin via two point mutations in the ptx gene, by deleting dnt encoding dermonecrotic toxin, and by replacing the B. pertussis ampG gene by Escherichia coli ampG, resulting in the removal of tracheal cytotoxin. Here, we assessed the  genetic stability of BPZE1 after 20 and 27 weeks of continuous passaging in vitro and in vivo, respectively. BPZE1 was passaged 20 times in vitro and 9 times in vivo in Balb/C mice. After these passages, 8 hemolytic colonies were analyzed by  PCR for the absence of dnt and B. pertussis ampG and the presence of E. coli ampG, by DNA sequencing for the presence of the two ptx point mutations and by DNA microarrays for the global genomic stability. In addition, the protective capacity of BPZE1 was evaluated after the passages. No genetic or protective difference was detected between the passaged bacteria and non-passaged BPZE1, indicating that stability of the vaccine strain is not a concern for BPZE1 to be  considered as an attenuated live vaccine against whooping cough. 
18729477	 A novel bacterial DNA sample preparation device for molecular diagnostics has been developed. On the basis of optimized conditions for bacterial adhesion, surface-modified silicon pillar arrays for bacterial cell capture were fabricated, and their ability to capture bacterial cells was demonstrated. The capture efficiency for bacterial cells such as Escherichia coli, Staphylococcus epidermidis, and Streptococcus mutans in buffer solution was over 75% with a flow rate of 400 microL/min. Moreover, the proposed method captured E. coli cells present in 50% whole blood effectively. The captured cells from whole blood were  then in- situ lyzed on the surface of the microchip, and the eluted DNA was successfully amplified by qPCR. These results demonstrate that the full process of pathogen capture to DNA isolation from whole blood could be automated in a single microchip. 
18854009	 BACKGROUND: Regulation of sulphur metabolism in Corynebacterium glutamicum ATCC 13032 has been studied intensively in the last few years, due to its industrial as well as scientific importance. Previously, the gene cg0156 was shown to belong to the regulon of McbR, a global transcriptional repressor of sulphur metabolism  in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting candidate for study to further the understanding of the regulation of sulphur metabolism in C. glutamicum. RESULTS: Deletion of cg0156, now designated cysR, results in the inability of the mutant to utilise sulphate and aliphatic sulphonates. DNA microarray hybridisations revealed 49 genes with significantly increased and 48 with decreased transcript levels in presence of the native CysR compared to a cysR deletion mutant. Among the genes positively controlled by CysR were the gene cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel retardation experiments demonstrated that binding of CysR to DNA depends in vitro on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of  the transcription start points of five transcription units helped to identify a 10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests proved this motif to be necessary for CysR-dependent transcriptional regulation. CONCLUSION: CysR acts as the functional analogue of the unrelated LysR-type regulator CysB from Escherichia coli, controlling sulphide production in response to acceptor availability. In both bacteria, gene duplication events seem to have  taken place which resulted in the evolution of dedicated regulators for the control of sulphonate utilisation. The striking convergent evolution of network topology indicates the strong selective pressure to control the metabolism of the essential but often toxic sulphur-containing (bio-)molecules. 
18828895	 BACKGROUND: A knowledge-based network, which is constructed by extracting as many relationships identified by experimental studies as possible and then superimposing them, is one of the promising approaches to investigate the associations between biological molecules. However, the molecular relationships change dynamically, depending on the conditions in a living cell, which suggests  implicitly that all of the relationships in the knowledge-based network do not always exist. Here, we propose a novel method to estimate the consistency of a given network with the measured data: i) the network is quantified into a log-likelihood from the measured data, based on the Gaussian network, and ii) the probability of the likelihood corresponding to the measured data, named the graph consistency probability (GCP), is estimated based on the generalized extreme value distribution. RESULTS: The plausibility and the performance of the present procedure are illustrated by various graphs with simulated data, and with two types of actual gene regulatory networks in Escherichia coli: the SOS DNA repair system with the  corresponding data measured by fluorescence, and a set of 29 networks with data measured under anaerobic conditions by microarray. In the simulation study, the procedure for estimating GCP is illustrated by a simple network, and the robustness of the method is scrutinized in terms of various aspects: dimensions of sampling data, parameters in the simulation study, magnitudes of data noise, and variations of network structures. In the actual networks, the former example  revealed that our method operates well for an actual network with a size similar  to those of the simulated networks, and the latter example illustrated that our method can select the activated network candidates consistent with the actual data measured under specific conditions, among the many network candidates. CONCLUSION: The present method shows the possibility of bridging between the static network from the literature and the corresponding measurements, and thus will shed light on the network structure variations in terms of the changes in molecular interaction mechanisms that occur in response to the environment in a living cell. 
18713320	 Ribonucleases J1 and J2 of Bacillus subtilis are evolutionarily conserved enzymes combining an endoribonucleolytic and a 5'-3' exoribonucleolytic activity in a single polypeptide. Their endoribonucleolytic cleavage specificity resembles that of RNase E, a key player in the processing and degradation of RNA in Escherichia  coli. The biological significance of the paralogous RNases J1 and J2 in Bacillus  subtilis is still unknown. Based on the premise that cleavage of an mRNA might alter its stability and hence its abundance, we have analysed the transcriptomes  and proteomes of single and double mutant strains. The absence or decrease of both RNases J1 and J2 together profoundly alters the expression level of hundreds of genes. By contrast, the effect on global gene expression is minimal in single  mutant strains, suggesting that the two nucleases have largely overlapping substrate specificities. Half-life measurements of individual mRNAs show that RNases J1/J2 can alter gene expression by modulating transcript stability. The absence/decrease of RNases J1 and J2 results in similar numbers of transcripts whose abundance is either increased or decreased, suggesting a complex role of these ribonucleases in both degradative and regulatory processing events that have an important impact on gene expression. 
18713317	 The carbon storage regulator CsrA is an RNA binding protein that controls carbon  metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF  domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobility shift assays confirmed the direct and specific binding of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH resulted in a more than 20-fold increase in the cellular concentration of the second messenger cyclic di-GMP (c-di-GMP), implying that both proteins possess diguanylate cyclase activity. Phenotypic characterization revealed that both proteins are involved in the regulation of motility in a c-di-GMP-dependent manner. CsrA was also found to regulate the expression of five additional GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels  of c-di-GMP. All together, these data demonstrate a global role for CsrA in the regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins  at the post-transcriptional level. 
18699868	 Thermus thermophilus SdrP is one of four cyclic AMP receptor protein (CRP)/fumarate and nitrate reduction regulator (FNR) family proteins from the extremely thermophilic bacterium T. thermophilus HB8. Expression of sdrP mRNA increased in the stationary phase during cultivation at 70 degrees C. Although the sdrP gene was non-essential, an sdrP-deficient strain showed growth defects,  particularly when grown in a synthetic medium, and increased sensitivity to disulphide stress. The expression of several genes was altered in the sdrP disruptant. Among them, we found eight SdrP-dependent promoters using in vitro transcription assays. A predicted SdrP binding site similar to that recognized by Escherichia coli CRP was found upstream of each SdrP-dependent promoter. In the wild-type strain, expression of these eight genes tended to increase upon entry into the stationary phase. Transcriptional activation in vitro was independent of any added effector molecule. The hypothesis that apo-SdrP is the active form of the protein was supported by the observation that the three-dimensional structure of apo-SdrP is similar to that of the DNA-binding form of E. coli CRP. Based on the properties of the SdrP-regulated genes found in this study, it is speculated  that SdrP is involved in nutrient and energy supply, redox control, and polyadenylation of mRNA. 
18813342	 We describe a novel, simple and low-cost protein microarray strategy wherein the  microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a  high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (approximately 3-7 x 10 (-13) M) of E. coli Tus protein to  Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication.  In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This "all DNA" microarray can be converted to a protein microarray on-demand without need for any additional capture reagent. 
18591240	 Pseudouridine, a non-classical nucleoside present in human urine as a degradation product of RNAs, is one of the few molecules that has a glycosidic C-C bond. Through a data base mining approach involving transcriptomic data, we have molecularly identified two enzymes that are involved in the metabolism of pseudouridine in uropathogenic Escherichia coli, the principal agent of urinary tract infections in humans. The first enzyme, coded by the gene yeiC, specifically phosphorylates pseudouridine to pseudouridine 5'-phosphate. Accordingly, yeiC(-) mutants are unable to metabolize pseudouridine, in contrast  to wild-type E. coli UTI89. The second enzyme, encoded by the gene yeiN belonging to the same operon as yeiC, catalyzes the conversion of pseudouridine 5'-phosphate to uracil and ribose 5-phosphate in a divalent cation-dependent manner. Remarkably, the glycosidic C-C bond of pseudouridine is cleaved in the course of this reaction, indicating that YeiN is the first molecularly identified enzyme able to hydrolyze a glycosidic C-C bond. Though this reaction is easily reversible, the association of YeiN with pseudouridine kinase indicates that it serves physiologically to metabolize pseudouridine 5'-phosphate rather than to form it. YeiN is homologous to Thermotoga maritima IndA, a protein with a new fold, which we now show to act also as a pseudouridine-5'-phosphate glycosidase.  Data base mining indicates that most eukaryotes possess homologues of pseudouridine kinase and pseudouridine-5'-phosphate glycosidase and that these are most often associated in a single bifunctional protein. The gene encoding this bifunctional protein is absent from the genomes of man and other mammals, indicating that the capacity for metabolizing pseudouridine has been lost late in evolution. 
18493744	 The enolase superfamily (COG4948) contains proteins with very different biological functions including regulators like the Escherichia coli RspA and metabolic enzymes like enolase. To unravel the biological function of an archaeal family member, an in frame deletion mutant of a gene encoding a COG4948 protein of Haloferax volcanii was generated. The mutant had a lag phase of 3 days after transition from a richer to a poorer medium, in contrast to the wild-type, and the gene was therefore named "important for transition" (iftA). After inoculation of fresh casamino acids or complex medium with stationary phase wild-type cells,  the transcript level of iftA was transiently induced at the onset of growth. In contrast, in minimal (or "poor") glucose medium, both transcript and protein were present throughout growth, even in late stationary phase. A comparison of the transcriptomes of deletion mutant and wild-type revealed that transcript levels of a very restricted set of genes were differentially regulated, including genes  encoding proteins involved in phosphate metabolism, regulators and stress response proteins. Taken together, the results indicate that IftA might have a dual function, i.e., transiently after transition to fresh medium and permanently during growth in glucose medium. 
18415079	 The methanogenic archaeon Methanosarcina mazei strain Gö1 contains two homologues of NrpR, the transcriptional repressor of nitrogen assimilation genes recently discovered and characterized in Methanococcus maripaludis. Insertion of a puromycin-resistance conferring cassette into MM1085 encoding a single NrpR domain with an N-terminal helix-turn-helix domain (NrpRI) lead to a significant reduction of the lag-phase after a shift from nitrogen sufficiency to nitrogen limitation. Consistent with this finding, loss of NrpRI resulted in significantly increased transcript levels of genes involved in nitrogen fixation or nitrogen assimilation though growing under nitrogen sufficiency as demonstrated by quantitative reverse transcriptional PCR analysis. Genome-wide analysis using DNA-microarrays confirmed that transcript levels of 27 ORFs were significantly elevated in the M. mazei MM1085::pac mutant under nitrogen sufficiency, including genes known to be up-regulated under nitrogen limitation (e.g., nifH, glnA(1), glnK(1)), and 17 additional genes involved in metabolism (4), encoding a flagella related protein (1) and genes encoding hypothetical proteins (12). Using cell extracts of Escherichia coli expressing MM1085 fused to the maltose binding protein (MBP-NrpRI) and employing promoter binding studies by DNA-affinity chromatography demonstrated that MBP-NrpRI binds specifically to the nifH-promoter. Deletion of various bases in the promoter region of nifH confirmed that the regulatory element ACC-N(7)-GGT is required for specific binding of NrpRI to the promoter. 
18640014	 Urinary tract infections (UTIs) are among the most frequently occurring infections and are mostly caused by extraintestinal pathogenic Escherichia coli.  DNA microarrays are potent molecular diagnostic tools for rapid diagnosis of bacterial infections with high relevance for UTIs. In this study, we present the  integration and application of two DNA chip modules for the simultaneous detection of single nucleotide polymorphisms in gyrA (quinolone resistance) and fimH (increased adhesion to urinary tract epithelium). The performance of the combined diagnostic chip was assessed by genotyping 140 E. coli strains. Resistance-causing mutations could only be identified in UTI isolates. A complete genotyping assay could be performed in <4h after DNA extraction. Together with the excellent genotyping results, this constitutes a competitive alternative as a standard tool for routine clinical diagnostics. 
18567656	 N-ethylmaleimide (NEM) has been used as a specific reagent of Cys modification in proteins and thus is toxic for cell growth. On the Escherichia coli genome, the nemA gene coding for NEM reductase is located downstream of the gene encoding an  as-yet-uncharacterized transcription factor, YdhM. Disruption of the ydhM gene results in reduction of nemA expression even in the induced state, indicating that the two genes form a single operon. After in vitro genomic SELEX screening,  one of the target recognition sequences for YdhM was identified within the promoter region for this ydhM-nemA operon. Both YdhM binding in vitro to the ydhM promoter region and transcription repression in vivo of the ydhM-nemA operon by YdhM were markedly reduced by the addition of NEM. Taken together, we propose that YdhM is the repressor for the nemA gene, thus hereafter designated NemR. The repressor function of NemR was inactivated by the addition of not only NEM but also other Cys modification reagents, implying that Cys modification of NemR renders it inactive. This is an addition to the mode of controlling activity of transcription factors by alkylation with chemical agents. 
18714349	 BACKGROUND: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. METHODOLOGY/PRINCIPAL FINDINGS: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression  of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation. CONCLUSION/SIGNIFICANCE: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart. 
18713062	 Escherichia coli O157:H7 poses a threat to humans through food- and water-borne transmission. To investigate how environmental stresses affect the Escherichia coli O157:H7 transcriptome, we designed a targeted microarray consisting of stress response and virulence genes (n = 125) to analyze the impact of acidified  (pH 3.5), cold (7.5 degrees C), and fresh tryptic soy broth (TSB) (37 degrees C)  on E. coli O157:H7 stress response and virulence gene expression. Nutrient replenishment with fresh TSB resulted in 72 differentially expressed genes (> or  = 1.5-fold change; p < 0.05), with 65 induced. All queried global and specific stress regulators were affected, as were 12 virulence genes. Cold-shocked cells displayed 17 differentially expressed genes, with 10 being induced. Induction of  rpoS, members of the sigma(H) regulon (clpB, dnaK, ftsH), and acid resistance (AR) genes (gadA, gadX) was observed. Porin transcript (ompC, ompF) and gapA and  tufA ancillary genes were repressed. Acid shock resulted in 24 differentially expressed genes, with 21 induced. No induction of any stationary phase AR system  was observed, though acid-coping mechanisms were recruited, including mar and phoB induction, and repression of ompC and ompF. Stress regulators were induced,  including relA, soxS, rpoE, and rpoH. The microarray data were validated by quantitative real-time polymerase chain reaction. Exposure to sublethal stress events led to the induction of diverse stress response networks. In the food chain, sublethal events may render cells increasingly resistant to future stresses, potentially leading to increased survival. 
18673071	 Transcriptome analysis using microarrays has become a powerful tool to better understand the process of disease and other complex biological processes such as  food spoilage and biofilm formation. This review is divided into two basic sections: 1) a short history and description of microarrays and 2) a discussion of studies involving bacterial food safety pathogens that focused on whole genome transcript analysis. Not included are the many studies using microarrays to identify, diagnose, or genetically characterize these organisms. This review focuses on studies involving Escherichia coli O157:H7, Salmonella spp., Campylobacter jejuni, Listeria monocytogenes, and Yersinia enterocolitica. Many of the studies involve altering the growth environment to simulate stress conditions and the use of host-pathogen model systems to explore virulence mechanisms. Few studies use conditions that might be considered unique to the food industry. Exceptions are studies of biofilm-specific transcriptome changes and analysis following pressure treatment. This review should not be considered as a comprehensive review, and where appropriate, species-specific reviews are cited that are more complete. 
18541659	 Shiga toxin 1 (Stx1) and Stx2 produced by Escherichia coli O157 are known to be cytotoxic to Vero and HeLa cells by inhibiting protein synthesis and by inducing  apoptosis. In the present study, we have demonstrated that 10 ng/ml Stx2 induced  DNA fragmentation in human brain microvascular endothelial cells (HBMEC), with cleavage activation of caspase-3, -6, -8, and -9. A microarray approach used to search for apoptotic potential signals in response to Stx2 revealed that Stx2 treatment induced a marked upregulation of C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible protein 153 (GADD153). Increased CHOP expression  was dependent on enzymatically active Stx1. Knockdown of CHOP mRNA reduced the activation of caspase-3 and prevented apoptotic cell death. These results suggest that Stx2-induced apoptosis is mediated by CHOP in HBMEC and involves activation  of both the intrinsic and extrinsic pathways of apoptosis. 
18539742	 The higher affinity of Cd(2+) for sulfur compounds than for nitrogen and oxygen led to the theoretical consideration that cadmium toxicity should result mainly from the binding of Cd(2+) to sulfide, thiol groups, and sulfur-rich complex compounds rather than from Cd(2+) replacement of transition-metal cations from nitrogen- or oxygen-rich biological compounds. This hypothesis was tested by using Escherichia coli for a global transcriptome analysis of cells synthesizing  glutathione (GSH; wild type), gamma-glutamylcysteine (DeltagshB mutant), or neither of the two cellular thiols (DeltagshA mutant). The resulting data, some of which were validated by quantitative reverse transcription-PCR, were sorted using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system, which  groups genes hierarchically with respect to the cellular functions of their respective products. The main difference among the three strains concerned tryptophan biosynthesis, which was up-regulated in wild-type cells upon cadmium shock and strongly up-regulated in DeltagshA cells but repressed in DeltagshB cells containing gamma-glutamylcysteine instead of GSH. Overall, however, all three E. coli strains responded to cadmium shock similarly, with the up-regulation of genes involved in protein, disulfide bond, and oxidative damage  repair; cysteine and iron-sulfur cluster biosynthesis; the production of proteins containing sensitive iron-sulfur clusters; the storage of iron; and the detoxification of Cd(2+) by efflux. General energy conservation pathways and iron uptake were down-regulated. These findings indicated that the toxic action of Cd(2+) indeed results from the binding of the metal cation to sulfur, lending support to the hypothesis tested. 
18397308	 Thermotoga maritima, an anaerobic hyperthermophilic bacterium, was found able to  grow in the presence of low concentrations of oxygen of up to 0.5% (v/v). Differential proteomics and transcripts analysis by qRT-PCR were used to identify the defence strategies used by T. maritima to protect itself against oxygen. A flavoprotein, homologous to rubredoxin oxygen oxidoreductase was found to be overproduced when cells were cultured in oxidative conditions. The recombinant protein, produced in Escherichia coli, exhibited an oxygen reductase activity, which could account for the observed decrease in oxygen concentration during growth. The gene encoding this oxygen reductase belongs to a multicistronic unit  that includes genes encoding proteins involved in exopolysaccharide biosynthesis, which may be related to a biofilm formation induced by the presence of oxygen. Enzymes involved in reactive oxygen species detoxification, iron-sulfur centre synthesis/repair and the cysteine biosynthesis pathway were also overproduced. All these enzymatic systems together contribute to the defence strategy of T. maritima against oxygen. Because of the position of T. maritima in deep branches  of the phylogenetic tree, we suggest that these strategies can be considered as ancestral mechanisms first developed by anaerobic microorganisms on the early Earth to protect themselves against primary abiotic or biotic oxygen production. 
18515344	 The Escherichia coli RutR protein is the master regulator of genes involved in pyrimidine catabolism. Here we have used chromatin immunoprecipitation in combination with DNA microarrays to measure the binding of RutR across the chromosome of exponentially growing E. coli cells. Twenty RutR-binding targets were identified and analysis of these targets generated a DNA consensus logo for  RutR binding. Complementary in vitro binding assays showed high-affinity RutR binding to 16 of the 20 targets, with the four low-affinity RutR targets lacking  predicted key binding determinants. Surprisingly, most of the DNA targets for RutR are located within coding segments of the genome and appear to have little or no effect on transcript levels in the conditions tested. This contrasts sharply with other E. coli transcription factors whose binding sites are primarily located in intergenic regions. We suggest that either RutR has yet undiscovered function or that evolution has been slow to eliminate non-functional DNA sites for RutR because they do not have an adverse effect on cell fitness. 
18214547	 Renal scarring is a serious complication of chronic pyelonephritis that occurs due to vesicoureteral reflux. In our study, we performed global expression profiling of the kidney during renal scarring formation in a rat pyelonephritis model. An inoculum of Escherichia coli was injected directly into the renal cortex. Histologically, renal scarring developed during the 3-to-4 week period after injection. The time-course expression profile of 18,442 genes was then analyzed using microarrays, followed by validation with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the genes found to be up-regulated during renal scarring are associated with immune and defense responses, including cytokines, chemokines and their receptors, complement factors, adhesion molecules and extracellular matrix proteins. These genes were up-regulated as early as 1 week after injection, when no fibrotic changes were yet evident, peaked at 2 weeks, and gradually decreased thereafter. However, a subset of cytokine genes was found to be persistently activated even at 6 weeks after injection, including interleukin (IL)-1beta, transforming growth factor (TGF)-beta, and IL-3. Further statistical analysis indicated that the pathways mediated by these cytokines are activated concomitantly with renal scarring formation. The products of these genes may thus potentially be novel non-invasive diagnostic or prognostic biomarkers of renal scarring. 
18560561	 The coordinated expression of the different genes in an organism is essential to  sustain functionality under the random external perturbations to which the organism might be subjected. To cope with such external variability, the global dynamics of the genetic network must possess two central properties. (a) It must  be robust enough as to guarantee stability under a broad range of external conditions, and (b) it must be flexible enough to recognize and integrate specific external signals that may help the organism to change and adapt to different environments. This compromise between robustness and adaptability has been observed in dynamical systems operating at the brink of a phase transition between order and chaos. Such systems are termed critical. Thus, criticality, a precise, measurable, and well characterized property of dynamical systems, makes  it possible for robustness and adaptability to coexist in living organisms. In this work we investigate the dynamical properties of the gene transcription networks reported for S. cerevisiae, E. coli, and B. subtilis, as well as the network of segment polarity genes of D. melanogaster, and the network of flower development of A. thaliana. We use hundreds of microarray experiments to infer the nature of the regulatory interactions among genes, and implement these data into the Boolean models of the genetic networks. Our results show that, to the best of the current experimental data available, the five networks under study indeed operate close to criticality. The generality of this result suggests that  criticality at the genetic level might constitute a fundamental evolutionary mechanism that generates the great diversity of dynamically robust living forms that we observe around us. 
18026935	 A human monocytes cell line, U-937, incubated in the presence of filtered medium  from Escherichia coli culture (FS) has been previously reported to degrade man made mineral fiber and it has been indicated as a good paradigm of in vivo fiber  biopersistence evaluation (manuscript accepted for publication). In the present paper, a study is reported aimed to define the molecular modification occurring in the U-937 monocytes during in vitro fiber degradation. The induction of gene expression was investigated in U-937 exposed to rock wool fibers (HDN) in the presence of FS by transcriptome analysis using 20 K DNA microarrays and quantitative RT-PCR. The over-expression of genes related to mobility and cellular adhesion, oxidative stress, immune system stimulation, enzymes, and ions transport protein systems were identified. Among them NCF1 gene, the gene encoding a subunit of NADPH oxidase, over-expression was detected. As the product of this gene allows the formation of superoxide anion that could lead to oxidative stress, HDN fibers were exposed to hydrogen peroxide. Fiber degradation similar to those observed upon incubation with U-937 in the presence of FS was obtained thus suggesting that reactive oxygen species production may be responsible for fiber degradation by U-937 monocytes. 
18362131	 DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the  LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector  pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains. 
18428126	 The aim of the study was to develop a multiplex PCR-based DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA microarrays. The detection limits were 10(1) copies/microl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E.  coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA microarray technology, which is rapid, specific and  sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses. 
18465893	 We considered, on a global scale, the relationship between the predicted fraction of protein disorder and the RNA and protein expression in Escherichia coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was  highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of  several groups of proteins. 
18487194	 Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed  by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes. 
18344336	 Differential gene expression in biofilm cells suggests that adding the derepressed conjugative plasmid R1drd19 increases biofilm formation by affecting  genes related to envelope stress (rseA and cpxAR), biofilm formation (bssR and cstA), energy production (glpDFK), acid resistance (gadABCEX and hdeABD), and cell motility (csgBEFG, yehCD, yadC, and yfcV); genes encoding outer membrane proteins (ompACF), phage shock proteins (pspABCDE), and cold shock proteins (cspACDEG); and phage-related genes. To investigate the link between the identified genes and biofilm formation upon the addition of R1drd19, 40 isogenic  mutants were classified according to their different biofilm formation phenotypes. Cells with class I mutations (those in rseA, bssR, cpxA, and ompA) exhibited no difference from the wild-type strain in biofilm formation and no increase in biofilm formation upon the addition of R1drd19. Cells with class II mutations (those in gatC, yagI, ompC, cspA, pspD, pspB, ymgB, gadC, pspC, ymgA, slp, cpxP, cpxR, cstA, rseC, ompF, and yqjD) displayed increased biofilm formation compared to the wild-type strain but decreased biofilm formation upon the addition of R1drd19. Class III mutants showed increased biofilm formation compared to the wild-type strain and increased biofilm formation upon the addition of R1drd19. Cells with class IV mutations displayed increased biofilm formation compared to the wild-type strain but little difference upon the addition of R1drd19, and class V mutants exhibited no difference from the wild-type strain but increased biofilm formation upon the addition of R1drd19. Therefore, proteins encoded by the genes corresponding to the class I mutant phenotype are involved in R1drd19-promoted biofilm formation, primarily through their impact on cell motility. We hypothesize that the pili formed upon the addition of the conjugative plasmid disrupt the membrane (induce ompA) and activate the two-component system CpxAR as well as the other envelope stress response system, RseA-sigma(E), both of which, along with BssR, play a key role in bacterial biofilm formation. 
18451862	 RNA localization is important for the establishment and maintenance of polarity in multiple cell types. Localized RNAs are usually transported along microtubules or actin filaments and become anchored at their destination to some underlying subcellular structure. Retention commonly involves actin or actin-associated proteins, although cytokeratin filaments and dynein anchor certain RNAs. RNA localization is important for diverse processes ranging from cell fate determination to synaptic plasticity; however, so far there have been few comprehensive studies of localized RNAs in mammalian cells. Here we have addressed this issue, focusing on migrating fibroblasts that polarize to form a leading edge and a tail in a process that involves asymmetric distribution of RNAs. We used a fractionation scheme combined with microarrays to identify, on a  genome-wide scale, RNAs that localize in protruding pseudopodia of mouse fibroblasts in response to migratory stimuli. We find that a diverse group of RNAs accumulates in such pseudopodial protrusions. Through their 3' untranslated  regions these transcripts are anchored in granules concentrated at the plus ends  of detyrosinated microtubules. RNAs in the granules associate with the adenomatous polyposis coli (APC) tumour suppressor and the fragile X mental retardation protein (FMRP). APC is required for the accumulation of transcripts in protrusions. Our results suggest a new type of RNA anchoring mechanism as well as a new, unanticipated function for APC in localizing RNAs. 
18405378	 BACKGROUND: Bacterial genome sequences are being determined rapidly, but few species are physiologically well characterized. Predicting regulation from genome sequences usually involves extrapolation from better-studied bacteria, using the  hypothesis that a conserved regulator, conserved target gene, and predicted regulator-binding site in the target promoter imply conserved regulation between  the two species. However many compared organisms are ecologically and physiologically diverse, and the limits of extrapolation have not been well tested. In E. coli K-12 the leucine-responsive regulatory protein (Lrp) affects expression of approximately 400 genes. Proteus mirabilis and Vibrio cholerae have highly-conserved lrp orthologs (98% and 92% identity to E. coli lrp). The functional equivalence of Lrp from these related species was assessed. RESULTS: Heterologous Lrp regulated gltB, livK and lrp transcriptional fusions in an E. coli background in the same general way as the native Lrp, though with significant differences in extent. Microarray analysis of these strains revealed  that the heterologous Lrp proteins significantly influence only about half of the genes affected by native Lrp. In P. mirabilis, heterologous Lrp restored swarming, though with some pattern differences. P. mirabilis produced substantially more Lrp than E. coli or V. cholerae under some conditions. Lrp regulation of target gene orthologs differed among the three native hosts. Strikingly, while Lrp negatively regulates its own gene in E. coli, and was shown to do so even more strongly in P. mirabilis, Lrp appears to activate its own gene in V. cholerae. CONCLUSION: The overall similarity of regulatory effects of the Lrp orthologs supports the use of extrapolation between related strains for general purposes. However this study also revealed intrinsic differences even between orthologous regulators sharing >90% overall identity, and 100% identity for the DNA-binding helix-turn-helix motif, as well as differences in the amounts of those regulators. These results suggest that predicting regulation of specific target genes based on genome sequence comparisons alone should be done on a conservative basis. 
18230670	 Although Escherichia coli LPS is known to elicit various proinflammatory responses in macrophages, its effect on the translational states of transcripts has not yet been explored on a genome-wide scale. To address this, we investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein  particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total cellular, polysomal, and free mRNP fractions enabled us to identify transcripts that were modulated at the translational level by LPS: among 19,791 transcripts,  115 and 418 were up- and downregulated at 1, 2, or 4 h after LPS stimulation (100 ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based analysis suggested that translation-dependent downregulated genes frequently include those encoding proteins in the mitochondrial respiratory chain. In fact,  the mRNA levels of some transcripts for complexes I, IV, and V in the mitochondrial respiratory chain were translationally downregulated, eventually contributing to the decline of their protein levels. Moreover, the amount of metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased without any change of its mRNA level in total cellular fraction after LPS stimulation. Consistently, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, and the attenuation was independent of nitric oxide. These results demonstrated that translational suppression may play a critical role in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric oxide-independent manner in J774.1 cells. 
18180286	 The carbon sources that support the growth of pathogenic Escherichia coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coli EDL933 grows primarily as single cells dispersed within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in  vitro, using a mixture of the sugars known to be present in cecal mucus, and found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time. We conducted systematic mutation analyses of E. coli EDL933 and E. coli MG1655 by using lesions in the pathways used for catabolism of 13 mucus-derived sugars and five other compounds for which the corresponding bacterial gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic backgrounds was fed to streptomycin-treated mice, together with the respective wild-type parent strain, and their colonization was monitored by fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations  that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In  addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota. 
18192383	 Gram-negative bacteria possess stress responses to maintain the integrity of the  cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a  single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition  of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay  increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics. 
18093856	 Classic strain engineering methods have previously been limited by the low-throughput of conventional sequencing technology. Here, we applied a new genomics technology, scalar analysis of library enrichments (SCALEs), to measure  >3 million Escherichia coli genomic library clone enrichment patterns resulting from growth selections employing three aspartic-acid anti-metabolites. Our objective was to assess the extent to which access to genome-scale enrichment patterns would provide strain-engineering insights not reasonably accessible through the use of conventional sequencing. We determined that the SCALEs method  identified a surprisingly large range of anti-metabolite tolerance regions (423,  865, or 909 regions for each of the three anti-metabolites) when compared to the  number of regions (1-3 regions) indicated by conventional sequencing. Genome-scale methods uniquely enable the calculation of clone fitness values by providing concentration data for all clones within a genomic library before and after a period of selection. We observed that clone fitness values differ substantially from clone concentration values and that this is due to differences in overall clone fitness distributions for each selection. Finally, we show that  many of the clones of highest fitness overlapped across all selections, suggesting that inhibition of aspartate metabolism, as opposed to specific inhibited enzymes, dominated each selection. Our follow up studies confirmed our  observed growth phenotypes and showed that intracellular amino-acid levels were also altered in several of the identified clones. These results demonstrate that  genome-scale methods, such as SCALEs, can be used to dramatically improve understanding of classic strain engineering approaches. 
18158608	 RpoS is a major regulator of genes required for adaptation to stationary phase in E. coli. However, the exponential phase expression of some genes is affected by rpoS mutation, suggesting RpoS may also have an important physiological role in growing cells. To test this hypothesis, we examined the regulatory role of RpoS in exponential phase using both genomic and biochemical approaches. Microarray expression data revealed that, in the rpoS mutant, the expression of 268 genes was attenuated while the expression of 24 genes was enhanced. Genes responsible for carbon source transport (the mal operon for maltose), protein folding (dnaK and mopAB), and iron acquisition (fepBD, entCBA, fecI, and exbBD) were positively controlled by RpoS. The importance of RpoS-mediated control of iron acquisition was confirmed by cellular metal analysis which revealed that the intracellular iron content of wild type cells was two-fold higher than in rpoS mutant cells. Surprisingly, many previously identified RpoS stationary-phase dependent genes were not controlled by RpoS in exponential phase and several genes were RpoS-regulated only in exponential phase, suggesting the involvement of other regulators. The expression of RpoS-dependent genes osmY, tnaA and malK was controlled by Crl, a transcriptional regulator that modulates RpoS activity. In summary, the identification of a group of exponential phase genes controlled by RpoS reveals a novel aspect of RpoS function. 
18208528	 6S RNA binds sigma70-RNA polymerase and downregulates transcription at many sigma70-dependent promoters, but others escape regulation even during stationary  phase when the majority of the transcription machinery is bound by the RNA. We report that core promoter elements determine this promoter specificity; a weak -35 element allows a promoter to be 6S RNA sensitive, and an extended -10 element similarly determines 6S RNA inhibition except when a consensus -35 element is present. These two features together predicted that hundreds of mapped Escherichia coli promoters might be subject to 6S RNA dampening in stationary phase. Microarray analysis confirmed 6S RNA-dependent downregulation of expression from 68% of the predicted genes, which corresponds to 49% of the expressed genes containing mapped E. coli promoters and establishes 6S RNA as a global regulator in stationary phase. We also demonstrate a critical role for region 4.2 of sigma70 in RNA polymerase interactions with 6S RNA. Region 4.2 binds the -35 element during transcription initiation; therefore we propose one mechanism for 6S RNA regulation of transcription is through competition for binding region 4.2 of sigma70. 
18302792	 BACKGROUND: Many E. coli genes show pH-dependent expression during logarithmic growth in acid (pH 5-6) or in base (pH 8-9). The effect of rapid pH change, however, has rarely been tested. Rapid acid treatment could distinguish between genes responding to external pH, and genes responding to cytoplasmic acidification, which occurs transiently following rapid external acidification. It could reveal previously unknown acid-stress genes whose effects are transient, as well as show which acid-stress genes have a delayed response. RESULTS: Microarray hybridization was employed to observe the global gene expression of E. coli K-12 W3110 following rapid acidification of the external medium, from pH 7.6 to pH 5.5. Fluorimetric observation of pH-dependent tetR-YFP  showed that rapid external acidification led to a half-unit drop in cytoplasmic pH (from pH 7.6 to pH 6.4) which began to recover within 20 s. Following acid treatment, 630 genes were up-regulated and 586 genes were down-regulated. Up-regulated genes included amino-acid decarboxylases (cadA, adiY, gadA), succinate dehydrogenase (sdhABCD), biofilm-associated genes (bdm, gatAB, and ymgABC), and the Gad, Fur and Rcs regulons. Genes with response patterns consistent with cytoplasmic acid stress were revealed by addition of benzoate, a  membrane-permeant acid that permanently depresses cytoplasmic pH without affecting external pH. Several genes (yagU, ygiN, yjeI, and yneI) were up-regulated specifically by external acidification, while other genes (fimB, ygaC, yhcN, yhjX, ymgABC, yodA) presented a benzoate response consistent with cytoplasmic pH stress. Other genes (the nuo operon for NADH dehydrogenase I, and  the HslUV protease) showed delayed up-regulation by acid, with expression rising  by 10 min following the acid shift. CONCLUSION: Transcriptomic profiling of E. coli K-12 distinguished three different classes of change in gene expression following rapid acid treatment: up-regulation with or without recovery, and delayed response to acid. For eight genes showing acid response and recovery (fimB, ygaC, yhcN, yhjX, ymgABC, yodA),  responses to the permeant acid benzoate revealed expression patterns consistent with sensing of cytoplasmic pH. The delayed acid response of nuo genes shows that NADH dehydrogenase I is probably induced as a secondary result of acid-associated metabolism, not as a direct response to cytoplasmic acidification. 
18245349	 During a 1000-generation evolution experiment, two types of morphologically and kinetically distinct bacteria repeatedly diverged from a common ancestor in a fully sympatric seasonal environment containing glucose and acetate. To investigate the metabolic modifications associated with this adaptive diversification, we compared transcription profiles of the two derived types and  the common ancestor. Both derived types share a suite of common metabolic changes that may represent adaptation to the environment preceding the diversification event. These include improved translation efficiency, glucose uptake capacity via the mal/lamB genes, upregulation of various transporters during stationary phase, and likely the disruption of the rbs operon. The diversification event is associated with the overexpression of genes involved in the TCA cycle, glyoxylate shunt, acetate consumption, and anaerobic respiration in one type and in acetate  excretion in the other. These results reveal that competition for both carbon and oxygen have likely played an important role in the adaptation of Escherichia coli during this adaptive diversification event, where one derived type mainly consumes glucose at a fast rate when oxygen is not limiting, and the other derived type consumes glucose and acetate at a slower rate, even when oxygen is limiting. 
18039831	 In some patients, Escherichia coli strains establish significant bacteriuria without causing symptoms of urinary tract infection (UTI). These asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence factors than the uropathogenic E. coli (UPEC) strains that cause severe, symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence genes, and the molecular basis of their low virulence therefore remains unclear.  This study examined whether ABU strains might evolve from UPEC by genome loss and virulence gene attenuation. The presence of conserved E. coli K-12 genes was examined using an E. coli K-12 strain MG1655-specific DNA array and the distribution of UPEC virulence-related genes was examined with the E. coli pathoarray. Two groups of strains could be distinguished. Several ABU strains were shown by multilocus sequence typing and by comparative genomic analyses to be related to UPEC but to have smaller genome sizes. There were significant alterations in essential virulence genes, including reductive evolution by point  mutations, DNA rearrangements, and deletions. Other strains were unrelated to UPEC and lacked most of the virulence-associated genes. The results suggest that  some ABU strains arise from virulent strains by attenuation of virulence genes while others are nonvirulent and resemble commensal strains. We propose that virulence attenuation might constitute a general mechanism for mucosal pathogens  to evolve toward commensalism. 
18237283	 Gram-negative bacteria-binding proteins (GNBPs) are pattern recognition receptors which contribute to the defensive response against Plasmodium infection in Anopheles. We have characterized the GNBP gene family in Anopheles gambiae at the molecular level, and show that they are functionally diverse components of the A. gambiae innate immune system. GNBPB4 is a major factor in the defence against a broad range of pathogens, while the other GNBPs have narrower defence specificities. GNBPB4 is associated with the regulation of immune signalling pathways and was found to interact with the Gram-negative Escherichia coli and weakly co-localized with Plasmodium berghei ookinetes in the mosquito midgut epithelium. 
18227264	 In Escherichia coli K-12 the expression of many genes is controlled by the oxygen-responsive transcription factor FNR and the nitrate- and nitrite-responsive two-component systems NarXL and NarPQ. Here, the ydhY gene is  shown to be the first gene of a six-gene operon (ydhYVWXUT) that encodes proteins predicted to be components of an oxidoreductase. Mapping the ydhY-T transcript start and site-directed mutagenesis confirmed that the ydhY-T genes are transcribed from an FNR-dependent class II promoter and showed that the FNR site  is centred at -42.5. In the presence of nitrate or nitrite, NarXL and NarPQ repressed ydhY-T expression. Analysis of the DNA sequence of the ydhY promoter region (PydhY) revealed the presence of four heptameric sequences similar to NarL/P binding sites centred at -42, -16, +6 and +15. The latter heptamers are arranged as a 7-2-7 inverted repeat, which is required for recognition by NarP. Accordingly, NarP protected the 7-2-7 region in DNase I footprints, and mutation  of either heptamer +6 or heptamer +15 impaired nitrite-mediated repression, whereas mutation of heptamer -42 and heptamer -16 did not affect the response to  nitrite. The NarL protein also protected the 7-2-7 region, but in contrast to NarP, the NarL footprint extended further upstream to encompass the -16 heptamer. The extended NarL footprint was consistent with the presence of multiple NarL-PydhY complexes in gel retardation assays. Mutation of heptamer -42, which is located within the FNR binding site, or heptamer +6 (but not heptamers -16 or  +15) impaired nitrate-mediated repression. Thus, although the region of the ydhY-T promoter containing the -16 and +15 heptamers was recognized by NarL in vitro, mutation of these heptamers did not affect NarL-mediated repression in vivo. 
18069965	 The Escherichia coli CpxAR two-component signal transduction system senses and responds to extracytoplasmic stress. The cpxA101* allele was previously found to  reduce F plasmid conjugation by post-transcriptional inactivation of the positive activator TraJ. Microarray analysis revealed upregulation of the protease-chaperone pair, HslVU, which was shown to degrade TraJ in an E. coli C600 cpxA101* background. Double mutants of cpxA101* and hslV or hslU restored TraJ and F conjugation to wild-type levels. The constitutive overexpression of nlpE, an outer membrane lipoprotein that induces the Cpx stress response, also led to HslVU-mediated degradation of TraJ and repression of F transfer. However,  Cpx-mediated TraJ degradation appears to be growth phase-dependent, as induction  of nlpE in mid-log phase cells did not appreciably alter TraJ levels. Further, His6-TraJ was sensitive to HslVU degradation in vitro only when it was purified from cells overexpressing nlpE. Thus, TraJ appears to become resistant to HslVU during normal growth, with this resistance mapping to the F transfer region. Extracytoplasmic stress prevents this modification of TraJ, leaving it susceptible to HslVU. Thus, the CpxAR stress response indirectly controls the synthesis of the F mating apparatus, a complex transenvelope type IV secretion system, by degrading TraJ. 
18215272	 BACKGROUND: The Yersinia enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of flhD and fliA mutants that result from the complex gene regulation. RESULTS: Phenotypes relating to central metabolism were investigated with Phenotype MicroArrays. Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source.  Several intermediates of the tricarboxylic acid and the urea cycle, as well as several dipeptides, provided differential growth conditions for the two mutants.  Gene expression was determined for selected genes and correlated with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay. The assay that was previously established for Escherichia coli strains was modified for Y. enterocolitica. The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria. In contrast, the fliA mutant caused wild-type lethality. This indicates that the virulence phenotype of the flhD mutant might be due to genes that are regulated by FlhD/FlhC but not FliA, such as those that encode the flagellar type III secretion system. CONCLUSION: Phenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation. 
18433631	 NsrR is a nitric oxide-sensitive regulator of transcription. In Escherichia coli, NsrR is a repressor of the hmp gene encoding the flavohemoglobin that detoxifies  nitric oxide. Three other transcription units (ytfE, ygbA, and hcp-hcr) are known to be subject to regulation by NsrR. This chapter describes experimental and statistical protocols used to identify NsrR-binding sites in the E. coli chromosome using chromatin immunoprecipitation and microarray analysis. The methods are applicable, with suitable modifications, to any regulatory protein and any organism. 
18174134	 Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 degrees C compared to 37 degrees C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 degrees C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 degrees C. Another category of genes more highly expressed at 23 degrees C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and DsrA-dependent for their transcription at 23 degrees C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 degrees C. Increased expression at 23 degrees C compared to 37 degrees C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels  within 1 h after a shift from 37 to 23 degrees C, indicating a rapid response to  this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 degrees C and viability at 4 degrees C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 degrees C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 degrees C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 degrees C to globally respond to this environmental change and that at least two thermoregulatory pathways are  involved in co-ordinating this response - the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators. 
18036138	 In Escherichia coli, DsbA introduces disulphide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulphides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulphides in proteins with multiple cysteines. These incorrect disulphides are  thought to be corrected by a protein disulphide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolated from the DsbA/DsbB pathway. We show that the DsbC and DsbA pathways are more intimately connected than previously thought. dsbA(-)dsbC(-) mutants have a number of phenotypes not exhibited by either dsbA(-), dsbC(-) or dsbA(-)dsbD(-) mutations: they exhibit an increased permeability of the outer membrane, are resistant to the lambdoid phage Phi80, and are unable to assemble the maltoporin LamB. Using differential two-dimensional liquid chromatographic tandem mass spectrometry/mass spectrometry analysis, we estimated the abundance of about 130 secreted proteins in various dsb(-) strains. dsbA(-)dsbC(-) mutants exhibit unique changes at the protein level that are not exhibited by dsbA(-)dsbD(-) mutants. Our data indicate that DsbC can assist DsbA in a DsbD-independent manner to oxidatively fold envelope proteins. The view that DsbC's function is limited to the disulphide isomerization pathway should therefore be reinterpreted. 
18414482	 We present the assembly of gene brushes by means of a photolithographic approach  that allows us to control the density of end-immobilized linear double-stranded DNA polymers coding for entire genes. For 2 kbp DNAs, the mean distance varies from 300 nm, where DNAs are dilute and assume relaxed conformations, down to 30 nm, where steric repulsion at dense packing forces stretching out. We investigated the gene-to-protein relationship of firefly luciferase under the T7/E.Coli-extract expression system, as well as transcription-only reactions with T7 RNA polymerase, and found both systems to be highly sensitive to brush density, conformation, and orientation. A 'structure-function' picture emerges in which extension of genes induced by moderate packing exposes coding sequences and improves their interaction with the transcription/translation machinery. However, tighter packing impairs the penetration of the machinery into the brush. The response of expression to two-dimensional gene crowding at the nanoscale identifies gene brushes as basic controllable units en route to multicomponent synthetic systems. In turn, these brushes could deepen our understanding of biochemical reactions taking place under confinement and molecular crowding in living cells. 
18093320	 BACKGROUND: Small untranslated RNAs (sRNAs) seem to be far more abundant than previously believed. The number of sRNAs confirmed in E. coli through various approaches is above 70, with several hundred more sRNA candidate genes under biological validation. Although the total number of sRNAs in any one species is still unclear, their importance in cellular processes has been established. However, unlike protein genes, no simple feature enables the prediction of the location of the corresponding sequences in genomes. Several approaches, of variable usefulness, to identify genomic sequences encoding sRNA have been described in recent years. RESULTS: We used a combination of in silico comparative genomics and microarray-based transcriptional profiling. This approach to screening identified ~60 intergenic regions conserved between Sinorhizobium meliloti and related members of the alpha-proteobacteria sub-group 2. Of these, 14 appear to correspond to novel non-coding sRNAs and three are putative peptide-coding or 5'  UTR RNAs (ORF smaller than 100 aa). The expression of each of these new small RNA genes was confirmed by Northern blot hybridization. CONCLUSION: Small non coding RNA (sra) genes can be found in the intergenic regions of alpha-proteobacteria genomes. Some of these sra genes are only present in S. meliloti, sometimes in genomic islands; homologues of others are present in related genomes including those of the pathogens Brucella and Agrobacterium. 
17869523	 A covalently modified heteroconjugate between linezolid and neomycin B leads to an enhanced and more specific binding affinity to hairpin RNA targets in comparison to neomycin B itself. This heteroconjugate was used as a lure to select linezolid-specific hairpin RNA from an Escherichia coli genome RNA. The selected RNA obtained after eight cycles not only has typical stem-loop structures but also includes known sequences of the linezolid binding site. The results of RNA footprinting show that the binding site of the heteroconjugate encompasses both stem and loop regions, suggesting that the possible binding site for linezolid is in the terminal loop. In addition, findings from application of  a surface plasmon resonance assay clearly demonstrate that linezolid binds to selected hairpin RNA in a highly specific manner with a low millimolar affinity.  The results suggest that heteroconjugates might represent a generally useful approach in studies aimed at uncovering loop-specific RNA binding ligands that would be otherwise difficult to identify owing to their weak affinities. 
17921263	 Transcriptome data for a xylitol-producing recombinant Escherichia coli were obtained and used to tune up its productivity. Structural genes of NADPH-dependent D-xylose reductase and D-xylose permease were inserted into an Escherichia coli chromosome to construct a recombinant strain producing xylitol from D-xylose for use as a model system for NADPH-dependent bioconversion. Transcriptome analysis of xylitol-producing and nonproducing conditions for the recombinant revealed that xylitol production down-regulated 56 genes. These genes were then selected as candidate factors for suppression of the NADPH supply and were disrupted to validate their functions. Of the gene disruptants, that resulting from the deletion of yhbC showed the best bioconversion rate. Also, the deletion accelerated cell growth during log phase. The features of the mutant could be maintained in jar fermenter-scale production of xylitol. Thus, our novel molecular host strain breeding method using transcriptome analysis was fully effective and could be applied to improving various industrial strains. 
17890330	 DNA microarray analyses revealed that clusters of repetitive extragenic palindromic PCR-related Escherichia coli isolates were isogenic only within interstitial Lake Huron beach water samples and not in surrounding waters. This suggested that adaptation and growth occurred within the interstitial water sites tested. All isolates were nonpathogenic, and three lake isolates possessed tetracycline resistance genes. 
17933893	 In contrast to the vast majority of the members of the domain Bacteria, several Pseudomonas and Xanthomonas species have two lexA genes, whose products have been shown to recognize different LexA binding motifs, making them an interesting target for studying the interplay between cohabiting LexA regulons in a single species. Here we report an analysis of the genetic composition of the two LexA regulons of Pseudomonas putida KT2440 performed with a genomic microarray. The data obtained indicate that one of the two LexA proteins (LexA1) seems to be in control of the conventional Escherichia coli-like SOS response, while the other LexA protein (LexA2) regulates only its own transcriptional unit, which includes  the imuA, imuB, and dnaE2 genes, and a gene (PP_3901) from a resident P. putida prophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA damage-mediated induction of several P. putida resident prophage genes. In silico searches suggested that this marked asymmetry in regulon contents also occurs in  other Pseudomonas species with two lexA genes, and the implications of this asymmetry in the evolution of the SOS network are discussed. 
17921288	 The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH  10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma  E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement  of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580). 
17766423	 Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating  RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is  unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of Escherichia coli strains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA  mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes  are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect. 
17462951	 The species Escherichia coli comprises not only non-pathogenic or commensal variants that belong to the normal intestinal flora of most mammals, but also various pathogenic strains causing diverse intestinal and extraintestinal infections in man and animals. Virulence factors and mechanisms involved in pathogenesis have been successfully analyzed for many years resulting in a wealth of knowledge about many E. coli pathotypes. However, our knowledge on the genome  content, diversity and variability between pathogenic and also non-pathogenic subtypes is only slowly accumulating. Pathotypes have been largely defined by the presence or absence of particular DNA segments that in most cases appear to have  been acquired via horizontal gene transfer events. As these regions are frequently subjected to excisions, rearrangements, and transfers they contribute  to the previously unexpected and underestimated rapid evolution of E. coli variants resulting in the development of novel strains and even pathotypes. In these studies various novel aspects of genome diversity and plasticity in extraintestinal and intestinal pathogenic E. coli pathotypes have been addressed  and the results have been directly applied for the improvement of diagnostic methods. 
17720788	 The facultative aerobe Escherichia coli K-12 can use respiratory nitrate ammonification to generate energy during anaerobic growth. The toxic compound nitric oxide is a by-product of this metabolism. Previous transcript microarray studies identified the yeaR-yoaG operon, encoding proteins of unknown function, among genes whose transcription is induced in response to nitrate, nitrite, or nitric oxide. Nitrate and nitrite regulate anaerobic respiratory gene expression  through the NarX-NarL and NarQ-NarP two-component systems. All known Nar-activated genes also require the oxygen-responsive Fnr transcription activator. However, previous studies indicated that yeaR-yoaG operon transcription does not require Fnr activation. Here, we report results from mutational analyses demonstrating that yeaR-yoaG operon transcription is activated by phospho-NarL protein independent of the Fnr protein. The phospho-NarL protein binding site is centered at position -43.5 with respect to the transcription initiation site. Expression from the Shewanella oneidensis MR-1 nnrS gene promoter, cloned into E. coli, similarly was activated by phospho-NarL  protein independent of the Fnr protein. Recently, yeaR-yoaG operon transcription  was shown to be regulated by the nitric oxide-responsive NsrR repressor (N. Filenko et al., J. Bacteriol. 189:4410-4417, 2007). Our mutational analyses reveal the individual contributions of the Nar and NsrR regulators to overall yeaR-yoaG operon expression and document the NsrR operator centered at position -32. Thus, control of yeaR-yoaG operon transcription provides an example of overlapping regulation by nitrate and nitrite, acting through the Nar regulatory  system, and nitric oxide, acting through the NsrR repressor. 
17919280	 Using the genomic SELEX, a total of six Escherichia coli DNA fragments have been  identified, which formed complexes with transcription factor RutR. The RutR regulon was found to include a large number of genes encoding components for not  only degradation of pyrimidines but also transport of glutamate, synthesis of glutamine, synthesis of pyrimidine nucleotides and arginine, and degradation of purines. DNase I footprinting indicated that RutR recognizes a palindromic sequence of TTGACCAnnTGGTCAA. The RutR box in P1 promoter of carAB encoding carbamoyl phosphate synthetase, a key enzyme of pyrimidine synthesis, overlaps with the PepA (CarP) repressor binding site, implying competition between RutR and PepA. Adding either uracil or thymine abolished RutR binding in vitro to the  carAB P1 promoter. Accordingly, in the rutR-deletion mutant or in the presence of uracil, the activation in vivo of carAB P1 promoter was markedly reduced. Northern blot analysis of the RutR target genes indicated that RutR represses the Gad system genes involved in glutamate-dependent acid resistance and allantoin degradation. Altogether we propose that RutR is the pyrimidine sensor and the master regulator for a large set of the genes involved in the synthesis and degradation of pyrimidines. 
17967175	 BACKGROUND: Global patterns of gene expression of Escherichia coli K-12 during growth transitions have been deeply investigated, however, comparable studies of  E. coli O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7  Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at  10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays. Expression levels of 14 genes, including those encoding Shiga toxins and other virulence factors associated with the locus of enterocyte effacement (LEE), were  confirmed by Q-PCR. RESULTS: Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time. QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern. ORFs from the largest cluster (n = 1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth. Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased approximately 4-fold. Most ORFs of the LEE pathogenicity island also decreased approximately 2-fold by early stationary phase. The ORFs encoding  proteins secreted via the LEE encoded type III secretion system, such as tccP and espJ, also decreased in expression from exponential to stationary phase. Three of the clusters (n = 154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging. Upregulated genes with an increase in mRNA levels from late exponential to early stationary phase belonged to one cluster (n = 923) which includes genes involved in stress responses (e.g. gadAB, osmBC, and dps). These transcript levels remained relatively high for > 3 h in stationary phase. The Shiga toxin genes (stx1AB and stx2B) were significantly induced after transition  into stationary phase. CONCLUSION: Expression of more than 300 O157-specific ORFs, many implicated in virulence of the O157 pathogen, was modulated in a growth dependent manner. These results provide a baseline transcriptional profile that can be compared to patterns of gene expression of this important foodborne pathogen under adverse environmental conditions. 
17765265	 The Escherichia coli gene cluster ymgABC was identified in transcriptome studies  to have a role in biofilm development and stability. In this study, we showed that YmgB represses biofilm formation in rich medium containing glucose, decreases cellular motility, and protects the cell from acid indicating that YmgB has a major role in acid-resistance in E. coli. Our data show that these phenotypes are potentially mediated through interactions with the important cell  signal indole. In addition, gel mobility-shift assays suggest that YmgB may be a  non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we showed that YmgB binds, either directly or indirectly, via a probable ligand, genes important for biofilm formation. To advance our understanding of the function of YmgB, we used X-ray crystallography to solve the structure of the protein to 1.8 A resolution. YmgB is a biological dimer that is structurally homologous to the E. coli gene regulatory protein Hha, despite having only 5% sequence identity. This supports our DNA microarray data showing that YmgB is a gene regulatory protein. Therefore, this protein, which clearly has a critical role in acid-resistance in E. coli, has been renamed as AriR for regulator of acid resistance influenced by indole. 
17827305	 The effects of feed supplementation with the approved antimicrobial agents bambermycin, penicillin, salinomycin, and bacitracin or a combination of salinomycin plus bacitracin were evaluated for the incidence and distribution of  antibiotic resistance in 197 commensal Escherichia coli isolates from broiler chickens over 35 days. All isolates showed some degree of multiple antibiotic resistance. Resistance to tetracycline (68.5%), amoxicillin (61.4%), ceftiofur (51.3%), spectinomycin (47.2%), and sulfonamides (42%) was most frequent. The levels of resistance to streptomycin, chloramphenicol, and gentamicin were 33.5,  35.5, and 25.3%, respectively. The overall resistance levels decreased from day 7 to day 35 (P < 0.001). Comparing treatments, the levels of resistance to ceftiofur, spectinomycin, and gentamicin (except for resistance to bacitracin treatment) were significantly higher in isolates from chickens receiving feed supplemented with salinomycin than from the other feeds (P < 0.001). Using a DNA  microarray analysis capable of detecting commonly found antimicrobial resistance  genes, we characterized 104 tetracycline-resistant E. coli isolates from 7- to 28-day-old chickens fed different growth promoters. Results showed a decrease in  the incidence of isolates harboring tet(B), bla(TEM), sulI, and aadA and class 1  integron from days 7 to 35 (P < 0.01). Of the 84 tetracycline-ceftiofur-resistant E. coli isolates, 76 (90.5%) were positive for bla(CMY-2). The proportions of isolates positive for sulI, aadA, and integron class 1 were significantly higher  in salinomycin-treated chickens than in the control or other treatment groups (P  < 0.05). These data demonstrate that multiantibiotic-resistant E. coli isolates can be found in broiler chickens regardless of the antimicrobial growth promoters used. However, the phenotype and the distribution of resistance determinants in E. coli can be modulated by feed supplementation with some of the antimicrobial agents used in broiler chicken production. 
17662562	 A base stacking hybridization-based microarray method was developed for rapid identification of clinical isolates within 2 h. The oligonucleotide probe sequences for species or genus-level identification were targeted against ribosomal RNA. Isolates were lysed and directly hybridized to the microarray-bound capture probes without conventional DNA or RNA isolation and prior polymerase chain reaction amplification. Five bacterial species encountered frequently in the clinical setting, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae, and one genus Enterococcus, could be discriminated by the microarray-based assay. Identification by this method matched biochemical identification for 150 of 152 clinical strains. This base-stacking hybridization microarray offers a simple, fast (</=2 h), and accurate identification of bacterial cultures and is a potential tool for routine clinical diagnosis. 
17644597	 RNA degradation is an important factor in the regulation of gene expression. It allows organisms to quickly respond to changing environmental conditions by adapting the expression of individual genes. The stability of individual mRNAs within an organism varies considerably, contributing to differential amounts of proteins expressed. In this study we used DNA microarrays to analyze mRNA degradation in exponentially growing cultures of the extremely halophilic euryarchaeon Halobacterium salinarum NRC-1 on a global level. We determined mRNA  half-lives for 1,717 open reading frames, 620 of which are part of known or predicted operons. Under the tested conditions transcript stabilities ranged from 5 min to more than 18 min, with 79% of the evaluated mRNAs showing half-lives between 8 and 12 min. The overall mean half-life was 10 min, which is considerably longer than the ones found in the other prokaryotes investigated thus far. As previously observed in Escherichia coli and Saccharomyces cerevisiae, we could not detect a significant correlation between transcript length and transcript stability, but there was a relationship between gene function and transcript stability. Genes that are known or predicted to be transcribed in operons exhibited similar mRNA half-lives. These results provide initial insights into mRNA turnover in a euryarchaeon. Moreover, our model organism, H. salinarum NRC-1, is one of just two archaea sequenced to date that are missing the core subunits of the archaeal exosome. This complex orthologous to the RNA degrading exosome of eukarya is found in all other archaeal genomes sequenced thus far. 
17850262	 All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non-essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in  highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a  dominant factor controlling the genetic footprint of transposons in living cells. 
17591798	 During infection in the gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) O157:H7 is exposed to a wide range of signaling molecules, including  the eukaryotic hormones epinephrine and norepinephrine, and bacterial signal molecules such as indole. Since these signaling molecules have been shown to be involved in the regulation of phenotypes such as motility and virulence that are  crucial for EHEC infections, we hypothesized that these molecules also govern the initial recognition of the large intestine environment and attachment to the host cell surface. Here, we report that, compared to indole, epinephrine and norepinephrine exert divergent effects on EHEC chemotaxis, motility, biofilm formation, gene expression, and colonization of HeLa cells. Using a novel two-fluorophore chemotaxis assay, it was found that EHEC is attracted to epinephrine and norepinephrine while it is repelled by indole. In addition, epinephrine and norepinephrine also increased EHEC motility and biofilm formation while indole attenuated these phenotypes. DNA microarray analysis of surface-associated EHEC indicated that epinephrine/norepinephrine up-regulated the expression of genes involved in surface colonization and virulence while exposure to indole decreased their expression. The gene expression data also suggested that autoinducer 2 uptake was repressed upon exposure to epinephrine/norepinephrine but not indole. In vitro adherence experiments confirmed that epinephrine and norepinephrine increased attachment to epithelial  cells while indole decreased adherence. Taken together, these results suggest that epinephrine and norepinephrine increase EHEC infection while indole attenuates the process. 
17631985	 We studied the mechanisms of resistance to fluoroquinolones in two Citrobacter freundii strains (1.44 and 1.38) isolated from the same patient and belonging to  the same clone by pulsed-field gel electrophoresis. This study allowed partial characterisation of the acrA and acrB genes of this microorganism. As previously  reported, the two strains showed the same substitutions in the GyrA and ParC proteins (Thr-83-->Ile and Asp-87-->Tyr in GyrA and Ser-83-->Ile in ParC). However, differences were observed in the amount of ciprofloxacin accumulated, with strain 1.38 showing less accumulation. Expression of genes in both strains was analysed using DNA microarrays for Escherichia coli. Ten genes were overexpressed in strain 1.38 compared with strain 1.44, including genes acrA and  acrB. Nucleotide similarity between the partially sequenced acrA and acrB genes of C. freundii and E. coli was 80.7% and 85%, respectively. The acrA and acrB genes of C. freundii are similar to those described in E. coli and their overexpression may play an important role in modulating the final minimum inhibitory concentration of fluoroquinolones in collaboration with mutations in the gyrA and parC genes. 
17768236	 Fis is a nucleoid-associated protein in Escherichia coli that is abundant during  early exponential growth in rich medium but is in short supply during stationary  phase. Its role as a transcriptional regulator has been demonstrated for an increasing number of genes. In order to gain insight into the global effects of Fis on E. coli gene expression during different stages of growth in rich medium,  DNA microarray analyses were conducted in fis and wild-type strains during early, mid-, late-exponential and stationary growth phases. The results uncovered 231 significantly regulated genes that were distributed over 15 functional categories. Regulatory effects were observed at all growth stages examined. Coordinate upregulation was observed for a number of genes involved in translation, flagellar biosynthesis and motility, nutrient transport, carbon compound metabolism, and energy metabolism at different growth stages. Coordinate down-regulation was also observed for genes involved in stress response, amino acid and nucleotide biosynthesis, energy and intermediary metabolism, and nutrient transport. As cells transitioned from the early to the late-exponential  growth phase, different functional categories of genes were regulated, and a gradual shift occurred towards mostly down-regulation. The results demonstrate that the growth phase-dependent Fis expression triggers coordinate regulation of  15 categories of functionally related genes during specific stages of growth of an E. coli culture. 
17573936	 Various forms of host stresses (e.g. physiological, psychological) are thought to influence susceptibility to pathogenic microorganisms. Catecholamines such as norepinephrine are released into the GI environment during acute stress and may influence the infective process of bacterial pathogens associated with the GI tract. To examine the effects of norepinephrine on expression of virulence factors in Escherichia coli O157:H7, the clinical-type isolate EDL933 (ATCC 43895) was grown in serum-Standard American Petroleum Institute media in the presence or absence of norepinephrine. After 5 h of exposure to norepinephrine, treatment and control cultures (not exposed to norepinephrine) were harvested, their RNA isolated, and gene expression evaluated. There was a dramatic increase  in the expression of virulence factor transcripts including stx1, stx2, and eae.  Also induced were transcripts involved in iron metabolism. Conversely, there was  comparative repression of iron acquisition and phage shock protein-related transcripts in the presence of norepinephrine. Novel observations from these data suggested that exposure to norepinephrine induced glutamate decarboxylase acid resistance as well as an SOS response in E. coli O157:H7. The results corroborate many of the previous findings detailed in the literature and provide new observations that could expand the scope of microbial endocrinology. 
17557822	 The folA gene was identified as a new member of the TyrR regulon by genomic SELEX. Binding of TyrR to two sites in folA activated its transcription. Mutations in the N-terminal or central domain of TyrR, the alpha subunit of RNA polymerase, or integration host factor all abolished activation of the folA promoter. 
17526692	 Csr (carbon storage regulation) of Escherichia coli is a global regulatory system that consists of CsrA, a homodimeric RNA binding protein, two noncoding small RNAs (sRNAs; CsrB and CsrC) that function as CsrA antagonists by sequestering this protein, and CsrD, a specificity factor that targets CsrB and CsrC for degradation by RNase E. CsrA inhibits translation initiation of glgC, cstA, and pgaA by binding to their leader transcripts and preventing ribosome binding. Translation inhibition is thought to contribute to the observed mRNA destabilization. Each of the previously known target transcripts contains multiple CsrA binding sites. A position-specific weight matrix search program was developed using known CsrA binding sites in mRNA. This search tool identified a potential CsrA binding site that overlaps the Shine-Dalgarno sequence of hfq, a gene that encodes an RNA chaperone that mediates sRNA-mRNA interactions. This putative CsrA binding site matched the SELEX-derived binding site consensus sequence in 8 out of 12 positions. Results from gel mobility shift and footprint  assays demonstrated that CsrA binds specifically to this site in the hfq leader transcript. Toeprint and cell-free translation results indicated that bound CsrA  inhibits Hfq synthesis by competitively blocking ribosome binding. Disruption of  csrA caused elevated expression of an hfq'-'lacZ translational fusion, while overexpression of csrA inhibited expression of this fusion. We also found that hfq mRNA is stabilized upon entry into stationary-phase growth by a CsrA-independent mechanism. The interaction of CsrA with hfq mRNA is the first example of a CsrA-regulated gene that contains only one CsrA binding site. 
17513468	 The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR,  a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh, encoding NADH dehydrogenase II, and cyoABCDE, encoding the cytochrome bo-type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in  vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system. 
17608794	 The phage-shock-protein (Psp) system is essential for Yersinia enterocolitica virulence. Mislocalized secretins induce psp gene expression, and kill psp null strains. We used transposon mutagenesis to investigate whether other genes are required to tolerate secretin-induced stress. Our motivation included the possibility of identifying signal transducers required to activate psp gene expression. Besides Psp, only defects in the RpoE system and the TrkA potassium transporter caused secretin sensitivity. These mutations did not cause the same specific/severe sensitivity as defects in the Psp system, nor did they affect psp gene expression. The Escherichia coli Psp system was reported to be induced via the ArcB redox sensor and to activate anaerobic metabolism. Our screen did not identify arcB, or any genes involved in anaerobic metabolism/regulation. Therefore, we investigated the role of ArcB in Y. enterocolitica and E. coli. ArcB was not required for secretin-dependent induction of psp gene expression. Furthermore, microarray analysis uncovered a restricted transcriptional response  to prolonged secretin stress in Y. enterocolitica. Taken together, these data do  not support the proposal that the Psp system is induced via ArcB and activates anaerobic metabolism. Rather, they suggest that Psp proteins may sense an inducing trigger and mediate their physiological output(s) directly. 
17652176	 The influence of the high intracellular concentration of macromolecules on cell physiology is increasingly appreciated, but its impact on system-level cellular functions remains poorly quantified. To assess its potential effect, here we develop a flux balance model of Escherichia coli cell metabolism that takes into  account a systems-level constraint for the concentration of enzymes catalyzing the various metabolic reactions in the crowded cytoplasm. We demonstrate that the model's predictions for the relative maximum growth rate of wild-type and mutant  E. coli cells in single substrate-limited media, and the sequence and mode of substrate uptake and utilization from a complex medium are in good agreement with subsequent experimental observations. These results suggest that molecular crowding represents a bound on the achievable functional states of a metabolic network, and they indicate that models incorporating this constraint can systematically identify alterations in cellular metabolism activated in response  to environmental change. 
17653283	 Many studies involving interacting microorganisms would benefit from simple devices able to deposit cells in precisely defined patterns. We describe an inexpensive bacterial piezoelectric inkjet printer (adapted from the design of the POSaM oligonucleotide microarrayer) that can be used to "print out" different strains of bacteria or chemicals in small droplets onto a flat surface at high resolution. The capabilities of this device are demonstrated by printing ordered  arrays comprising two bacterial strains labeled with different fluorescent proteins. We also characterized several properties of this piezoelectric printer, such as the droplet volume (of the order of tens of pl), the distribution of number of cells in each droplet, and the dependence of droplet volume on printing frequency. We established the limits of the printing resolution, and determined that the printed viability of Escherichia coli exceeded 98.5%. 
17512922	 The ability of three Rhamnus alaternus leaves extracts on antigenotoxic and gene  expression level effects was respectively investigated in a bacterial assay system, i.e. the SOS chromotest with Escherichia coli PQ37 and in human K562 lymphoblast cell line. Total oligomers flavonoids (TOF) enriched, methanol and ethyl acetate extracts were prepared from powdered R. alaternus leaves and characterized quantitatively for the presence of polyphenolic compounds. We explored the response to oxidative stress using the transcriptional profile of genes in K562 cells stressed with H2O2 after incubation with plant extracts. For  this purpose, we used a cDNA microarrays containing 82 genes related to cell defense, essentially represented by antioxidant and DNA repair genes. Analysis revealed that SOD1, AOE 372, TXN genes involved in the antioxidant defense system and XPC, LIG4, POLD2, PCNA genes implied in the DNA repair system were among the  most expressed ones in the presence of the tested extracts. These results were in accordance with those obtained when we tested the antigenotoxic and antioxidant effects of the same extracts with, respectively the SOS chromotest and the xanthine/xanthine oxidase enzymatic assay system. The effect of the tested extracts on SOS response induced by both Aflatoxin B1 (AFB1: 10 microg/assay) and nifuroxazide (20 microg/assay) showed that the TOF extract exhibited the highest  antimutagenic level towards the indirect mutagen AFB1. Whereas ethyl acetate extract showed the highest antimutagenic effect towards the direct mutagen, nifuroxazide. None of the tested extracts induced mutagenic activity. However all the tested extracts exhibited xanthine oxidase inhibiting and superoxide anions scavenging effects. R. alaternus extracts contain compounds with significant antioxidant and antigenotoxic activities. These compounds modulate gene expression as detected by using cDNA arrays. 
17468243	 RstBA, a two-component regulatory system of Escherichia coli with an unidentified regulatory function, is under the control of a Mg(2+)-sensing PhoQP two-component system. In order to identify the network of transcription regulation downstream of RstBA, we isolated a set of RstA-binding sequences from the E. coli genome by  using the genomic SELEX system. A gel mobility shift assay indicated the binding  of RstA to two SELEX DNA fragments, one including the promoter region of asr (acid shock RNA) and another including the promoter for csgD (a regulator of the  curli operon). Using a DNase I footprinting assay, we determined the RstA-binding sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of the asr gene was induced 10- to 60-fold either in low-pH (pH 4.5) LB medium or in low-phosphate minimal medium as detected by promoter assay. The acid-induced in vivo transcription of asr was reduced after the deletion of rstA. In vivo transcription of the asr promoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg(2+) led to a severe reduction of the asr promoter activity, and the disruption of phoP also reduced the asr promoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator of asr transcription. In contrast,  transcription of csgD was repressed by overexpression of RstA, indicating that RstA is a repressor for csgD. With these data taken together, we conclude that the expression of both asr and csgD is under the direct control of the PhoQP-RstBA signal relay cascade. 
17437063	 Analysis of over 200,000 expressed sequence tags from a range of Medicago truncatula cDNA libraries resulted in the identification of over 150 different family 1 glycosyltransferase (UGT) genes. Of these, 63 were represented by full length clones in an EST library collection. Among these, 19 gave soluble proteins when expressed in E. coli, and these were screened for catalytic activity against a range of flavonoid and isoflavonoid substrates using a high-throughput HPLC assay method. Eight UGTs were identified with activity against isoflavones, flavones, flavonols or anthocyanidins, and several showed high catalytic specificity for more than one class of (iso)flavonoid substrate. All tested UGTs  preferred UDP-glucose as sugar donor. Phylogenetic analysis indicated that the Medicago (iso)flavonoid glycosyltransferase gene sequences fell into a number of  different clades, and several clustered with UGTs annotated as glycosylating non-flavonoid substrates. Quantitative RT-PCR and DNA microarray analysis revealed unique transcript expression patterns for each of the eight UGTs in Medicago organs and cell suspension cultures, and comparison of these patterns with known phytochemical profiles suggested in vivo functions for several of the  enzymes. 
17570850	 BACKGROUND: The sequencing of many genomes and tiling arrays consisting of millions of DNA segments spanning entire genomes have made high-resolution copy number analysis possible. Microarray-based comparative genomic hybridization (array CGH) has enabled the high-resolution detection of DNA copy number aberrations. While many of the methods and algorithms developed for the analysis  microarrays have focused on expression analysis, the same technology can be used  to detect genetic alterations, using for example standard commercial Affymetrix arrays. Due to the nature of the resultant data, standard techniques for processing GeneChip expression experiments are inapplicable. RESULTS: We have developed a robust and flexible methodology for high-resolution  analysis of DNA copy number of whole genomes, using Affymetrix high-density expression oligonucleotide microarrays. Copy number is obtained from fluorescence signals after processing with novel normalization, spatial artifact correction, data transformation and deletion/duplication detection. We applied our approach to identify deleted and amplified regions in E. coli mutants obtained after prolonged starvation. CONCLUSION: The availability of Affymetrix expression chips for a wide variety of organisms makes the proposed array CGH methodology useful more generally. 
17559662	 BACKGROUND: Glucose is the preferred carbon and energy source for Escherichia coli. A complex regulatory network coordinates gene expression, transport and enzyme activities in response to the presence of this sugar. To determine the extent of the cellular response to glucose, we applied an approach combining global transcriptome and regulatory network analyses. RESULTS: Transcriptome data from isogenic wild type and crp- strains grown in Luria-Bertani medium (LB) or LB + 4 g/L glucose (LB+G) were analyzed to identify  differentially transcribed genes. We detected 180 and 200 genes displaying increased and reduced relative transcript levels in the presence of glucose, respectively. The observed expression pattern in LB was consistent with a gluconeogenic metabolic state including active transport and interconversion of small molecules and macromolecules, induction of protease-encoding genes and a partial heat shock response. In LB+G, catabolic repression was detected for transport and metabolic interconversion activities. We also detected an increased capacity for de novo synthesis of nucleotides, amino acids and proteins. Cluster  analysis of a subset of genes revealed that CRP mediates catabolite repression for most of the genes displaying reduced transcript levels in LB+G, whereas Fis participates in the upregulation of genes under this condition. An analysis of the regulatory network, in terms of topological functional units, revealed 8 interconnected modules which again exposed the importance of Fis and CRP as directly responsible for the coordinated response of the cell. This effect was also seen with other not extensively connected transcription factors such as FruR and PdhR, which showed a consistent response considering media composition. CONCLUSION: This work allowed the identification of eight interconnected regulatory network modules that includes CRP, Fis and other transcriptional factors that respond directly or indirectly to the presence of glucose. In most cases, each of these modules includes genes encoding physiologically related functions, thus indicating a connection between regulatory network topology and related cellular functions involved in nutrient sensing and metabolism. 
17547756	 BACKGROUND: Genomic studies on bacteria have clearly shown the existence of chromosomal organization as regards, for example, to gene localization, order and orientation. Moreover, transcriptomic analyses have demonstrated that, in free-living bacteria, gene transcription levels and chromosomal organization are  mutually influenced. We have explored the possible conservation of relationships  between mRNA abundances and chromosomal organization in the highly reduced genome of Buchnera aphidicola, the primary endosymbiont of the aphids, and a close relative to Escherichia coli. RESULTS: Using an oligonucleotide-based microarray, we normalized the transcriptomic data by genomic DNA signals in order to have access to inter-gene  comparison data. Our analysis showed that mRNA abundances, gene organization (operon) and gene essentiality are correlated in Buchnera (i.e., the most expressed genes are essential genes organized in operons) whereas no link between mRNA abundances and gene strand bias was found. The effect of Buchnera genome evolution on gene expression levels has also been analysed in order to assess the constraints imposed by the obligate symbiosis with aphids, underlining the importance of some gene sets for the survival of the two partners. Finally, our results show the existence of spatial periodic transcriptional patterns in the genome of Buchnera. CONCLUSION: Despite an important reduction in its genome size and an apparent decay of its capacity for regulating transcription, this work reveals a significant correlation between mRNA abundances and chromosomal organization of the aphid-symbiont Buchnera. 
17574901	 Small noncoding RNAs have been discovered at a staggering rate in Escherichia coli and many other bacteria. Most of the sRNAs of known function regulate gene expression by binding to specific mRNAs or proteins. Given the scores of sRNAs of unknown function, the identification of their cellular targets has become urgent. Here, we review the diverse strategies that have been used to identify and validate bacterial sRNA targets. These include the pulse-expression of sRNAs followed by global transcriptome analysis (microarrays), new biocomputational prediction algorithms, and novel gfp reporter gene fusions to validate candidate  target gene regulation. 
17553733	 Almost 140 bacterial small RNAs (sRNAs; sometimes referred to as non-coding RNAs) have been discovered in the past six years. The majority of these sRNAs were discovered in Escherichia coli, and a smaller subset was characterized in other bacteria, many of which were pathogenic. Many of these genes were identified as a result of systematic screens using computational prediction of sRNAs and experimental-based approaches, including microarray and shotgun cloning. A smaller number of sRNAs were discovered by direct labeling or by functional genetic screens. Many of the discovered genes, ranging in size from 50 to 500 nucleotides, are conserved and located in intergenic regions, in-between open reading frames. The expression of many of these genes is growth phase dependent or stress related. As each search employed specific parameters, this led to the identification of genes with distinct characteristics. Consequently, unique sRNAs such as those that are species-specific, sRNA genes that are transcribed under unique conditions or genes located on the antisense strand of protein-encoding genes, were probably missed. 
17449618	 Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defense mechanisms or as products of their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of transcription factors) plays key roles in this stress response. Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were previously shown to be regulated by NsrR. Novel NsrR targets revealed by this study include hcp-hcr (which were predicted in a recent bioinformatic study to be NsrR regulated) and the well-studied nrfA promoter that directs the expression of the periplasmic respiratory nitrite reductase. Conversely, transcription from the ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR is consistent with the ability of the periplasmic nitrite reductase to reduce nitric oxide and hence protect against reactive nitrogen species. Gel retardation assays were used to show that both FNR and NarL bind to the hcp promoter. The expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr, hmpA, and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein, HCP, might also be part of a defense mechanism against reactive nitrogen stress. 
17287131	 Recently identified interferon-epsilon (IFN-epsilon) belongs to type I interferons. IFN-epsilon is highly and constitutively expressed in the brain, but its biochemical and biological characteristics are poorly understood. In this study, full-length IFN-epsilon cDNA was cloned from human peripheral blood lymphocyte by RT-PCR, and was expressed in Escherichia coli (E. coli). Reverse phase high pressure liquid chromatography was used to purify recombinant human IFN-epsilon (rhIFN-epsilon) and to facilitate refolding of the protein. About 0.8mg of highly purified rhIFN-epsilon protein was obtained from 100ml of E. coli culture. Functional study of rhIFN-epsilon demonstrated that the antiviral activity of rhIFN-epsilon was 6+/-0.5x10(5)IU/mg, which was lower than that of rhIFN-alpha-2b in the WISH-VSV (WISH cells infected with vesicular stomatitis virus) assay system. As for the activity to promote NK cytotoxicity and antiproliferation activities, rhIFN-epsilon was about 60 times less potent than rhIFN-alpha-2b. However, oligonucleotide microarray analyses revealed dramatic differences in gene expression profiles of cultured human cells treated with IFN-epsilon and IFN-alpha-2b. Particularly, differential regulation of genes related to central nervous system by rhIFN-epsilon suggests a role for IFN-epsilon in maintenance of the structure and function of brain. 
16934953	 Here, we describe a novel microarray-based approach for investigating the genomic diversity of Escherichia coli O157:H7 in a semi-high throughput manner using a high density, oligonucleotide-based microarray. This microarray, designed to detect polymorphisms at each of 60,000 base-pair (bp) positions within an E. coli genome, is composed of overlapping 29-mer oligonucleotides specific for 60 equally spaced, 1000-bp loci of the E. coli O157:H7 strain EDL933 chromosome. By  use of a novel 12-well microarray that permitted the simultaneous investigation of 12 strains, the genomes of 44 individual isolates of E. coli O157:H7 were interrogated. These analyses revealed more than 150 single nucleotide polymorphisms (SNPs) and several deletions and amplifications in the test strains. Pyrosequencing was used to confirm the usefulness of the novel SNPs by determining their allelic frequency among a collection of diverse isolates of E.  coli O157:H7. The tiling DNA microarray system would be useful for the tracking and identification of individual strains of E. coli O157:H7 needed for forensic investigations. 
17506902	 BACKGROUND: Genetic analysis of Escherichia coli O157:H7 strains has shown divergence into two distinct lineages, lineages I and II, that appear to have distinct ecological characteristics, with lineage I strains more commonly associated with human disease. In this study, microarray-based comparative genomic hybridization (CGH) was used to identify genomic differences among 31 E.  coli O157:H7 strains that belong to various phage types (PTs) and different lineage-specific polymorphism assay (LSPA) types. RESULTS: A total of 4,084 out of 6,057 ORFs were detected in all E. coli O157:H7  strains and 1,751 were variably present or absent. Based on this data, E. coli O157:H7 strains were divided into three distinct clusters, which consisted of 15  lineage I (LSPA type 111111), four lineage I/II (designated in this study) (LSPA  type 211111) and 12 lineage II strains (LSPA 222222, 222211, 222212, and 222221), respectively. Eleven different genomic regions that were dominant in lineage I strains (present in > or =80% of lineage I and absent from > or = 92% of lineage  II strains) spanned segments containing as few as two and up to 25 ORFs each. These regions were identified within E. coli Sakai S-loops # 14, 16, 69, 72, 78,  83, 85, 153 and 286, Sakai phage 10 (S-loops # 91, 92 and 93) and a genomic backbone region. All four lineage I/II strains were of PT 2 and possessed eight of these 11 lineage I-dominant loci. Several differences in virulence-associated  loci were noted between lineage I and lineage II strains, including divergence within S-loop 69, which encodes Shiga toxin 2, and absence of the non-LEE encoded effector genes nleF and nleH1-2 and the perC homologue gene pchD in lineage II strains. CONCLUSION: CGH data suggest the existence of two dominant lineages as well as LSPA type and PT-related subgroups within E. coli O157:H7. The genomic composition of these subgroups supports the phylogeny that has been inferred from other methods and further suggests that genomic divergence from an ancestral form and lateral gene transfer have contributed to their evolution. The genomic features identified in this study may contribute to apparent differences in the epidemiology and ecology of strains of different E. coli O157:H7 lineages. 
17351046	 The Escherichia coli Rsd protein forms complexes with the RNA polymerase sigma(70) factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative sigma(38) factor, and this  was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with sigma(70). Our experiments support a model in which the role of Rsd is primarily to sequester sigma(70), thereby increasing the levels of RNA polymerase containing the alternative sigma(38) factor. 
17320232	 We have analyzed gene expression of Escherichia coli MG1655 expressing native and engineered bacterial globin proteins, in order to identify the molecular mechanisms leading to the improved phenotypical traits relative to control cells  under oxygen-limited conditions. Regulated expression of hemoglobin and flavohemoglobin proteins postponed the onset of rpoS expression relative to plasmid bearing control cells. This change in expression pattern coincided with the expression pattern of stationary-phase specific genes including sigma(S)-dependent and sigma(S)-independent genes. Furthermore, several genes known to affect rpoS transcription, rpoS mRNA stability and sigma(S) turnover were regulated in such a manner as to ultimately lower the cellular level of sigma(S) in all globin-expressing strains. In a strain harboring an rpoS-lacZ fusion, lacZ expression correlated with acetate accumulation, a metabolite that is known to activate rpoS transcription, but not with growth. Therefore, we hypothesize that reduced excretion of acetate in globin expressing cells prevents induction of stationary phase specific genes. Additionally, several genes responding to carbon starvation (e.g. csrAB, cstA, sspA) were expressed at lower  levels in globin-expressing cells. These findings are in good agreement with previous reports showing a more efficient energy household, i.e. also reduced glucose consumption, in hemoglobin- and flavohemoglobin-expressing cells relative to controls. 
17464049	 Previous work has shown that locus of enterocyte effacement (LEE)-encoded effector proteins such as Tir and Map can be exported via the type III secretion  system (T3SS) of Escherichia coli O157 : H7. Additionally, a family of non-LEE-encoded (Nle) effector proteins has been shown to be secreted from Citrobacter rodentium, homologues of which are located on the E. coli O157 chromosome. While NleA has been shown to be secreted from pathogenic E. coli, the secretion of other Nle effector proteins has only been detected under induced conditions, or using a mutated T3SS. This study aimed to determine: (1) which nle genes are expressed in E. coli O157 : H7 under secretion-permissive conditions; (2) if Nle proteins are secreted from wild-type E. coli O157 : H7 under secretion-permissive conditions; and (3) if nle gene expression is regulated co-ordinately with other LEE-encoded effectors. Using data generated from a combination of transcriptome arrays, reporter fusions and proteomics, it was demonstrated that only nleA is expressed co-ordinately with the LEE. Secretion and expression of NleA were regulated directly or indirectly by ler, a key activator of the LEE. MS confirmed the secretion of NleA into the culture supernatant, while NleB-F were not detected. 
17379695	 Rice microarray analysis showed that a number of stress-related genes are induced by external addition of L-ascorbic acid (AsA). The gene designated as AK073843 which is homologous to class capital SHA, Cyrillic chitinase was found to exhibit the highest induction among these genes. However, its crucial residues within the chitinase active site are substituted with other residues, suggesting that the protein has no chitinase activity. The recombinant protein which is encoded by the AK073843 gene produced in Escherichia coli has xylanase inhibitor activity, indicating that the gene encodes a novel rice XIP-type xylanase inhibitor protein (OsXIP). The expression of OsXIP was enhanced not only by exogenous AsA treatment but also by various stresses such as citrate and sodium chloride treatments, and  wounding; however, it was not influenced by increasing endogenous AsA content. External AsA treatment caused a significant increase in electrolyte leakage from  rice root. These results suggested that OsXIP was induced by stress which is caused by external AsA treatment. Rice XIP-family genes, OsXIP, riceXIP and RIXI, showed differential organ-specific expression. Also, these genes were differentially induced by stress and stress-related phytohormones. The transcripts of OsXIP and riceXIP were undetectable under normal conditions, and were drastically induced by wounding and methyl jasmonate (MeJA) treatment in the root. RIXI was constitutively expressed in the shoot but not induced by wounding  and stress-related phytohormones. Thus, XIP-type xylanase inhibitors were suggested to be specialized in their function and involved in defense mechanisms  in rice. 
17379776	 Analysis of cellular components at multiple levels of biological information can  provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations. 
16891109	 Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay  resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between  these two different detection techniques. A lower detection limit of 0.75 pM was  obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus). 
17293527	 A bovine-specific cDNA microarray was used to characterize gene expression in the bovine rectoanal junction mucosa in response to Escherichia coli O157:H7 colonization, and results were confirmed using quantitative real-time PCR. The results showed involvement of cell processes including immune response, cell structure/dynamics, signal transduction, intercellular communication, and metabolism. 
17216460	 To measure the toxicity experienced by superoxide-generating compounds, two plasmids were constructed in which the superoxide-inducible fpr and zwf promoters from Escherichia coli were fused to promoterless Vibrio fischeri luxCDABE operon  present in plasmid pUCD615. The bioluminescent response of E. coli harboring these constructs was studied as a function of the toxicity and was shown to be specific for superoxide generating chemicals. The two promoters employed, fpr and zwf, responded differentially to the redox-chemicals tested. Furthermore, a DeltamarA strain bearing the fpr::luxCDABE fusion had a weaker response to paraquat (methyl viologen) than its isogenic parent strain, whereas zwf induction was not inhibited in DeltamarA or Deltarob strains. The fpr and zwf promoters were also induced by alkylating agents but were unresponsive in DeltamarA or Deltarob strains. Using optimized assay conditions, the abilities of these strains to differentially respond to superoxide stress and alkylating agents that may be present in contaminants proves them to be good biosensor candidates for monitoring toxicity. 
17280868	 Extraintestinal pathogenic Escherichia coli (ExPEC) are a major cause of urinary  tract infections, sepsis, and neonatal meningitis. A variety of virulence factors in these strains is encoded by mobile genetic elements, such as transposons or pathogenicity islands (PAIs). Using subtractive cloning of ExPEC genomes, we recently detected short DNA fragments, which were significantly associated with the extraintestinal virulent phenotype. In this study, we identified four novel genomic DNA regions of the highly virulent uropathogenic E. coli strain JS299 carrying these previously identified DNA fragments. Characterization of the partial sequences of the genomic DNA regions revealed complex DNA arrangements with variable genetic compositions regarding the G+C contents and codon usage patterns. The prevalence of 15 previously uncharacterized genes was determined in a collection of clinical ExPECs and commensal E. coli strains by means of DNA microarray analyses. From this, 13 novel DNA sequences were demonstrated to be significantly associated with extraintestinal virulent strains, and thus may represent new virulence traits. Beside genes predicted to play a role in metabolic functions, such as sucrose utilization (scr), we identified DNA sequences shared by both ExPEC and enteropathogenic E. coli (EPEC). These sequences were significantly more prevalent among ExPECs when compared to commensal E. coli isolates. Our results support the idea of a considerable genetic variability among ExPEC strains and suggest that the novel genomic determinants described in this study may contribute to the ExPEC virulence. 
17397547	 BACKGROUND: Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a  unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. RESULTS: Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical  pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity  Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target  validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting  prolonged activation of p38 MAPK and increased cytokine production. In contrast,  transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli. CONCLUSION: The combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis. 
17227774	 Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are  produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria  Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation  of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a  novel role for Destruxin A in specific suppression of the humoral immune response in insects. 
17331609	 The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in  genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ insignificantly in genome structure. The growth patterns of these two sub-strains were very similar in a well-equipped bioreactor, and their genome structures were shown to be almost identical by DNA microarray. However, detailed profiling of protein and gene expression by 2-dimensional gel electrophoresis and microarray analysis showed many differentially expressed genes and proteins, combinations of which were highly correlated. The differentially regulated genes and proteins belonged to the following functional categories: genes regulated by sigma subunit of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in central metabolism. Genes involved in central cell metabolism - the glycolysis pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were differentially regulated at both the mRNA and proteome levels. The strains differ significantly in central metabolism and thus in the generation of precursor metabolites and energy. This high plasticity probably represents a universal feature of metabolic activities in closely related species, and has the potential to reveal differences in regulatory networks. We suggest that unless care is taken in the choice of strains for any validating experiment, the results might be misleading. 
17343762	 BACKGROUND: Extra-cellular shear force is an important environmental parameter that is significant both medically and in the space environment. Escherichia coli cells grown in a low-shear modeled microgravity (LSMMG) environment produced in a high aspect rotating vessel (HARV) were subjected to transcriptional and physiological analysis. RESULTS: Aerobic LSMMG cultures were grown in rich (LB) and minimal (MOPS + glucose) medium with a normal gravity vector HARV control. Reproducible changes in transcription were seen, but no specific LSMMG responsive genes were identified. Instead, absence of shear and a randomized gravity vector appears to  cause local extra-cellular environmental changes, which elicit reproducible cellular responses. In minimal media, the majority of the significantly up- or down-regulated genes of known function were associated with the cell envelope. In rich medium, most LSMMG down-regulated genes were involved in translation. No observable changes in post-culture stress responses and antibiotic sensitivity were seen in cells immediately after exposure to LSMMG. Comparison with earlier studies of Salmonella enterica serovar Typhimurium conducted under similar growth conditions, revealed essentially no similarity in the genes that were significantly up- or down-regulated. CONCLUSION: Comparison of these results to previous studies suggests that different organisms may dramatically differ in their responses to medically significant low-shear and space environments. Depending on their specific response, some organisms, such as Salmonella, may become preadapted in a manner that predisposes them to increased virulence. 
17342207	 N6-Adenine methylation is an important epigenetic signal, which regulates various processes, such as DNA replication and repair and transcription. In gamma-proteobacteria, Dam is a stand-alone enzyme that methylates GATC sites, which are non-randomly distributed in the genome. Some of these overlap with transcription factor binding sites. This work describes a global computational analysis of a published Dam knockout microarray alongside other publicly available data to throw insights into the extent to which Dam regulates transcription by interfering with protein binding. The results indicate that DNA  methylation by DAM may not globally affect gene transcription by physically blocking access of transcription factors to binding sites. Down-regulation of Dam during stationary phase correlates with the activity of TFs whose binding sites are enriched for GATC sites. 
17087734	 The probiotic Escherichia coli strain Nissle 1917 (EcN) has been used for decades in human medicine in Central Europe for the treatment and prevention of intestinal disorders and diseases. However, the molecular mechanisms underlying its beneficial effects are only partially understood. To identify molecular responses induced by EcN that might contribute to its probiotic properties polarized T84 cells were investigated employing DNA microarrays, quantitative RT-PCR, Western blotting, immunofluorescence and specific protein kinase C (PKC)  inhibitors. Polarized T84 epithelial cell monolayers were used as a model to monitor barrier disruption by infection with the enteropathogenic E. coli (EPEC)  strain E2348/69. Co-incubation of EPEC with EcN or addition of EcN following EPEC infection abolished barrier disruption and, moreover, restored barrier integrity  as monitored by transepithelial resistance. DNA-microarray analysis of T84 cells  incubated with EcN identified 300+ genes exhibiting altered expression. EcN altered the expression, distribution of zonula occludens-2 (ZO-2) protein and of  distinct PKC isotypes. ZO-2 expression was enhanced in parallel to its redistribution towards the cell boundaries. This study provides evidence that EcN induces an overriding signalling effect leading to restoration of a disrupted epithelial barrier. This is transmitted via silencing of PKCzeta and the redistribution of ZO-2. We suggest that these properties contribute to the reported efficacy in the treatment of inflammatory bowel diseases and in part rationalize the probiotic nature of EcN. 
17220219	 Proteins whose synthesis is enhanced by polyamines at the level of translation were identified in a polyamine-requiring mutant cultured in the presence of 0.1%  glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under these conditions, enhancement of cell growth by polyamines was almost the same as that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra, and H-NS was enhanced by polyamines at the level of translation at the early logarithmic phase of growth (A(540) of 0.15). The effects of polyamines on synthesis of RpoN, H-NS, and Cra were due to the existence of unusual Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon  (Cra) in their mRNAs. Thus, rpoN, cra, and hns genes were identified as new members of the polyamine modulon. Because most of the polyamine modulon genes thus far identified encode transcription factors (RpoS [sigma(38)], Cya, FecI [sigma(18)], Fis, RpoN [sigma(54)], Cra, and H-NS), DNA microarray analysis of mRNA expressed in cells was performed. At the early logarithmic phase of growth,  a total of 97 species of mRNAs that were up-regulated by polyamines more than twofold were under the control of seven polyamine modulon genes mentioned above. 
17302821	 Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate  F-actin 'pedestal' structures that elevate them above surrounding cell surfaces.  Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell  and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECDeltadam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild-type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP  fusions, Northern blotting, reverse transcription polymerase chain reaction, and  microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable  of robust intestinal colonization of experimentally infected animals. 
17207772	 We identified the 1.6-kb region of Thermus thermophilus plasmid pTT8 capable of autonomous replication, which shows a significant sequence similarity to the replicon regions of the ColE2-related plasmids. We showed the requirement of DNA  polymerase I for pTT8 replication. The putative rep gene coding for the replication initiator protein, Rep, similar to those of the ColE2-related plasmids was cloned into an expression vector. The 6xHis-Rep protein expressed in Escherichia coli was successfully purified by stepwise denaturing with urea and refolding in the presence of glycerol on Ni-resin. We identified the nucleotide sequence recognized by the pTT8 Rep protein by the SELEX experiment using the purified protein, and proposed the existence of the third origin of pTT8 replication different from those predicted previously. 
17273855	 During aerobic growth on glucose, Escherichia coli produces acetate in the so-called overflow metabolism. DNA microarray analysis was used to determine the  global gene expression patterns of chemostat cultivations of E. coli MG1655 that  were characterized by different acetate formation rates during aerobic growth on  glucose. A correlation analysis identified that expression of ten genes (sdhCDAB, sucB, sucC, acnB, lpdA, fumC and mdh) encoding the TCA cycle enzymes succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, aconitase, fumarase and malate dehydrogenase, respectively, and of the acs-yjcH-actP operon for acetate utilization correlated negatively with acetate formation. Relieving transcriptional control of the sdhCDAB-b0725-sucABCD operon  by chromosomal promoter exchange mutagenesis yielded a strain with increased specific activities of the TCA cycle enzymes succinate dehydrogenase, alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase, which are encoded  by this operon. The resulting strain produced less acetate and directed more carbon towards carbon dioxide formation than the parent strain MG1655 while maintaining high growth and glucose consumption rates. 
17222132	 Analysis of the temporal development of Escherichia coli K-12 biofilms in complex medium indicates the greatest differential gene expression between biofilm and suspension cells occurred in young biofilms at 4 and 7 h (versus 15 and 24 h). The main classes of genes differentially expressed (biofilm versus biofilm and biofilm versus suspension cells) include 42 related to stress response (e.g. cspABFGI), 66 related to quorum sensing (e.g. ydgG, gadABC, hdeABD), 20 related to motility (e.g. flgBCEFH, fliLMQR, motB), 13 related to fimbriae (e.g. sfmCHM,  fimZ, csgC), 24 related to sulfur and tryptophan metabolism (e.g. trpLBA, tnaLA,  cysDNCJH), 80 related to transport (e.g. gatABC, agaBC, ycjJ, ydfJ, phoU, phnCJKM), and six related to extracellular matrix (e.g. wcaBDEC). Of the 93 mutants identified and studied, 76 showed altered biofilm formation. Biofilm architecture changed from thin and dense to globular and dispersed to dense and smooth. The quorum-sensing signal AI-2 controls gene expression most clearly in mature biofilms (24 h) when intracellular AI-2 levels are highest. Sulfate transport and metabolism genes (cysAUWDN) and genes with unknown functions (ymgABCZ) were repressed in young (4, 7 h) biofilms, induced in developed biofilms (15 h), and repressed in mature (24 h) biofilms. Genes related to both motility and fimbriae were induced in biofilms at all sampling time points and colanic acid genes were induced in mature biofilms (24 h). Genes related to dihydroxyacetone phosphate synthesis from galactitol and galactosamine (e.g. gatZABCDR, agaBCY) were highly regulated in biofilms. Genes involved in the biosynthesis of indole and sulfide (tnaLA) are repressed in biofilms after 7 h (corroborated by decreasing intracellular indole concentrations in biofilms). Cold-shock protein transcriptional regulators (cspABFGI) appear to be positive biofilm regulators, and deletions in respiratory genes (e.g. hyaACD, hyfCG, appC, narG) increased biofilm formation sevenfold. 
17233676	 The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set  of genes which show changes in expression profile following the removal of rpoZ.  We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together  we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level. 
17125967	 Escherichia coli K (JM109) and E. coli B (BL21) are strains used routinely for recombinant protein production. These two strains grow and respond differently to environmental factors such as glucose and oxygen concentration. The differences have been attributed to differential expression of individual genes that constitute certain metabolic pathways that are part of the central carbon metabolism. By implementing a semiparametric algorithm, which is based on a density ratio model, it was possible to compare and quantify the expression patterns of groups of genes involved in several central carbon metabolic pathways. The groups comprising the glyoxylate shunt, TCA cycle, fatty acid, and  gluconeogenesis and anaplerotic pathways were expressed differently between the two strains, whereas no differences were apparent for the groups comprising either glycolysis or the pentose phosphate pathway. These results further characterized differences between the two E. coli strains and illustrated the potency of the semiparametric algorithm. 
17145952	 Urinary tract infection (UTI) is an important health problem worldwide, with many millions of cases each year, and Escherichia coli is the most common organism causing UTI in humans. Also, E. coli is responsible for most infections in patients with chronic indwelling bladder catheter. The two asymptomatic bacteriuria (ABU) E. coli strains 83972 and VR50 are significantly better biofilm formers in their natural growth medium, human urine, than the two uropathogenic E. coli isolates CFT073 and 536. We used DNA microarrays to monitor the expression profile during biofilm growth in urine of the two ABU strains 83972 and VR50. Significant differences in expression levels were seen between the biofilm expression profiles of the two strains with the corresponding planktonic  expression profiles in morpholinepropanesulfonic acid minimal laboratory medium and human urine; 417 and 355 genes were up- and down-regulated, respectively, during biofilm growth in urine of 83972 and VR50. Many genes involved in transcription and stress were up-regulated in biofilm-grown cells. The role in biofilm formation of four of the up-regulated genes, i.e., yceP, yqgA, ygiD, and  aaeX, was investigated by creating single-knockout mutant versions of 83972 and VR50; all mutants showed reduced biofilm formation in urine by 18 to 43% compared with the wild type (P < 0.05). Also, the expression profile of strain 83972 in the human urinary tract partially overlaps with the biofilm expression profile. 
17114269	 The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce  meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS II complex. We  found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (alpha3) and AccD6 (beta6) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the alpha3-beta6 complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this  carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the alpha3-beta6 complex was inhibited by the epsilon subunit. The alpha3-beta6 carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the beta6 subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex. 
16815005	 A DNA hybridization based optical detection platform for the detection of foodborne pathogens has been developed with virtually zero probability of the false negative signal. This portable, low-cost and real-time assaying detection platform utilizes the color changing molecular beacon as a probe for the optical  detection of the target sequence. The computer-controlled detection platform exploits the target hybridization induced change of fluorescence color due to the Förster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon (oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection characteristic for the fluorophore-quencher beacon where bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. In perspective, the two-color design allows also to quantify the concentration of the target DNA in a sample down to < =1 ng/microl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format. 
17129579	 In vitro antioxidant and antimutagenic activities of two polyphenols isolated from the fruits of Pistacia lentiscus was assessed. Antioxidant activity was determined by the ability of each compound to scavenge the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH*), to inhibit xanthine oxidase and to inhibit the lipid peroxidation induced by H(2)O(2) in K562 cell line. Antimutagenic activity was assayed with SOS chromotest using Escherichia coli PQ37 as tester strain and Comet assay using K562 cell line. 1,2,3,4,6-Pentagalloylglucose was found to be more effective to scavenge DPPH* radical and protect against lipid peroxidation. Moreover, these two compounds induced an inhibitory activity against nifuroxazide and aflatoxin B1 mutagenicity. The protective effect exhibited by these molecules was also determined by analysis of gene expression as response to an oxidative stress. For this purpose, we used a cDNA-microarray containing 82 genes related to cell defense, essentially represented by antioxidant and DNA repair proteins. We found that 1,2,3,4,6-pentagalloylglucose  induced a decrease in the expression of 11 transcripts related to antioxidant enzymes family (GPX1, TXN, AOE372, SHC1 and SEPW1) and DNA repair (POLD1, APEX, POLD2, MPG, PARP and XRCC5). The use of Gallic acid, induced expression of TXN, TXNRD1, AOE372, GSS (antioxidant enzymes) and LIG4, POLD2, MPG, GADD45A, PCNA, RPA2, DDIT3, HMOX2, XPA, TDG, ERCC1 and GTF2H1 (DNA repair) as well as the repression of GPX1, SEPW1, POLD1 and SHC1 gene expression. 
17085696	 Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation.  DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene  profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban  site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and  D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources. 
17071797	 The effect of target size on microarray hybridization efficiencies and specificity was investigated using a set of 166 oligonucleotide probes targeting  the 16S rRNA gene of Escherichia coli. The targets included unfragmented native rRNA, fragmented rRNA ( approximately 20 to 100 bp), PCR amplicons (93 to 1,480 bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp). Fluorescence intensities of probes hybridized with targets were categorized into  classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to  60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization efficiency was defined for those probes conferring intensities in classes I to IV; those in classes V and VI were regarded as weak and false-negative signals, respectively. Using unfragmented native rRNA, 13.9% of the probes had fluorescence intensities in classes I to IV, whereas the majority (57.8%) exhibited false-negative signals. Similar trends were observed for the 1,480-bp PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after hybridization of fragmented rRNA, the percentage of probes in classes I to IV rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45 bp, this percentage increased approximately 14-fold. Overall, microarray hybridization efficiencies and specificity were improved with fragmented rRNA (20 to 100 bp), short PCR amplicons (<150 bp), and synthetic targets (45 to 56 bp). Such an understanding is important to the application of DNA microarray technology in microbial community studies. 
16949636	 Escherichia coli is known to respond to certain toxic chemicals through an increased expression of various stress genes. In this study, therefore, the expression of recA, katG, fabA and grpE genes was used as a representative for DNA, oxidative, membrane and protein damage, respectively, after E. coli was exposed to different polycyclic aromatic hydrocarbons (PAHs), i.e., phenanthrene, naphthalene and benzo[a]pyrene. To accomplish this, the expression levels of these four genes were quantified using a real-time RT-PCR analysis when E. coli cultures were under stressful conditions, such as those caused by an exposure to  mitomycin C, hydrogen peroxide and phenol. It was found that the primary toxic effect of each chemical is clearly seen when the expression levels of the different genes are compared. Tests with the PAHs showed naphthalene and benzo[a]pyrene to be genotoxic, while phenanthrene had no clear effect on the expression of any of these genes. Based on these results, the effects due to these toxic chemicals and the extent of each stress can be evaluated with ease using the expression levels of different stress responsive genes. 
17169986	 Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low  temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process. 
17164287	 Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have  measured the distribution of the global transcription regulator protein, FNR, across the entire Escherichia coli chromosome in exponentially growing cells. Sixty-three binding targets, each located at the 5' end of a gene, were identified. Some targets are adjacent to poorly transcribed genes where FNR has little impact on transcription. In stationary phase, the distribution of FNR was  largely unchanged. Control experiments showed that, like FNR, the distribution of the nucleoid-associated protein, IHF, is little altered when cells enter stationary phase, whilst RNA polymerase undergoes a complete redistribution. 
17014972	 RpoS, the sigma S subunit of RNA polymerase, is vital during the growth and survival of Geobacter sulfurreducens under conditions typically encountered in its native subsurface environments. We investigated the conservation of sites that may be important for RpoS function in G. sulfurreducens. We also employed sequence information and expression microarray data to predict G. sulfurreducens  genome sites that may be related to RpoS regulation. Hierarchical clustering identified three clusters of significantly downregulated genes in the rpoS deletion mutant. The search for conserved overrepresented motifs in co-regulated  operons identified likely -35 and -10 promoter elements upstream of a number of functionally important G. sulfurreducens operons that were downregulated in the rpoS deletion mutant. Putative -35/-10 promoter elements were also identified in  the G. sulfurreducens genome using sequence similarity searches to matrices of -35/-10 promoter elements found in G. sulfurreducens and in Escherichia coli. Due to a sufficient degree of sequence similarity between -35/-10 promoter elements for RpoS, RpoD, and other sigma factors, both the sequence similarity searches and the search for conserved overrepresented motifs using microarray data may identify promoter elements for both RpoS and other sigma factors. 
17157257	 We perform a genome-wide analysis of the transition between transcriptional initiation and elongation in Escherichia coli by determining the association of core RNA polymerase (RNAP) and the promoter-recognition factor sigma70 with respect to RNA transcripts. We identify 1286 sigma70-associated promoters, including many internal to known operons, and demonstrate that sigma70 is usually released very rapidly from elongating RNAP complexes. On average, RNAP density is higher at the promoter than in the coding sequence, although the ratio is highly  variable among different transcribed regions. Strikingly, a significant fraction  of RNAP-bound promoters is not associated with transcriptional activity, perhaps  due to an intrinsic energetic barrier to promoter escape. Thus, the transition from transcriptional initiation to elongation is highly variable, often rate limiting, and in some cases is essentially blocked such that RNAP is effectively  "poised" to transcribe only under the appropriate environmental conditions. The genomic pattern of RNAP density in E. coli differs from that in yeast and mammalian cells. 
17021058	 Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotype-specific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 10(4) CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping  Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance. 
16954205	 Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. P(aniA) is activated by FNR (regulator of fumarate and nitrate reduction), the two-component regulatory system NarQ-NarP, and induced by nitrite; P(norB) is induced by NO independently of FNR by an uncharacterized mechanism. We report the results of microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation, which revealed that only five genes with readily identified NarP-binding sites are differentially expressed in narP(+) and narP strains. These include three genes implicated in the truncated gonococcal denitrification pathway: aniA, norB, and narQ. We also report that (i) nitrite induces aniA transcription in a narP mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of  a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study;  (iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive nitrogen species response protein) were expressed constitutively in the absence of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E. coli and gonococcal NarQ are ligand-insensitive and constitutively active: a "locked-on" phenotype. We conclude that genes involved in the truncated denitrification pathway of N. gonorrhoeae are key components of the small NarQP regulon, that NarP indirectly regulates P(norB) by stimulating NO production by AniA, and that NsrR plays a critical role in enabling gonococci to evade NO generated as a host defense mechanism. 
17046599	 Aphids exhibit a complex life cycle in which their phenotypes vary in response to environmental changes. Wing polyphenism is one of the phenotypes that have not been adequately studied. We developed a cDNA microarray from the green peach aphid Myzus persicae (Sulzer), and compared mRNA samples derived from alate and apterous adults. The microarray experiment resulted in 108 significantly changing clones that represented 31 unique ESTs. Among the highest and significantly expressed clones in alate adults, seven clones showed portions of a new adenine nucleotide translocase (ANT) gene from M. persicae and four clones contained sequences of the OS-D gene. Another clone showed significant homology with the takeout (TO) and TO-like proteins described from other insects. A full-length cDNA clone of M. persicae ANT (Mp ANT) was isolated and characterized. An reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant differences in Mp ANT, OS-D and Mp takeout-like genes (Mp TOL) expression in alate compared to apterous adults. A polyclonal antibody against the beef heart mitochondrial ANT reacted with a recombinant Mp ANT expressed in Escherichia coli, as it did with aphid extracts, showing higher amounts of the expected 33kDa band in alate adults. Spatial expression analysis showed higher Mp ANT expression in the thorax compared to head, abdomen, leg and antennae. OS-D and Mp TOL showed ubiquitous expression across the body, with OS-D being significantly higher in legs and antennae while Mp TOL being significantly higher in the head and abdomen. The possible causes for higher expression of ANT, OS-D and TOL in alates and the physiological effects on the aphid flight and dispersal are discussed. 
16936033	 The sigma transcription factor confers the promoter recognition specificity of RNA polymerase (RNAP) in eubacteria. Chlamydia trachomatis has three known sigma  factors, sigma(66), sigma(54), and sigma(28). We developed two methods to facilitate the characterization of promoter sequences recognized by C. trachomatis sigma(28) (sigma(28)(Ct)). One involved the arabinose-induced expression of plasmid-encoded sigma(28)(Ct) in a strain of Escherichia coli defective in the sigma(28) structural gene, fliA. The second was an analysis of transcription in vitro with a hybrid holoenzyme reconstituted with E. coli RNAP core and recombinant sigma(28)(Ct). These approaches were used to investigate the interactions of sigma(28)(Ct) with the sigma(28)(Ct)-dependent hctB promoter and  selected E. coli sigma(28) (sigma(28)(Ec))-dependent promoters, in parallel, compared with the promoter recognition properties of sigma(28)(EC). Our results indicate that RNAP containing sigma(28)(Ct) has at least three characteristics: (i) it is capable of recognizing some but not all sigma(28)(EC)-dependent promoters; (ii) it can distinguish different promoter structures, preferentially  activating promoters with upstream AT-rich sequences; and (iii) it possesses a greater flexibility than sigma(28)(EC) in recognizing variants with different spacing lengths separating the -35 and -10 elements of the core promoter. 
16943349	 Analysis of the complete genomic sequence of Campylobacter jejuni strain RM1221 identified four large genomic elements, Campylobacter jejuni-integrated elements  (CJIEs), that were absent from C. jejuni strain NCTC 11168. To further investigate the genomic diversity of Campylobacter, we conducted a comparative genomic analysis from a collection of 67 C. jejuni and 12 Campylobacter coli strains isolated from various geographical locations and clinical and veterinary  sources. Utilizing PCR, we demonstrated that 55% of the C. jejuni strains examined were positive for at least one RM1221-like genomic element and 27% were  positive for two or more of these CJIEs. Furthermore, many C. coli strains were positive for either genomic element CJIE1 or CJIE3. To simultaneously assess for  the presence or absence of several genes that comprise the various CJIEs, we developed a multistrain C. jejuni DNA microarray that contained most of the putative coding sequences for strains NCTC 11168 and RM1221. A comparative genomic hybridization (CGH) analysis of 35 of the 67 C. jejuni strains confirmed  the presence of genomic elements similar to those in strain RM1221. Interestingly, the DNA microarray analysis demonstrated that these genomic elements in the other C. jejuni strains often exhibited modular patterns with some regions of the CJIEs present and other regions either absent or highly divergent compared to strain RM1221. Our CGH method also identified 18 other intraspecies hypervariable regions, such as the capsule and lipooligosaccharide biosynthesis regions. Thus, the inclusion of genes from these integrated genomic  elements and the genes from the other intraspecies hypervariable regions contributes to a better assessment of the diversity in C. jejuni and may increase the usefulness of DNA microarrays as an epidemiological genotyping tool. Finally, we also showed that in CJIE1, a Campylobacter Mu-like phage, is located differentially in other strains of C. jejuni, suggesting that it may integrate essentially randomly. 
17010156	 Bis-(3'-5')-cyclic-di-guanosine monophosphate (c-di-GMP) is a bacterial signalling molecule produced by diguanylate cyclases (DGC, carrying GGDEF domains) and degraded by specific phosphodiesterases (PDE, carrying EAL domains). Neither its full physiological impact nor its effector mechanisms are currently understood. Also, the existence of multiple GGDEF/EAL genes in the genomes of most species raises questions about output specificity and robustness of c-di-GMP signalling. Using microarray and gene fusion analyses, we demonstrate that at least five of the 29 GGDEF/EAL genes in Escherichia coli are not only stationary  phase-induced under the control of the general stress response master regulator sigma(S) (RpoS), but also exhibit differential control by additional environmental and temporal signals. Two of the corresponding proteins, YdaM (GGDEF only) and YciR (GGDEF + EAL), which in vitro show DGC and PDE activity, respectively, play an antagonistic role in the expression of the biofilm-associated curli fimbriae. This control occurs at the level of transcription of the curli and cellulose regulator CsgD. Moreover, we show that H-NS positively affects curli expression by inversely controlling the expression  of ydaM and yciR. Furthermore, we demonstrate a temporally fine-tuned GGDEF cascade in which YdaM controls the expression of another GGDEF protein, YaiC. By  genome-wide microarray analysis, evidence is provided that YdaM and YciR strongly and nearly exclusively control CsgD-regulated genes. We conclude that specific GGDEF/EAL proteins have very distinct expression patterns, and when present in physiological amounts, can act in a highly precise, non-global and perhaps microcompartmented manner on a few or even a single specific target(s). 
17001495	 Previous studies have identified two distinct O-methyltransferases (OMTs) implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating  the A-ring 7-hydroxyl of the isoflavone daidzein and a 4'-OMT methylating the B-ring 4'-hydroxyl of 2,7,4'-trihydroxyisoflavanone. Genes related to these OMTs  from the model legume Medicago truncatula cluster as separate branches of the type I plant small molecule OMT family. To better understand the possible functions of these related OMTs in secondary metabolism in M. truncatula, seven of the OMTs were expressed in E. coli, purified, and their in vitro substrate preferences determined. Many of the enzymes display promiscuous activities, and some exhibit dual regio-specificity for the 4' and 7-hydroxyl moieties of the isoflavonoid nucleus. Protein structure homology modeling was used to help rationalize these catalytic activities. Transcripts encoding the different OMT genes exhibited differential tissue-specific and infection- or elicitor-induced expression, but not always in parallel with changes in expression of confirmed genes of the isoflavonoid pathway. The results are discussed in relation to the potential in vivo functions of these OMTs based on our current understanding of the phytochemistry of M. truncatula, and the difficulties associated with gene annotation in plant secondary metabolism. 
16988236	 We have previously shown that outer membrane protein A (OmpA) and type 1 fimbriae are the bacterial determinants involved in Escherichia coli K1 binding to human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In investigating the role of OmpA in E. coli K1 binding to HBMEC, we showed for the first time that ompA deletion decreased the expression of type 1 fimbriae in E. coli K1. Decreased expression of type 1 fimbriae in the ompA deletion mutant was largely the result of driving the fim promoter toward the type 1 fimbrial phase-OFF orientation. mRNA levels of fimB and fimE were found to be decreased with the OmpA mutant compared to the parent strain. Of interest, the ompA deletion further decreased the abilities of E. coli K1 to bind to and invade HBMEC under the conditions of fixing type 1 fimbria expression in the phase-ON or phase-OFF status. These findings suggest that the decreased ability of the OmpA mutant to interact with HBMEC is not entirely due to its decreased type 1 fimbrial expression and that OmpA and type 1 fimbriae facilitate the interaction  of E. coli K1 with HBMEC at least in an additive manner. 
17021100	 The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood diarrhea is controversial. The aim of the present study was to search for genes linked with diarrhea in atypical EPEC strains from a case-control study among Norwegian children. Using DNA microarray analysis, genomic DNAs from strains isolated from children with (n = 37) and without (n = 20) diarrhea were hybridized against 242 different oligonucleotide probes specific for 182 virulence genes or markers from all known E. coli pathotypes. PCR was performed to test the strains for seven putative virulence genes not included in the microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest statistical association with diarrhea (P = 0.0008). Other OI-122 genes (set/ent,  nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were also associated with diarrheal disease. The phylogenetic marker gene yjaA was negatively associated with diarrhea (P = 0.0004). Atypical EPEC strains could be  classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In conclusion, using DNA microarray analysis to determine the virulence gene profile of atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Based on their composition of virulence genes, the majority of strains could be classified in two virulence groups, of which one was seen mainly in children with diarrhea. 
16680459	 Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a  dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual  stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were  no genes that only responded to the dual stress. The most interesting difference  between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since, tRNAs facilitate protein synthesis via translation,  this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses. 
17020583	 Hfq plays an important role in cellular physiology by regulating the expression of several genes. Hfq synthesis in Escherichia coli is subject to auto-repression at translational level. Studies with Shigella flexneri show that hfq transcription is regulated by a pleiotropic regulator, DksA. Comparison of gene expression profiles of wild type and dksA mutant S. flexneri determined that hfq  expression was reduced in the dksA mutant. As DksA is required for stress resistance and plaque formation in cultured cell monolayers, a measure of virulence, we assessed the role of Hfq in the dksA virulence phenotype. Expression of hfq in the dksA mutant restored plaque formation, and an hfq mutant failed to form plaques. Thus, DksA plays a role in regulating hfq gene expression and this regulation is important for S. flexneri virulence. In an in vitro transcription assay, addition of DksA increased transcription of hfq and this effect was greatest with one of the known hfq promoters. Addition of ppGpp, a stringent response molecule, along with DksA in the in vitro transcription assay  resulted in a further increase in transcription of hfq, indicating that DksA is required for maximal transcription of hfq during both exponential and stringent response growth conditions. 
16961923	 BACKGROUND: The stringent response is the initial reaction of microorganisms to nutritional stress. During stringent response the small nucleotides (p)ppGpp act  as global regulators and reprogram bacterial transcription. In this work, the genetic network controlled by the stringent response was characterized in the amino acid-producing Corynebacterium glutamicum. RESULTS: The transcriptome of a C. glutamicum rel gene deletion mutant, unable to synthesize (p)ppGpp and to induce the stringent response, was compared with that  of its rel-proficient parent strain by microarray analysis. A total of 357 genes  were found to be transcribed differentially in the rel-deficient mutant strain. In a second experiment, the stringent response was induced by addition of DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of  the maximal effect on transcription was determined by real-time RT-PCR using the  histidine and serine biosynthetic genes. Transcription of all of these genes reached a maximum at 10 minutes after SHX addition. Microarray experiments were performed comparing the transcriptomes of SHX-induced cultures of the rel-proficient strain and the rel mutant. The differentially expressed genes were grouped into three classes. Class A comprises genes which are differentially regulated only in the presence of an intact rel gene. This class includes the non-essential sigma factor gene sigB which was upregulated and a large number of  genes involved in nitrogen metabolism which were downregulated. Class B comprises genes which were differentially regulated in response to SHX in both strains, independent of the rel gene. A large number of genes encoding ribosomal proteins  fall into this class, all being downregulated. Class C comprises genes which were differentially regulated in response to SHX only in the rel mutant. This class includes genes encoding putative stress proteins and global transcriptional regulators that might be responsible for the complex transcriptional patterns detected in the rel mutant when compared directly with its rel-proficient parent  strain. CONCLUSION: In C. glutamicum the stringent response enfolds a fast answer to an induced amino acid starvation on the transcriptome level. It also showed some significant differences to the transcriptional reactions occurring in Escherichia coli and Bacillus subtilis. Notable are the rel-dependent regulation of the nitrogen metabolism genes and the rel-independent regulation of the genes encoding ribosomal proteins. 
16949419	 OBJECTIVE: The purpose of this study was to identify changes in gene expression that are associated with preterm labor induced by either bacteria or ovariectomy. STUDY DESIGN: Pregnant mice (14.5 days of gestation) were allocated to: (1) intrauterine injection of heat-inactivated Escherichia coli; (2) media alone; (3) ovariectomy; or (4) sham operation. The uterine transcriptome was studied with photolithographic, very short oligonucleotide-based microarrays, and arachidonate metabolism genes were assayed with quantitative reverse transcriptase-polymerase  chain reaction. Significance was determined by analysis of variance. RESULTS: Microarray-based gene expression changes in the arachidonate metabolism  pathway are associated globally with bacteria-induced preterm labor (P < or = .0031) and ovariectomy-induced preterm labor (P < or = .00036). Quantitative real-time reverse transcriptase-polymerase chain reaction measurements demonstrated that bacteria-induced preterm labor substantially increased the expression of genes involved in prostaglandin synthesis. In contrast, ovariectomy-induced preterm labor increased the expression of genes involved in lipoxin, leukotriene, and hydroxyeicosatetraenoic acid synthesis. CONCLUSION: Bacteria-induced and ovariectomy-induced preterm labor each express a different balance of genes that are required for the synthesis of prostaglandins, lipoxins, leukotrienes, and hydroxyeicosatetraenoic acids. 
16913923	 Escherichia coli growing in continuous culture under continuous UVA irradiation exhibits growth inhibition with a subsequent adaptation to the stress. Transcriptome analysis was performed during transient growth inhibition and in the UVA light-adapted growth state. The results indicate that UVA light induces stringent response and an additional response that includes the upregulation of the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and glutamate. The induction of several SOS response-genes strongly points to DNA damage as a result of UVA exposure. The involvement of oxidative stress was observed with the induction of ahpCF. Taken together it supports the hypothesis of the production of reactive oxygen species by UVA light. In the UVA-adapted cell population strong repression of the acid tolerance response was found. We identified the enzyme chorismate mutase as a possible chromophore for UVA light-inactivation and found strong repression of the pyrBI operon and the gene mgtA encoding for an ATP-dependent Mg2+ transporter. Furthermore, our results indicate that the role of RpoS may not be as important in the adaptation of E. coli to UVA light as it was implicated by previous results with starved cells, but that RpoS might be of crucial importance for the resistance under transient light exposure. 
16952965	 DNA microarrays were used to probe the transcriptional response of Escherichia coli to N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Fifty-five transcripts were significantly up-regulated, including all of the genes that are  regulated by Zur and many that are regulated by Fur. In the same TPEN-treated cells, 46 transcripts were significantly down-regulated. 
17046956	 Heat-stable nucleoid-structuring protein (H-NS) is one of the main nucleoid proteins expressed in exponentially growing Escherichia coli cells. In addition to a role in nucleoid organization, H-NS functions as a pleiotropic regulator of  gene expression. The genome-wide distribution of H-NS, compared with the distribution of RNA polymerase and transcriptionally active genes, was investigated using a high-density oligonucleotide chip. The new approach utilized in this study revealed that H-NS binds specifically to approximately 250 loci, covering >1000 genes, to maintain transcriptional inactivation. RNA polymerase was detected in >65% of H-NS binding sites with low or no transcriptional activity, indicating that the association of RNA polymerase to promoter regions is a general mode of transcription repression by H-NS. This study also revealed that most H-NS bound DNA have been horizontally acquired, which indicates that repression of inappropriate gene expression by H-NS plays an important role in the diversification of the E. coli genome. This study presents a comprehensive assessment of the distribution of H-NS within the E. coli genome, sheds light on  the mechanism underlying the transcriptional regulation by H-NS, and provides new insight into bacterial genome evolution. 
17028724	 An electronic oligonucleotide microarray technique was developed for detection and differentiation of the viable Campylobacter species, C. jejuni, C. coli, and  C. lari. This development consisted of four major components: identification of single nucleotide polymorphisms (SNPs) within the hsp60 gene as species markers,  design of fluorescently labelled SNP-based reporters, development of an electronic microarray detection, and application of the integrated technique to analysis of Campylobacter species in food samples. A unique capability of this technique is the specific detection of viable cells and not dead ones. This is achieved by using mRNA of the 60 kDa heat-shock protein as the viability marker.  The identification of two unique SNPs closely located at positions 291 and 294 of the hsp60 gene enabled the differentiation of the three Campylobacter species. This technique was able to detect as few as two viable Campylobacter cells. The analysis of 19 blind Campylobacter samples showed 100% agreement with their identities obtained using pulsed-field gel electrophoresis. The analysis of six chicken samples revealed the presence of C. coli in one of the samples. 
16885300	 Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that emt1 is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of mat1 resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in U. maydis. 
16538485	 In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that  were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes. 
16816893	 Metabolically-engineered Escherichia coli strains were developed by cloning poly-gamma-glutamic acid (gamma-PGA) biosynthesis genes, consisting of pgsB, pgsC and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of gamma-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible  trc promoter and a constitutive promoter (P(HCE)) derived from the D-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The constitutive HCE promoter was more efficient than inducible trc promoter for the  expression of gamma-PGA biosynthesis genes. DNA microarray analysis showed that the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY, yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were down-regulated. When (NH(4))(2)SO(4 )was added at 40 g/l into the feeding solution, the final gamma-PGA concentration reached 3.7 g/l in the fed-batch culture of recombinant E. coli/pCOpgs. 
16849790	 Escherichia coli strain PHL628 was subjected to saturating Tn5 transposon mutagenesis and then grown under competitive planktonic or biofilm conditions. The locations of transposon insertions from the remaining cells were then mapped  on a gene array. The results from the array mapping indicated that 4.5 % of the E. coli genome was important under these conditions. Specifically, 114 genes were identified as important for the biofilm lifestyle, whereas 80 genes were important for the planktonic lifestyle. Four broad functional categories were identified as biofilm-important. These included genes encoding cell structures, small-molecule transport, energy metabolism and regulatory functions. For one of  these genes, arcA, an insertion mutant was generated and its biofilm-related phenotype was examined. Results from both the transposon array and insertion mutagenesis indicated that arcA, which is known to be a negative response regulator of genes in aerobic pathways, was important for competitiveness in E. coli PHL628 biofilms. This work also demonstrated that ligation-mediated PCR, coupled with array-based transposon mapping, was an effective tool for identifying a large variety of candidate genes that are important for biofilm fitness. 
16733264	 Multiple factors are involved in the translation of functional genomic results into proteins for proteome research and target validation on tumoral tissues. In  this report, genes were selected by using DNA microarrays on a panel of colorectal cancer (CRC) paired samples. A large number of up-regulated genes in colorectal cancer patients were investigated for cellular location, and those corresponding to membrane or extracellular proteins were used for a non-biased expression in Escherichia coli. We investigated different sources of cDNA clones  for protein expression as well as the influence of the protein size and the different tags with respect to protein expression levels and solubility in E. coli. From 29 selected genes, 21 distinct proteins were finally expressed as soluble proteins with, at least, one different fusion protein. In addition, seven of these potential markers (ANXA3, BMP4, LCN2, SPARC, SPP1, MMP7, and MMP11) were tested for antibody production and/or validation. Six of the seven proteins (all  except SPP1) were confirmed to be overexpressed in colorectal tumoral tissues by  using immunoblotting and tissue microarray analysis. Although none of them could  be associated to early stages of the tumor, two of them (LCN2 and MMP11) were clearly overexpressed in late Dukes' stages (B and C). This proteomic study reveals novel clues for the assembly of a robust and highly efficient high throughput system for the validation of genomic data. Moreover it illustrates the different difficulties and bottlenecks encountered for performing a quick conversion of genomic results into clinically useful proteins. 
16841897	 Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate  the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable  the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of  the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR. 
16645822	 Expression profiles of genes related to stress responses, substrate assimilation, acetate metabolism, and biosynthesis were obtained by monitoring growth of Escherichia coli MG1655 in Luria-Bertani (LB) medium with transcriptional microarrays. Superimposing gene expression profiles on a plot of specific growth  rate demonstrates that the cells pass through four distinct physiological states  during fermentation before entering stationary phase. Each of these states can be characterized by specific patterns of substrate utilization and cellular biosynthesis corresponding to the nutrient status of the medium. These data allow the growth phases of the classical microbial growth curve to be redefined in terms of the physiological states and environmental changes commonly occurring during bacterial growth in batch culture on LB medium. 
16628448	 Microorganisms respond to environmental changes by reprogramming their metabolism primarily through altered patterns of gene expression. DNA microarrays provide a  tool for exploiting microorganisms as living sensors of their environment. The potential of DNA microarrays to reflect availability of nutrient components during fermentations on complex media was examined by monitoring global gene expression throughout batch cultivation of Escherichia coli MG1655 on Luria-Bertani (LB) medium. Gene expression profiles group into pathways that clearly demonstrate the metabolic changes occurring in the course of fermentation. Functional analysis of the gene expression related to metabolism of sugars, alcohols, and organic acids revealed that E. coli growing on LB medium switches from a sequential mode of substrate utilization to the simultaneous one  in the course of the growth. Maltose and maltodextrins are the first of these substrates to support growth. Utilization of these nutrients associated with the  highest growth rate of the culture was followed by simultaneous induction of enzymes involved in assimilation of a large group of other carbon sources including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol, amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of these nutrients to the cells was monitored by induction of corresponding transport and/or catabolic systems specific for each of the compounds. 
16575570	 Analysis of gene expression data related to assimilation and biosynthesis of nitrogen-containing compounds amino acids, peptides, and nucleotides was used to  monitor availability of these nutrients to Escherichia coli MG1655 growing on Luria-Bertani medium. The data indicate that free amino acids and nucleotides only transiently support the nitrogen requirement for growth and are no longer available by 3.5 h of fermentation. The resulting shortage of available nitrogen  sources induces the Ntr response, which involves induction of the glnALG, glnK-amtB, dppABCDF, and oppABCDF operons as well as the genes coding for outer membrane proteins, porins OmpA and OmpC, and proteases OmpP and OmpT. The increased uptake of peptides facilitated by the products of dppABCDF, oppABCDF, ompA, ompC, ompP, and ompT alleviates nitrogen limitation of the growth. 
16799466	 Regulation of cellular growth implies spatiotemporally coordinated programmes of  gene transcription. A central question, therefore, is how global transcription is coordinated in the genome. The growth of the unicellular organism Escherichia coli is associated with changes in both the global superhelicity modulated by cellular topoisomerase activity and the relative proportions of the abundant DNA-architectural chromatin proteins. Using a DNA-microarray-based approach that  combines mutations in the genes of two important chromatin proteins with induced  changes of DNA superhelicity, we demonstrate that genomic transcription is tightly associated with the spatial distribution of supercoiling sensitivity, which in turn depends on chromatin proteins. We further demonstrate that essential metabolic pathways involved in the maintenance of growth respond distinctly to changes of superhelicity. We infer that a homeostatic mechanism organizing the supercoiling sensitivity is coordinating the growth-phase-dependent transcription of the genome. 
16713644	 The aim of the present study was to study different strains of bifidobacteria for adhesion to Caco-2 intestinal epithelial cells (IECs) and to test for the mRNA response of these cells following interaction with bifidobacteria. Adhesion was tested at different pH conditions using model epithelia consisting of transwell cultures of fully differentiated Caco-2 cells. Microarrays were used to characterize changes in global expression profiles of Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) and challenged with non-pathogenic Escherichia coli D2241 or four different strains of bifidobacteria. Furthermore, cytokine mRNA of IECs in responses to challenge with Bifidobacterium bifidum S17 or E. coli D2241 was tested in PBMC-sensitised Caco-2 cells using RT-PCR. Bifidobacteria showed strain-specific adhesion to Caco-2. Shift of apical pH from 7 to 4.5 resulted in strain-specific changes of adhesion. Global expression profiles of PBMC-sensitised Caco-2 cells revealed differential  expression of a significant number of genes only after challenge with E. coli D2241 while cells were essentially unresponsive to challenge with four strains of bifidobacteria showing different adhesion properties. Using a RT-PCR approach, in the same system a similar differential expression after challenge with E. coli D2241 or B. bifidum S17 was observed for various immune markers. The presented results suggest that Caco-2 cells might be specifically unresponsive to challenge with bifidobacteria irrespective of the level of adhesion. 
16511888	 2006 Wiley Periodicals, Inc. 
16768798	 BACKGROUND: Bacterial populations contain persisters, phenotypic variants that constitute approximately 1% of cells in stationary phase and biofilm cultures. Multidrug tolerance of persisters is largely responsible for the inability of antibiotics to completely eradicate infections. Recent progress in understanding  persisters is encouraging, but the main obstacle in understanding their nature was our inability to isolate these elusive cells from a wild-type population since their discovery in 1944. RESULTS: We hypothesized that persisters are dormant cells with a low level of translation, and used this to physically sort dim E. coli cells which do not contain sufficient amounts of unstable GFP expressed from a promoter whose activity depends on the growth rate. The dim cells were tolerant to antibiotics and exhibited a gene expression profile distinctly different from those observed  for cells in exponential or stationary phases. Genes coding for toxin-antitoxin module proteins were expressed in persisters and are likely contributors to this  condition. CONCLUSION: We report a method for persister isolation and conclude that these cells represent a distinct state of bacterial physiology. 
16723563	 A DNA microarray was developed to detect bacterial genes conferring resistance to macrolides and related antibiotics. A database containing 65 nonredundant genes selected from publicly available DNA sequences was constructed and used to design 100 oligonucleotide probes that could specifically detect and discriminate all 65 genes. Probes were spotted on a glass slide, and the array was reacted with DNA templates extracted from 20 reference strains of eight different bacterial species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and Bacteroides fragilis) known to harbor 29 different macrolide resistance genes. Hybridization results showed that probes reacted with, and only with, the expected DNA templates and allowed discovery of three unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not yet been described for gram-negative bacteria. 
16555073	 Indole-3-acetic acid (IAA) is a ubiquitous molecule playing regulatory roles in many living organisms. To elucidate the physiological changes induced by IAA treatment, we used Escherichia coli K-12 as a model system. By microarray analysis we found that 16 genes showed an altered expression level in IAA-treated cells. One-third of these genes encode cell envelope components, or proteins involved in bacterial adaptation to unfavourable environmental conditions. We thus investigated the effect of IAA treatment on some of the structural components of the envelope that may be involved in cellular response to stresses. This showed that IAA-treated cells had increased the production of trehalose, lipopolysaccharide (LPS), exopolysaccharide (EPS) and biofilm. We demonstrated further that IAA triggers an increased tolerance to several stress conditions (heat and cold shock, UV-irradiation, osmotic and acid shock and oxidative stress) and different toxic compounds (antibiotics, detergents and dyes) and this correlates with higher levels of the heat shock protein DnaK. We suggest that IAA triggers an increased level of alert and protection against external adverse conditions by coordinately enhancing different cellular defence systems. 
16515535	 We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted  to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however,  the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor.  The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity. 
16527537	 The meaning of the word affinity in the context of protein separation has undergone evolutionary changes over the years. The exploitation of molecular recognition phenomenon is no longer limited to affinity chromatography modes. Affinity based separations today include precipitation, membrane based purification and two-phase/three-phase extractions. Apart from the affinity ligands, which have biological relationship (in vivo) with the target protein, a  variety of other ligands are now used in the affinity based separations. These include dyes, chelated metal ions, peptides obtained by phage display technology, combinatorial synthesis, ribosome display methods and by systematic evolution of  ligands by exponential enrichment (SELEX). Molecular modeling techniques have also facilitated the designing of biomimetic ligands. Fusion proteins obtained by recombinatorial methods have emerged as a powerful approach in bioseparation. Overexpression in E. coli often result in inactive and insoluble inclusion bodies. A number of interesting approaches are used for simultaneous refolding and purification in such cases. Proteomics also needs affinity chromatography to  reduce the complexity of the system before analysis by electrophoresis and mass spectrometry are made. At industrial level, validation, biosafety and process hygiene are also important aspects. This overview looks at these evolving paradigms and various strategies which utilize affinity phenomenon for protein separations. 
16789824	 Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us  to determine how operons form, how they become closely spaced, and how they die.  Our findings suggest that operon evolution may be driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysA and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although genes within operons are usually closely spaced because of a neutral bias toward deletion and because of selection against large overlaps, genes in highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution may be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed. 
16596705	 The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here, we present a unique method to attach a single DNA address to proteins in one step during the purification from the E. coli lysate by fusion to human O6-alkylguanine-DNA-alkyltransferase (SNAP-tag) and the Avitag. Use of the conjugates in converting a DNA chip into a protein chip by self assembly is demonstrated. 
16490787	 Prostate cancer cell lines were examined for proteins that partnered with the transcription factor C/EBPalpha by use of a pull-down assay with S-tagged C/EBPalpha combined with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis. Ku70, Ku80, and poly(ADP-ribose) polymerase-1 (PARP-1) were identified as proteins that associated with C/EBPalpha. The physical interaction of C/EBPalpha with these partner proteins was further demonstrated by glutathione S-transferase (GST) pull-downs using purified protein expressed in Escherichia coli. The strongest binding was between C/EBPalpha and PARP-1. Immunoprecipitation of C/EBPalpha expressed in prostate cancer cells co-precipitated Ku70, Ku80, and PARP-1. Deletion analysis of C/EBPalpha indicated that the C terminus of C/EBPalpha was essential for the interaction of C/EBPalpha with Ku70, Ku80, and PARP-1. Functional analysis of the interaction between C/EBPalpha and the Ku proteins as well as PARP-1 showed that  cells exhibiting these interactions had increased radiation sensitivity and decreased ability to repair double strand DNA breaks. Deficient DNA repair was dependent on the prostate cancer cell line tested, suggesting a complex process.  We conclude that the association of C/EBPalpha with Ku proteins and PARP-1 raises the likelihood that C/EBPalpha-expressing prostate cancer cells may be more sensitive to DNA-damaging agents and may be important in the design of new prostate cancer therapies. 
16614443	 Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to  detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis  of gene expression and does not discriminate against short or structured RNAs. 
16597971	 Seventeen Escherichia coli O157:H7 strains were treated with ultrahigh pressure at 500 MPa and 23 +/- 2 degrees C for 1 min. This treatment inactivated 0.6 to 3.4 log CFU/ml, depending on the strain. The diversity of these strains was confirmed by pulsed-field gel electrophoresis (PFGE) analysis, and there was no apparent association between PFGE banding patterns and pressure resistance. The pressure-resistant strain E. coli O157:H7 EC-88 (0.6-log decrease) and the pressure-sensitive strain ATCC 35150 (3.4-log decrease) were treated with a sublethal pressure (100 MPa for 15 min at 23 +/- 2 degrees C) and subjected to DNA microarray analysis using an E. coli K-12 antisense gene chip. High pressure  affected the transcription of many genes involved in a variety of intracellular mechanisms of EC-88, including the stress response, the thiol-disulfide redox system, Fe-S cluster assembly, and spontaneous mutation. Twenty-four E. coli isogenic pairs with mutations in the genes regulated by the pressure treatment were treated with lethal pressures at 400 MPa and 23 +/- 2 degrees C for 5 min. The barotolerance of the mutants relative to that of the wild-type strains helped to explain the results obtained by DNA microarray analysis. This study is the first report to demonstrate that the expression of Fe-S cluster assembly proteins and the fumarate nitrate reductase regulator decreases the resistance to pressure, while sigma factor (RpoE), lipoprotein (NlpI), thioredoxin (TrxA), thioredoxin reductase (TrxB), a trehalose synthesis protein (OtsA), and a DNA-binding protein (Dps) promote barotolerance. 
16597943	 We previously discovered that yliH and yceP are induced in Escherichia coli biofilms (D. Ren, L. A. Bedzyk, S. M. Thomas, R. W. Ye, and T. K. Wood, Appl. Microbiol. Biotechnol. 64:515-524, 2004). Here, it is shown that deletion of yceP (b1060) and yliH (b0836) increases biofilm formation in continuous-flow chambers  with minimal glucose medium by increasing biofilm mass (240- to 290-fold), surface coverage (16- to 31-fold), and mean thickness (2,800-fold). To determine  the genetic basis of the increase in biofilm formation, we examined the differential gene expression profile in biofilms for both the mutants relative to the wild-type strain in rich medium with glucose and found that 372 to 882 genes  were induced and that 76 to 337 were repressed consistently >2-fold (P < or = 0.05). The increase in biofilm formation was related to differential expression of genes related to stress response (8 to 64 genes) for both mutants, including rpoS and sdiA. More importantly, 42 to 130 genes related to autoinducer 2 cell signaling were also differentially expressed, including gadAB and flgBCEGHIJLMN,  as well as signaling through indole, since 17 to 26 indole-related genes were differentially expressed, including phoAER, gltBD, mtr (encodes protein for indole import), and acrEF (encodes proteins for indole export). Increased biofilm formation in the yliH and yceP mutants in LB supplemented with 0.2% glucose (LB glu) occurred through a reduction in extracellular and intracellular indole concentrations in both mutants (50- to 140-fold), and the addition of indole to the culture restored the wild-type biofilm phenotype; hence, indole represses biofilms. Additionally, both mutants regulate biofilms through quorum sensing, since deletion of either yliH or yceP increased extracellular autoinducer 2 concentrations 50-fold when grown in complex medium (most notably in the stationary phase). Both proteins are involved in motility regulation, since YliH  (127 amino acids) and YceP (84 amino acids) repressed motility two to sevenfold (P < or = 0.05) in LB, and YceP repressed motility sevenfold (P < or = 0.05) in LB glu. Heightened motility in the yceP mutant occurred, due to increased transcription of the flagella and motility loci, including fliC, motA, and qseB (3- to 86-fold). We propose new names for these two loci: bssR for yliH and bssS  for yceP, based on the phrase "regulator of biofilm through signal secretion." 
16549663	 The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids. 
16540542	 The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37 degrees C. A strain carrying an ntrB(Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the  sole nitrogen source, whereas strains with lesions in the b1012 operon could not. Growth-yield experiments indicated that both nitrogens of uridine and thymidine were available. Studies with [(14)C]uridine indicated that a three-carbon waste product from the pyrimidine ring was excreted. After trimethylsilylation and gas  chromatography, the waste product was identified by mass spectrometry as 3-hydroxypropionic acid. In agreement with this finding, 2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of  available nitrogens and the waste products distinguished the pathway encoded by the b1012 operon from pyrimidine catabolic pathways described previously. We propose that the genes of this operon be named rutA-G for pyrimidine utilization. The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively. 
16418169	 Cyclic diguanylic acid (c-di-GMP; cGpGp) is a global second messenger controlling motility and adhesion in bacterial cells. Intracellular concentrations of c-di-GMP depend on two opposite activities: diguanylate cyclase, recently assigned to the widespread GGDEF domain, and c-di-GMP-specific phosphodiesterase, associated with proteins harboring the EAL domain. To date, little is known about the targets of c-di-GMP in the cell or if it affects transcriptional regulation of certain genes. In order to expand our knowledge of the effect of this molecule on the bacterial metabolism, here we report on the Escherichia coli transcriptional profile under high levels of c-di-GMP. We show that an important  number of genes encoding cell surface and membrane-bound proteins are altered in  their transcriptional activity. On the other hand, genes encoding several transcriptional factors, such as Fur, RcsA, SoxS, and ZraR, are up-regulated, and others, such as GadE, GadX, GcvA, and MetR, are down-regulated. Transcription of  motility and cell division genes were altered, and consistent with this was the physiological analysis of cells overexpressing yddV, a diguanylate cyclase; these cells displayed an abnormal cell division process when high levels of c-di-GMP were present. We also show evidence that the diguanylate cyclase gene yddV is co-transcribed with dos, a heme base oxygen sensor with c-di-GMP-specific phosphodiesterase activity. A delta dos::kan mutation rendered the cells unable to divide properly, suggesting that dos and yddV may be part of a fine-tuning mechanism for regulating the intracellular levels of c-di-GMP. 
16319065	 The ability of biological systems to adapt to genetic and environmental perturbations is a fundamental but poorly understood process at the molecular level. By quantifying metabolic fluxes and global mRNA abundance, we investigated the genetic and metabolic mechanisms that underlie adaptive evolution of four metabolic gene deletion mutants of Escherichia coli (delta pgi, delta ppc, delta  pta, and delta tpi) in parallel evolution experiments of each mutant. The initial response to the gene deletions was flux rerouting through local bypass reactions  or normally latent pathways. The principal effect of evolution was improved capacity of already active pathways, whereas new flux distributions were not observed. Combinatorial changes in capacity and pathway activation, however, led  to different intracellular flux states that enabled evolution in three of the four parallel cases tested. The molecular bases of the evolved phenotypes were then elucidated by global mRNA transcript analyses. Activation of latent pathways and flux changes in the tricarboxylic acid cycle were found to correlate well with molecular changes at the transcriptional level. Flux alterations in other central metabolic pathways, in contrast, were apparently not connected to changes in the transcriptional network. These results give new insight into the dynamics  of the evolutionary process by demonstrating the flexibility of the metabolic network of E. coli to compensate for genetic perturbations and the utility of combining multiple high throughput data sets to differentiate between causal and  noncausal mechanistic changes. 
16539702	 BACKGROUND: Previous research has identified the potential for the existence of two separate lineages of Escherichia coli O157:H7. Clinical isolates tended to cluster primarily within one of these two lineages. To determine if there are virulence related genes differentially expressed between the two lineages we chose to utilize microarray technology to perform an initial screening. RESULTS: Using a 610 gene microarray, designed against the E. coli O157 EDL 933 transcriptome, targeting primarily virulence systems, we chose 3 representative Lineage I isolates (LI groups mostly clinical isolates) and 3 representative Lineage II isolates (LII groups mostly bovine isolates). Using standard dye swap  experimental designs, statistically different expression (P < 0.05) of 73 genes between the two lineages was revealed. Result highlights indicate that under in vitro anaerobic growth conditions, there is up-regulation of stx2b, ureD, curli (csgAFEG), and stress related genes (hslJ, cspG, ibpB, ibpA) in Lineage I, which  may contribute to enhanced virulence or transmission potential. Lineage II exhibits significant up-regulation of type III secretion apparatus, LPS, and flagella related transcripts. CONCLUSION: These results give insight into comparative regulation of virulence genes as well as providing directions for future research. Ultimately, evaluating the expression of key virulence factors among different E. coli O157 isolates has inherent value and the interpretation of such expression data will continue to evolve as our understanding of virulence, pathogenesis and transmission improves. 
16513753	 RNA levels of flagellar genes in eight different genetic backgrounds were compared to that of the wild type by DNA microarray analysis. Cluster analysis identified new, potential flagellar genes, three putative methyl-accepting chemotaxis proteins, STM3138 (McpA), STM3152 (McpB), and STM3216(McpC), and a CheV homolog, STM2314, in Salmonella, that are not found in Escherichia coli. Isolation and characterization of Mud-lac insertions in cheV, mcpB, mcpC, and the previously uncharacterized aer locus of S. enterica serovar Typhimurium revealed  them to be controlled by sigma28-dependent flagellar class 3 promoters. In addition, the srfABC operon previously isolated as an SsrB-regulated operon clustered with the flagellar class 2 operon and was determined to be under FlhDC  control. The previously unclassified fliB gene, encoding flagellin methylase, clustered as a class 2 gene, which was verified using reporter fusions, and the fliB transcriptional start site was identified by primer extension analysis. RNA  levels of all flagellar genes were elevated in flgM or fliT null strains. RNA levels of class 3 flagellar genes were elevated in a fliS null strain, while deletion of the fliY, fliZ, or flk gene did not affect flagellar RNA levels relative to those of the wild type. The cafA (RNase G) and yhjH genes clustered with flagellar class 3 transcribed genes. Null alleles in cheV, mcpA, mcpB, mcpC, and srfB did not affect motility, while deletion of yhjH did result in reduced motility compared to that of the wild type. 
16553885	 In transcription initiation, all RNA polymerase molecules bound to a promoter have been conventionally supposed to proceed into elongation of transcript. However, for Escherichia coli RNA polymerase, evidence has been accumulated for a view that only its fraction can proceed into elongation and the rest is retained  at a promoter in non-productive form: a pathway branching in transcription initiation. Proteins such as GreA and GreB affect these fractions at several promoters in vitro. To reveal the ubiquitous existence of the branched mechanism  in E. coli, we searched for candidate genes whose transcription decreased by disruption of greA and greB using a DNA array. Among the arbitrarily selected 11  genes from over 100, the atpC, cspA and rpsA passed the test by Northern blotting. The Gre factors activated transcription initiation from their promoters in vitro, and the results demonstrated that the branched mechanism is exploited in vivo regulation. Consistently, decrease in the level of the GreA in an anaerobic stationary condition accompanied a decrease in the levels of transcripts of these genes. 
16377617	 The transcription factor FNR, the regulator of fumarate and nitrate reduction, regulates major changes as Escherichia coli adapts from aerobic to anaerobic growth. In an anaerobic glycerol/trimethylamine N-oxide/fumarate medium, the fnr  mutant grew as well as the parental strain, E. coli K12 MG1655, enabling us to reveal the response to oxygen, nitrate, and nitrite in the absence of glucose repression or artifacts because of variations in growth rate. Hence, many of the  discrepancies between previous microarray studies of the E. coli FNR regulon were resolved. The current microarray data confirmed 31 of the previously characterized FNR-regulated operons. Forty four operons not previously known to be included in the FNR regulon were activated by FNR, and a further 28 operons appeared to be repressed. For each of these operons, a match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 103, and possibly as many as 115, operons. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL, and a  further 41 operons are repressed. The narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP+ strain than in the mutant, and a further  37 were strongly repressed. This is the first report that NarP might function as  a global repressor as well as a transcription activator. The data also revealed possible new defense mechanisms against reactive nitrogen species. 
16417494	 RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives  defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic  growth in the presence or absence of nitrate or nitrite, in non-repressing media  in which both strain MG1655 and an fnr deletion mutant grew at similar rates. Glycerol was used as the non-repressing carbon source and both trimethylamine-N-oxide and fumarate were added as terminal electron acceptors. Microarray data supplemented with bioinformatic data revealed that the FNR (fumarate and nitrate reductase regulator) regulon includes at least 104, and possibly as many as 115, operons, 68 of which are activated and 36 are repressed  during anaerobic growth. A total of 51 operons were directly or indirectly activated by NarL in response to nitrate; a further 41 operons were repressed. Four subgroups of genes implicated in management of reactive nitrogen compounds,  NO and products of NO metabolism, were identified; they included proteins of previously unknown function. Global repression by the nitrate- and nitrite-responsive two-component system, NarQ-NarP, was shown for the first time. In contrast with the frdABCD, aspA and ansB operons that are repressed only by NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP. 
16428429	 The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella oneidensis MR-1 is lower by factors of 20 and 200 than those for Escherichia coli and Deinococcus radiodurans, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to UV radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis and the absence of differential regulation of tricarboxylic acid cycle activity, which occurs in IR-irradiated D. radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes  in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR, considering that MR-1 carries genes encoding a complex set of  DNA repair systems and 40 Gy IR induces less than one double-strand break in its  genome. Instead, a combination of oxidative stress, protein damage, and prophage-mediated cell lysis during irradiation and recovery might underlie this  organism's great sensitivity to IR. 
16282532	 Mast cells, which are associated with T helper cell type 2-dependent inflammation, have now been implicated in the innate immune response. To further  characterize how mast cells are programmed to respond to infectious organisms, we used expression profiling using DNA microarray analysis of gene expression by human mast cells (huMC) during ingestion of Escherichia coli and examined immunoglobulin E (IgE)-mediated degranulation. Analysis of data revealed that specific groups of genes were modulated, including genes encoding transcription factors, cell signaling molecules, cell cycle regulators, enzymes, cytokines, novel chemokines of the CC family, adhesion molecules, and costimulatory molecules. Enzyme-linked immunosorbent assay analysis confirmed the production of tumor necrosis factor and the chemokines CC chemokine ligand (CCL)-1/I-309, CCL-19/macrophage-inflammatory protein-3beta (MIP-3beta), and CCL-18/MIP-4; flow  cytometry confirmed the up-regulation of carcinoembryonic antigen-related cell adhesion molecule 1, the integrin CD49d, and CD80. Coincubation with E. coli down-regulated Fc receptor for IgE I (FcepsilonRI) expression and FcepsilonRI-mediated huMC degranulation. These data are consistent with the concept that bacterial exposure directs mast cell responses toward innate immunity and away from IgE-mediated effects. 
16257064	 We report the first identification, gene cloning, recombinant expression and biochemical characterization of an angiotensin converting enzyme (ACE) related dipeptidylcarboxypeptidase (DCP) in a protozoan parasite. The mammalian counterpart of this enzyme, peptidyl dipeptidase A (a carboxyl dipeptidase) also  known as ACE leads to the cleavage of angiotensin I to produce a potent vasopressor. The catalytic enzyme activity of its Escherichia coli DCP counter part can be inhibited by the antihypertensive drug captopril, suggesting that this class of enzymes constitutes a novel target for drugs and vaccines. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding a DCP enzyme for the kinetoplast protozoan Leishmania donovani (LdDCP) that was differentially expressed in promastigote and amastigote stages of the parasite life cycle. Both RNA and protein levels of LdDCP are higher in axenic amastigotes compared to promastigotes. Immuno-fluorescence analysis revealed the  cytosolic expression of the protein. Primary structure analysis of LdDCP revealed the presence of an active Zn binding site. When expressed in E. coli, the recombinant enzyme showed carboxy-dipeptidase activity with synthetic substrates. Replacement of two histidine and one glutamic acid at positions 466, 470 and 467, respectively, with alanine residues in its active site resulted in loss of enzyme activity. Captopril, an ACE specific inhibitor was able both to reduce significantly LdDCP enzyme activity and to inhibit promastigote growth. Both its  cytosolic location and close homology to DCPs from bacterial species suggests a role in parasite nutrition. Further, identification of LdDCP now provides an opportunity to investigate Leishmania peptidases for their potential as drug and  vaccine targets. 
16401087	 Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate  determination of secondary structure. 
16368479	 A novel approach for extracting living cells' genomic DNA materials utilizing functionalized magnetic particles (MPs) is reported in this investigation. This strategy is amenable to handle bio-samples in a miniaturized environment and it offers a possibility to separate and purify DNA from other cell lysate mixtures "on-chip", which is known to be a bottle-neck step in an integrated micro-total-analysis-system (muTAS). "Species-specific" genomic DNA of interest is captured by the MPs based on the hybridization interaction between the biotinylated probes modified MPs and a complementary region of the targeted genome. The genome DNA anchored on the particles can be separated from the rest of cellular mixtures by a simple buffer washing upon the exertion of external magnetic force. Surface modifications of MPs and hybridization conditions affecting the genome capturing efficiency are investigated. Extraction of genomic DNA from E. coli is demonstrated in a silicon/glass-based micro-reactor patterned with a platinum heater and sensors. On-chip extraction and manipulation of genomic DNAs illustrated in this study is a step forward toward a total integrated bioanalytical microsystem for crude cells/sample analysis. 
15951163	 A quartz crystal microbalance (QCM) DNA sensor, based on the nanoparticle amplification method, was developed for detection of Escherichia coli O157:H7. A  thiolated single-stranded DNA (ssDNA) probe specific to E. coli O157:H7 eaeA gene was immobilized onto the QCM sensor surface through self-assembly. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA, and resulted in the mass change and therefore frequency change of the QCM. Streptavidin conjugated Fe(3)O(4) nanoparticles (average diameter=145 nm) were used as "mass enhancers" to amplify the frequency change. Synthesized biotinylated oligonucleotides as well as E. coli O157:H7 eaeA gene fragments (151 bases) amplified using asymmetric PCR with biotin labeled primers were tested. As low as 10(-12)M synthesized oligonucleotides and 2.67 x 10(2) colony forming unit (CFU)/ml E. coli O157:H7 cells can be detected by the sensor. Linear correlation  between frequency change and logarithmic number of bacterial cell concentration was found for E. coli O157:H7 from 2.67 x 10(2) to 2.67 x 10(6)CFU/ml. 
16391141	 Invasion of intestinal epithelial cells by Salmonella enterica is decreased after exposure to butyric acid. To understand the molecular mechanisms of this phenomenon, a comparative transcriptomic analysis of Salmonella enterica serovar  Enteritidis and Salmonella enterica serovar Typhimurium grown in medium supplemented with butyrate was performed. We found that butyrate down-regulated the expression of 19 genes common to both serovars by a factor of twofold or more, and 17 of these genes localized to the Salmonella pathogenicity island 1 (SPI1). These included the SPI1 regulatory genes hilD and invF. Of the remaining  two genes, ampH has 91% homology to an Escherichia coli penicillin-binding protein and sopE2 encodes a type III-secreted effector protein associated with invasion but located at a separate site on the chromosome from SPI1. 
16392005	 DNA microarrays were employed to compare gene expression in a thermotolerant, nalidixic acid-resistant mutant of Escherichia coli with that of the parental strain. When grown at 37 degrees C, up-regulated genes in the mutant included those coding for multiple antibiotic resistance proteins and enzymes for the degradation of small molecules, whereas among the down-regulated genes were those coding for fimbrial, flagellar, and outer membrane proteins as well as sigma 38.  When the mutant grown at 42 degrees C was compared to the mutant grown at 37 degrees C, enhanced expression of several genes coding for flagellar proteins was detected. Reverse transcriptase-polymerase chain reaction analysis of selected genes confirmed results obtained with microarrays. 
16626502	 Genome-wide surveys of transcription depend on gene classifications for the purpose of data interpretation. We propose a new information-theoretical-based method to: assess significance of co-expression within any gene group; quantitatively describe condition-specific gene-class activity; and systematically evaluate conditions in terms of gene-class activity. We applied this technique to describe microarray data tracking Escherichia coli transcriptional responses to more than 30 chemical and physiological perturbations. We correlated the nature and breadth of the responses with the nature of perturbation, identified gene group proxies for the perturbation classes and quantitatively compared closely related physiological conditions. 
16344567	 We developed a new microscopic platform for the real-time analysis of molecular interactions by combining microbead-tagging techniques with total internal reflection fluorescent microscopy (TIRFM). The optical manipulation of probe microbeads, followed by photo immobilization on a solid surface, enabled us to generate arrays with extremely high density (>100 microbeads in a 25 microm x 25  microm area), and TIRFM made it possible to monitor the binding reactions of fluorescently labeled targets onto probe microbeads without removal of free targets. We demonstrated the high performance of this platform through analyses of interactions between antigen and antibody and between small compounds and proteins. Then, recombinant protein levels in total cellular lysates of Escherichia coli were quantified from the association kinetics using antibody-immobilized microbead arrays, which served as a model for a protein-profiling array. Furthermore, in combination with in vitro synthesis-coupled protein labeling, we could kinematically analyze the interaction of nuclear factor kappaB (p50) with DNA. These results demonstrated that this platform enabled us to: (1) monitor binding processes of fluorescently  labeled targets to multiple probes in real-time without removal of free targets,  (2) determine concentrations of free targets only from the association kinetics at an early phase, and (3) greatly reduce the required volume of the target solution, in principle to subnanoliter, for molecular interaction analysis. The unique features of this microbead-based microarray system open the way to explore molecular interactions with a wide range of affinities in extremely small volumes of target solutions, such as extracts from single cells. 
16938559	 A large variety of proteins bind to specific parts of the genome to regulate gene expression, DNA replication, and chromatin structure. DamID is a powerful method  used to map the genomic interaction sites of these proteins in vivo. It is based  on fusing a protein of interest to Escherichia coli DNA adenine methyltransferase (dam). Expression of this fusion protein in vivo leads to preferential methylation of adenines in DNA surrounding the native binding sites of the dam fusion partner. Because adenine methylation does not occur endogenously in most eukaryotes, it provides a unique tag to mark protein interaction sites. The adenine-methylated DNA fragments are isolated by selective polymerase chain reaction amplification and can be identified by microarray hybridization. We and  others have successfully applied DamID to the genome-wide identification of interaction sites of several transcription factors and other chromatin-associated proteins. This chapter discusses DamID technology in detail, and a step-by-step experimental protocol is provided for use in Drosophila cell lines. 
17040898	 Polyadenylation of RNAs by poly(A) polymerase I (PAP I) in Escherichia coli plays a significant role in mRNA decay and general RNA quality control. However, many important features of this system, including the prevalence of polyadenylated mRNAs in the bacterium, are still poorly understood. By comparing the transcriptomes of wild-type and pcnB deletion strains using macroarray analysis,  we demonstrate that >90% of E.coli open reading frames (ORFs) transcribed during  exponential growth undergo some degree of polyadenylation by PAP I, either as full-length transcripts or decay intermediates. Detailed analysis of over 240 transcripts suggests that Rho-independent transcription terminators serve as polyadenylation signals. Conversely, mRNAs terminated in a Rho-dependent fashion  are probably not substrates for PAP I, but can be modified by the addition of long polynucleotide tails through the biosynthetic activity of polynucleotide phosphorylase (PNPase). Furthermore, real-time PCR analysis indicates that the extent of polyadenylation of individual full-length transcripts such as lpp and ompA varies significantly in wild-type cells. The data presented here demonstrates that polyadenylation in E.coli occurs much more frequently than previously envisioned. 
16963779	 The Escherichia coli chromosome is condensed into an ill-defined structure known  as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase. 
16351151	 In this study, we describe a straightforward strategy to develop whole cell-based biosensors using fusions of the bacterial bioluminescence genes and the promoters from chemically responsive genes within Escherichia coli, in which chemical target-responsive genes were screened by using the information of gene expression data obtained from DNA microarray analysis. Paraquat was used as a model chemical to trigger gene expression changes of E. coli and to show the DNA microarray-assisted development of whole cell-based biosensors. Gene expression data from the DNA microarray were obtained by time course analysis (10, 30, and 60 min) after exposure to paraquat. After clustering gene expression data obtained by time course analysis, a group of highly expressed genes over the all  time courses could be classified. Within this group, three genes expressed highly for overall time points were selected and promoters of these genes were used as fusion partners with reporter genes, lux CDABE, to construct whole cell-based biosensors. The constructed biosensors recognized the presence of model inducer,  paraquat, and structural analogue chemicals of paraquat with a high specificity,  and the results were reconfirmed by using DNA microarray experiments for those structural analogues. This strategy to develop whole cell-based biosensors assisted by DNA microarray information should be useful in general for constructing chemical-specific or stress-specific biosensors with a high-throughput manner. 
16336117	 Small non-coding RNAs (sRNAs) have attracted considerable attention as an emerging class of gene expression regulators. In bacteria, a few regulatory RNA molecules have long been known, but the extent of their role in the cell was not  fully appreciated until the recent discovery of hundreds of potential sRNA genes  in the bacterium Escherichia coli. Orthologs of these E. coli sRNA genes, as well as unrelated sRNAs, were also found in other bacteria. Here we review the disparate experimental approaches used over the years to identify sRNA molecules  and their genes in prokaryotes. These include genome-wide searches based on the biocomputational prediction of non-coding RNA genes, global detection of non-coding transcripts using microarrays, and shotgun cloning of small RNAs (RNomics). Other sRNAs were found by either co-purification with RNA-binding proteins, such as Hfq or CsrA/RsmA, or classical cloning of abundant small RNAs after size fractionation in polyacrylamide gels. In addition, bacterial genetics  offers powerful tools that aid in the search for sRNAs that may play a critical role in the regulatory circuit of interest, for example, the response to stress or the adaptation to a change in nutrient availability. Many of the techniques discussed here have also been successfully applied to the discovery of eukaryotic and archaeal sRNAs. 
16321939	 The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of  the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2  as a true signal molecule for E. coli W3110 under the investigated conditions. 
16339806	 Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one functional proteins with a UBC domain and active-site cysteine are predicted in the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are predicted or shown to function with ubiquitin-like proteins. Only nine were previously characterized biochemically as ubiquitin E2s. We obtained soluble protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three previously characterized ubiquitin E2s, were also tested for the ability to catalyze ubiquitination in vitro in the presence of one of 65 really interesting  new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and  with all RING types. Expression analysis was performed to determine whether E2s or E3s were expressed in specific organs or under specific environmental conditions. Closely related E2s show unique patterns of expression and most express ubiquitously. Some RING E3s are also ubiquitously expressed; however, others show organ-specific expression. Of all the organs tested, RING mRNAs are most abundant in floral organs. This study demonstrates that E2 diversity includes examples with broad and narrow specificity toward RINGs, and that most ubiquitin E2s are broadly expressed with each having a unique spatial and developmental pattern of expression. 
16267290	 The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA,  which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage.  To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5'-CGAACATATGTTCG-3'. Although the number of genes controlled  by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli. 
16175260	 We have directly integrated a continuous-flow, electrokinetic method of bacterial cell concentration with room temperature, sequence-specific genetic detection. The system we have developed traps cells from a continuous-flow sample stream via dielectrophoresis, providing a 160-fold increase in cell concentration. PDMS microvalves then define a 100 nL volume around the trapped cells to which cell lysis buffer and genetic detection components are introduced. Direct, optical detection of Escherichia coli MC1061 cells is then accomplished via the sequence-specific hybridization of an rRNA-directed optical molecular beacon. This integrated microsystem is capable of sequence-specific genetic detection of  25 cells within 30 min. 
16207912	 Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and  promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes,  regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF  transition of the fim switch. Enhanced type 1 fimbrial expression as a result of  lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells.  Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also. 
16164569	 While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen-metabolic phosphotransferase system (nitrogen-metabolic PTS). To elucidate the physiological role of the nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first  enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIA(Ntr)) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I(Ntr)) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity  of LCPs was found to be due to leucine and the dephospho-form of enzyme IIA(Ntr)  was found to be necessary to neutralize leucine toxicity. Further studies showed  that the dephospho-form of enzyme IIA(Ntr) is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched-chain amino acids. 
16131593	 The global Csr regulatory system controls bacterial gene expression post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that plays a central role in repressing several stationary phase processes and activating certain exponential phase functions. CsrA regulates translation initiation of several genes by binding to the mRNA leaders and blocking ribosome  binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of sequestering CsrA and antagonizing its activity. Each of the known target transcripts contains multiple CsrA binding sites, although considerable sequence  variation exists among these RNA targets, with GGA being the most highly conserved element. High-affinity RNA ligands containing single CsrA binding sites were identified from a combinatorial library using systematic evolution of ligands by exponential enrichment (SELEX). The SELEX-derived consensus was determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and  the GU sequence being present in all but one ligand. The majority (51/55) of the  RNAs contained GGA in the loop of a hairpin within the most stable predicted structure, an arrangement similar to several natural CsrA binding sites. Strikingly, the identity of several nucleotides that were predicted to form base  pairs in each stem were 100% conserved, suggesting that primary sequence information was embedded within the base-paired region. The affinity of CsrA for  several selected ligands was measured using quantitative gel mobility shift assays. A mutational analysis of one selected ligand confirmed that the conserved ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary structure participates in CsrA-RNA recognition. 
16171339	 The immobilization of novel proteins onto addressable locations on a flat surface has potential applications in a range of biotechnologies. Here we describe the nanopatterning of a de novo protein onto a gold surface. Patterning was achieved  using a technique called nanografting, in which the tip of an atomic force microscope is used to disrupt a preexisting monolayer of alkanethiol molecules on a gold surface, thereby facilitating exchange with alternative thiol-linked molecules from the surrounding solution. The protein used for these studies was chosen from a designed combinatorial library of de novo sequences expressed in E. coli and was engineered to have a glycine-glycine-cysteine tag at its C-terminus, thereby enabling attachment to the gold surface through a single cysteine thiol.  The average height of the grafted protein patterns was found to be somewhat higher than expected from the known NMR structure of the protein. Compression of  the nanografted patches by an external force (below 10 nN) was reversible but showed some hysteresis. Interestingly, both the energy required to deform the immobilized protein patterns and the energy defined by the hysteresis loop were found to be of the same order as the energy required to unfold the monomeric protein in solution. These studies demonstrate the possibility of preparing nanometer scale protein arrays, lowering significantly the volume requirements of the protein samples necessary to fabricate protein-based biosensor arrays and thereby providing a base for increasing their sensitivity. 
16051189	 The overall composition upstream of start codons in Escherichia coli was evaluated and viewed in connection with global transcriptome data. Genes starting with AUG as initiation codon tended to be expressed at higher levels than the non-AUG genes, and the upstream region of the non-AUG genes showed negligible signs of Shine-Dalgarno sequences. The latter is in sharp contrast to the AUG genes. Viewing these findings in connection with the current literature, it is proposed that a distinct mechanism for initiation of translation might exist for  non-AUG genes that are not preceded by a Shine-Dalgarno sequence. A survey covering a range of other eubacteria (Firmicutes, Proteoacteria, and Actinobacteria) reveals that it is mainly among the Proteobacteria that non-AUG genes do not display clear signs of Shine-Dalgarno regions. 
16115199	 Cra (or FruR), a global transcription factor with both repression and activation  activities, controls a large number of the genes for glycolysis and gluconeogenesis. To get insights into the entire network of transcription regulation of the E. coli genome by Cra, we isolated a set of Cra-binding sequences using an improved method of genomic SELEX. From the DNA sequences of 97 independently isolated DNA fragments by SELEX, the Cra-binding sequences were identified in a total of ten regions on the E. coli genome, including promoters of six known genes and four hitherto-unidentified genes. All six known promoters  are repressed by Cra, but none of the activation-type promoters were cloned after two cyles of SELEX, because the Cra-binding affinity to the repression-type promoters is higher than the activation-type promoters, as determined by the quantitative gel shift assay. Of a total of four newly identified Cra-binding sequences, two are associated with promoter regions of the gapA (glyceraldehyde 3-phosphate dehydrogenase) and eno (enolase) genes, both involved in sugar metabolism. The regulation of newly identified genes by Cra was confirmed by the  in vivo promoter strength assay using a newly developed TFP (two-fluorescent protein) vector for promoter assay or by in vitro transcription assay in the presence of Cra protein. 
16113288	 Vibrio cholerae encodes a small RNA with homology to Escherichia coli RyhB. Like  E. coli ryhB, V. cholerae ryhB is negatively regulated by iron and Fur and is required for repression of genes encoding the superoxide dismutase SodB and multiple tricarboxylic acid cycle enzymes. However, V. cholerae RyhB is considerably longer (>200 nucleotides) than the E. coli RNA (90 nucleotides), and it regulates the expression of a variety of genes that are not known to be regulated by RyhB in E. coli, including genes involved in motility, chemotaxis, and biofilm formation. A mutant with a deletion in ryhB had reduced chemotactic motility in low-iron medium and was unable to form wild-type biofilms. The defect in biofilm formation was suppressed by growing the mutant in the presence of excess iron or succinate. The wild-type strain showed reduced biofilm formation in iron-deficient medium, further supporting a role for iron in normal biofilm formation. The ryhB mutant was not defective for colonization in a mouse model and appeared to be at a slight advantage when competing with the wild-type parental strain. Other genes whose expression was influenced by RyhB included those encoding the outer membrane porins OmpT and OmpU, several iron transport systems, and proteins containing heme or iron-sulfur clusters. These data indicate that V. cholerae RyhB has diverse functions, ranging from iron homeostasis to the regulation of biofilm formation. 
16109946	 The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus  strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One  ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I  footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family  involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms. 
16135230	 Chromosomes are divided into topologically independent regions, called domains, by the action of uncharacterized barriers. With the goal of identifying domain barrier components, we designed a genetic selection for mutants with reduced negative supercoiling of the Escherichia coli chromosome. We employed a strain that contained two chromosomally located reporter genes under the control of a supercoiling-sensitive promoter and used transposon mutagenesis to generate a wide range of mutants. We subjected the selected mutants to a series of secondary screens and identified five proteins as modulators of chromosomal supercoiling in vivo. Three of these proteins: H-NS, Fis and DksA, have clear ties to chromosome  biology. The other two proteins, phosphoglucomutase (Pgm) and transketolase (TktA), are enzymes involved in carbohydrate metabolism and have not previously been shown to affect DNA. Deletion of any of the identified genes specifically affected chromosome topology, without affecting plasmid supercoiling. We suggest  that at least H-NS, Fis and perhaps TktA assist directly in the supercoiling of domains by forming topological barriers on the E. coli chromosome. 
16041320	 Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells  that lack datA was indistinguishable from that in wild-type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow-cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle. 
15967361	 Three MutS fusion systems were designed as the mutation recognition and signal elements of DNA chips for detection of DNA mutations. The expression vectors containing the encoding sequences of three recombinant proteins, Trx-His6-GFP-(Ser-Gly)6-MutS (THGLM), Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM) and Trx-His6-(Ser-Gly)6-MutS (THLM), were constructed by gene slicing in vitro. THGLM, THLSLM and THLM were then expressed  in Escherichia coli AD494(DE3), respectively. SDS-PAGE analysis revealed that each of the expected proteins was approximately 30% of the total bacterial proteins. The recombinant proteins were purified to the purity over 90% by immobilized metal (Co2+) chelation affinity chromatography. Bioactivity assay indicated that three fusion proteins retained the mismatch-binding activity and the functions of other fusion partners. DNA chips arrayed both mismatched and unpaired DNA oligonucleotides as well as rpoB gene from Mycobacterium tuberculosis were prepared. THGLM, THLSLM and THLM that was labeled with Fluorolinktrade mark Cy3 reactive dye, were then used as both mutation recognition and labeling elements of DNA chips. The resulting DNA chips were used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides and single base mutation in rpoB gene of M. tuberculosis that is resistant to rifamycin. 
16000839	 Transcriptional analysis was used to examine the effect of a genomically encoded  artificial RNA on Escherichia coli in rich and minimal media. Only the expression of a single gene, deoC, was unequivocally affected under both conditions. E. coli marker strains of this type may be useful in monitoring the fate and transport of bacteria in various applications. 
16000817	 After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 microg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 microg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 microg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and  yrfHI). There were 31 and 17 genes repressed by 10 and 30 microg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid). 
15955309	 Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed. 
15995204	 Hierarchical control ensures that facultative bacteria preferentially use the available respiratory electron acceptor with the most positive standard redox potential. Thus, nitrate is used before other electron acceptors such as fumarate for anaerobic respiration. Nitrate regulation is mediated by the NarX-NarL two-component system, which activates the transcription of operons encoding nitrate respiration enzymes and represses the transcription of operons for other  anaerobic respiratory enzymes, including enzymes involved in fumarate respiration. These are fumarate reductase (encoded by the frdABCD operon), fumarase B, which generates fumarate from malate, and the DcuB permease for fumarate, malate, and aspartate. The transcription of the corresponding structural genes is activated by the DcuS-DcuR two-component system in response to fumarate or its dicarboxylate precursors. We report results from preliminary transcription microarray experiments that revealed two previously unknown members of the NarL regulon: the aspA gene encoding aspartate-ammonia lyase, which generates fumarate; and the dcuSR operon encoding the dicarboxylate-responsive regulatory system. We measured beta-galactosidase expression from monocopy aspA-lacZ, frdA-lacZ, and dcuS-lacZ operon fusions in response to added nitrate and fumarate and with respect to the dcuR and narL genotypes. Nitrate, acting through the NarX-NarL regulatory system, repressed the transcription of all three operons. Only frdA-lacZ expression, however, was responsive to added fumarate or  a dcuR(+) genotype. Phospho-NarL protein protected operator sites in the aspA and dcuS promoter regions from DNase I cleavage in vitro. The overall results are consistent with the hypothesis that nitrate represses frdA operon transcription not only directly, by repressing frdA promoter activity, but also indirectly, by  repressing dcuS promoter activity. 
16002309	 Interaction among different signalling pathways has been noted repeatedly. However, no systematic method has been developed to identify and quantify such interactions. Here we reported that network component analysis (NCA) was able to  determine interactions among various signalling pathways in Escherichia coli K-12 based on known transcription factor (TF)-promoter connectivity information and microarray data from genetic knockout strains. The TF activities determined from  NCA allow the quantitation of functional interactions, barring gross errors in the connectivity and microarray data. By using a robust statistical test, 37 pairs of functional interactions were identified. Eighteen interaction pairs confirmed previous implications, while 19 others represent new predictions. These results demonstrate that the functional interactions among various signalling pathways may be rather significant. With reasonable TF-promoter connectivity, NCA coupled with genetic knockouts and microarray experiments provides a systematic way to elucidate interaction networks. As this approach cannot distinguish between cross-talks and unidentified direct regulation, the results should provide incentives for further experimental testing. 
15768050	 The etiology of schizophrenia is unclear, although family, twin, and linkage studies implicate genetic factors. Here, we identified adenomatous polyposis coli (APC), a tumor suppressor gene, as a risk factor for schizophrenia. We compared leukocytic gene expression patterns of six pairs of patients with schizophrenia and healthy controls by microarray. APC expression levels were significantly increased in all patients compared to healthy controls. To confirm the findings of microarray analysis, we measured expression levels of APC in the leukocytes from 30 relapse patients taking antipsychotic medication, 29 first-episode drug-naïve patients, and 30 healthy controls using real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). APC expression levels were significantly increased in leukocytes of schizophrenics both taking and not taking antipsychotic medication and hence the increase of APC expression was not  due to antipsychotic medication. APC is located at 5q21-22, which has been previously reported to be linked with schizophrenia. Further, we performed the transmission disequilibrium test (TDT) and TDT based on haplotypes to search for  the association between schizophrenia and APC by examining 163 parent-offspring trios of Chinese descent. We analyzed three single-nucleotide polymorphisms (SNPs) (rs2229992, rs42427, rs465899) at the exon region of APC. TDT showed that  the three SNPs are significantly associated with schizophrenia (TDT chi(2)=4.23,  P<0.05; 4.15, P<0.05; 8.49 P<0.01, respectively; HHRRchi(2)=5.54, P<0.05; 4.40, P<0.05; 9.79, P<0.01, respectively). We found a significant association between the APC haplotypes from rs2229992-rs42427-rs465899 and schizophrenia (Global chi(2)=44.376,df=7, P<0.001). The C-A-T haplotype has a frequency of more than 57% and has a strong association with schizophrenia (chi(2)=15.04, P<0.001). These results indicate that the APC may be a candidate gene conferring susceptibility to schizophrenia and also may be associated with reduced vulnerability to cancer in schizophrenia. 
15965020	 Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray  analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced  the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat  shock genes. In addition, the cells with a mutant gene for Hik34 (DeltaHik34 cells) survived incubation at 48 degrees C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44 degrees C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in DeltaHik34 cells after incubation of cells at 42 degrees C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified  it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44 degrees C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis. 
15806547	 In a series of previous reports it was established by implementing metabolic flux, NMR/MS, and Northern blot analysis that the glyoxylate shunt, the TCA cycle, and acetate uptake by acetyl-CoA synthetase are more active in Escherichia coli BL21 than in Escherichia coli JM109. These differences were accepted as the  reason for the differences in the glucose metabolism and acetate excretion of these two strains. Examination of the bacterial metabolism by microarrays and time course Northern blot showed that in addition to the glyoxylate shunt, the TCA cycle and the acetate uptake, other metabolic pathways are active differently in the two strains. These are gluconeogenesis, sfcA shunt, ppc shunt, glycogen biosynthesis, and fatty acid degradation. It was found that in E. coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses  acetyl-CoA. The inactivation of the gluconeogenesis enzyme phosphoenolpyruvate synthetase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF) cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs, and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate  concentration increased, but at low glucose concentrations these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis pathway (fbp, pckA, and ppsA) which leads to glycogen accumulation is constitutively active in E. coli BL21 regardless of glucose feeding strategy. 
15963225	 BACKGROUND: DNA microarray technology is widely used to determine the expression  levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus. RESULTS: We describe an efficient procedure for selecting 40-60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting  standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling. CONCLUSION: This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions. 
15938745	 BACKGROUND: Although the organisation of the bacterial chromosome is an area of active research, little is known yet on that subject. The difficulty lies in the  fact that the system is dynamic and difficult to observe directly. The advent of  massive hybridisation techniques opens the way to further studies of the chromosomal structure because the genes that are co-expressed, as identified by microarray experiments, probably share some spatial relationship. The use of several independent sets of gene expression data should make it possible to obtain an exhaustive view of the genes co-expression and thus a more accurate image of the structure of the chromosome. RESULTS: For both Bacillus subtilis and Escherichia coli the co-expression of genes varies as a function of the distance between the genes along the chromosome. The long-range correlations are surprising: the changes in the level  of expression of any gene are correlated (positively or negatively) to the changes in the expression level of other genes located at well-defined long-range distances. This property is true for all the genes, regardless of their localisation on the chromosome. We also found short-range correlations, which suggest that the location of these co-expressed genes corresponds to DNA turns on the nucleoid surface (14-16 genes). CONCLUSION: The long-range correlations do not correspond to the domains so far identified in the nucleoid. We explain our results by a model of the nucleoid solenoid structure based on two types of spirals (short and long). The long spirals are uncoiled expressed DNA while the short ones correspond to coiled unexpressed DNA. 
15797912	 MOTIVATION: Function annotation of an unclassified protein on the basis of its interaction partners is well documented in the literature. Reliable predictions of interactions from other data sources such as gene expression measurements would provide a useful route to function annotation. We investigate the global relationship of protein-protein interactions with gene expression. This relationship is studied in four evolutionarily diverse species, for which substantial information regarding their interactions and expression is available: human, mouse, yeast and Escherichia coli. RESULTS: In E.coli the expression of interacting pairs is highly correlated in comparison to random pairs, while in the other three species, the correlation of  expression of interacting pairs is only slightly stronger than that of random pairs. To strengthen the correlation, we developed a protocol to integrate ortholog information into the interaction and expression datasets. In all four genomes, the likelihood of predicting protein interactions from highly correlated expression data is increased using our protocol. In yeast, for example, the likelihood of predicting a true interaction, when the correlation is > 0.9, increases from 1.4 to 9.4. The improvement demonstrates that protein interactions are reflected in gene expression and the correlation between the two is strengthened by evolution information. The results establish that co-expression of interacting protein pairs is more conserved than that of random ones. 
15882420	 Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription  factor belonging to the cAMP receptor protein (CRP) family caused growth defects  in laboratory medium, in bone marrow-derived macrophages and in a mouse model of  tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA  promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections. 
15927055	 BACKGROUND: Transcription is the first step in cellular information processing. It is regulated by cis-acting elements such as promoters and operators in the DNA, and trans-acting elements such as transcription factors and sigma factors. Identification of cis-acting regulatory elements on a genomic scale requires computational analysis. RESULTS: We have used oligonucleotide profiling to predict regulatory regions in  a bacterial genome. The method has been applied to the Escherichia coli K12 genome and the results analyzed. The information content of the putative regulatory oligonucleotides so predicted is validated through intra-genomic analyses, correlations with experimental data and inter-genome comparisons. Based on the results we have proposed a model for the bacterial promoter. The results show that the method is capable of identifying, in the E.coli genome, cis-acting  elements such as TATAAT (sigma70 binding site), CCCTAT (1 base relative of sigma32 binding site), CTATNN (LexA binding site), AGGA-containing hexanucleotides (Shine Dalgarno consensus) and CTAG-containing hexanucleotides (core binding sites for Trp and Met repressors). CONCLUSION: The method adopted is simple yet effective in predicting upstream regulatory elements in bacteria. It does not need any prior experimental data except the sequence itself. This method should be applicable to most known genomes. Profiling, as applied to the E.coli genome, picks up known cis-acting and regulatory elements. Based on the profile results, we propose a model for the bacterial promoter that is extensible even to eukaryotes. The model is that the core promoter lies within a plateau of bent AT-rich DNA. This bent DNA acts as a  homing segment for the sigma factor to recognize the promoter. The model thus suggests an important role for local landscapes in prokaryotic and eukaryotic gene regulation. 
15808307	 Through the process of phagocytosis, the macrophage is responsible for the clearance and destruction of both intracellular and extracellular pathogens. When stimulated, macrophages undergo a process of activation involving an increase in  size and motility, enhanced phagocytic, bactericidal, and tumoricidal activity, and up-regulation of several cell-surface markers. One well characterized method  of mammalian macrophage activation involves the Toll-like receptor (TLR) pathway. TLRs are surface molecules that function as direct receptors for microbial components. Binding of ligand to TLRs results in activation of transcription factors that regulate genes involved in microbial killing, apoptosis, and antigen recognition, as well as pro- and anti-inflammatory cytokines and chemokines. We have constructed a 4906-element (14,718 spot) avian macrophage-specific cDNA microarray (AMM). The AMM contains 16 of the approximately 44 genes identified within the mammalian TLR pathway. This array was used to examine the transcriptional response of avian macrophages to Gram-negative bacteria and their cell wall components and to evaluate the contribution of the avian TLR pathway to that response. Of the elements on the AMM, 981 (20%) exhibited significant (greater than two-fold, p < 0.01) changes in expression during phagocytosis of Escherichia coli and 243 (5%) exhibited significant expression changes during exposure to lipopolysaccharide (LPS). A unique set of overlapping elements (154), were observed to exhibit significant changes in expression for both phagocytosis  and LPS stimulation, representing a set of core response elements. Of these elements, 63% were commonly induced, while 32% were commonly repressed. Both LPS  and bacteria were found to induce NFkappabeta and several end products of the TLR pathway. 
15870370	 DNA microarray analysis showed that yfiD, yggB, and yggE genes were up-regulated  when superoxide dismutase (SOD)-deficient Escherichia coli IM303 (I4) was cultivated under the oxidative stress generated by photoexcited TiO(2), and pYFD, pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19 vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the culture of wild-type strain, E. coli MM294, in the medium with paraquat (10 micromol/l), maximum specific growth rate of the cells with pYGE was about five times higher than that of the control cells, with a decreased ROS content in the  former cells. The introduction of pYGE also suppressed the occurrence of the cells with altered amino acid requirement in the culture of MM294 cells with paraquat. 
15932244	 Temperature shift is often practiced in the cultivation of Escherichia coli to reduce undesired metabolite formation and to maximize synthesis of correctly folded heterologous protein. As the culture temperature is decreased below the optimal 37 degrees C, growth rate decreases and many physiological changes occur. In this study, we investigated the gene expression dynamics of E. coli on switching its cultivation temperature from 37 to 33 and 28 degrees C using whole  genome DNA microarrays. Approximately 9% of the genome altered expression level on temperature shift. Overall, the alteration of transcription upon the downshift of temperature is rapid and globally distributed over a wide range of gene classes. The general trends of transcriptional changes at 28 and 33 degrees C were similar. The largest functional class among the differentially expressed genes was energy metabolism. About 12% of genes in energy metabolism show a decrease in their level of expression, and approximately 6% show an increase. Consistent with the decrease in the glucose uptake rate, many genes involved in glycolysis and the PTS sugar transport systems show decreased expression. Genes encoding enzymes related to amino acid biosynthesis and transport also have reduced expression levels. Such decrease in expression probably reflects the reduced growth rate and the accompanying reduction in energy and amino acid demand at lower temperatures. However, nearly all genes encoding enzymes in the TCA cycle have increased expression levels, which may well be compensating the reduction of the activity of TCA cycle enzymes at lower temperatures. Temperature shift also results in shift of the cytochromes from the high affinity cytochrome  o system to the low affinity cytochrome d system. There is no evidence that protein processing genes are selectively altered to create favorable conditions for heterologous protein synthesis. Our results indicate that the beneficial effect of temperature shift in many biotechnological processes is likely to be attributed to the general effect of reduced growth and metabolism. 
15845502	 SoxR is a transcriptional regulator that controls an oxidative stress response in Escherichia coli. The regulator is primarily activated by superoxide anion-dependent oxidation. Activated SoxR turns on transcription of a single gene, soxS, which encodes a transcriptional regulator that activates a regulon that includes dozens of oxidative stress response genes. SoxR homologues have been identified in many bacterial species, including the opportunistic pathogen Pseudomonas aeruginosa. However, the expected SoxR partner, SoxS, has not been found in P. aeruginosa. Thus, the primary gene target(s) of P. aeruginosa SoxR is unknown and the involvement of this regulator in the oxidative stress response of the bacterium remains unclear. We utilized transcriptome profiling to identify the P. aeruginosa SoxR regulon and constructed and characterized an unmarked P. aeruginosa DeltasoxR mutant. We provide evidence indicating that P. aeruginosa SoxR activates a six-gene regulon in response to O(2)(.-)-induced stress. The regulon includes three transcriptional units: (i) the recently identified mexGHI-ompD four-gene operon, which encodes a multidrug efflux pump system involved in quorum-sensing signal homeostasis; (ii) gene PA3718, encoding a probable efflux pump; and (iii) gene PA2274, encoding a probable monooxygenase. We also demonstrate that P. aeruginosa SoxR is not a key regulatory player in the oxidative stress response. Finally, we show that P. aeruginosa SoxR is required for virulence in a mouse model of intrapulmonary infection. These results demonstrate that the E. coli-based SoxRS paradigm does not hold in P. aeruginosa  and foster new hypotheses for the possible physiological role of P. aeruginosa SoxR. 
15845498	 Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC. 
15838054	 Genome-wide analysis of temporal gene expression profiles in Escherichia coli following exposure to cadmium revealed a shift to anaerobic metabolism and induction of several stress response systems. Disruption in the transcription of  genes encoding ribosomal proteins and zinc-binding proteins may partially explain the molecular mechanisms of cadmium toxicity. 
15887077	 Transcription profiling (quantitative analysis of RNA abundance) can provide a genome-wide picture of gene expression changes that accompany organismal adaptation to a new environment. Here, we used DNA microarrays to characterize genome-wide changes in transcript abundance in three replicate lines of the bacterium E. coli grown for 2,000 generations at a stressful high temperature (41.5 degrees C). Across these lines, 12% of genes significantly changed expression during high-temperature adaptation; the majority of these changes (55%) were less than twofold increments or decrements. Thirty-nine genes, four times the number expected by chance alone, exhibited moderately or highly replicated expression changes across lines. Expression changes within a priori defined functional categories showed an even greater level of replication than did individual genes. Expression changes in the phenotypically defined stress genes and adaptation functional categories were important in evolutionary adaptation to high temperature. 
15699038	 The ArcAB two-component system of Escherichia coli regulates the aerobic/anaerobic expression of genes that encode respiratory proteins whose synthesis is coordinated during aerobic/anaerobic cell growth. A genomic study of E. coli was undertaken to identify other potential targets of oxygen and ArcA regulation. A group of 175 genes generated from this study and our previous study on oxygen regulation (Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield,  G. W., and Gunsalus, R. P. (2003) J. Biol. Chem. 278, 29837-29855), called our gold standard gene set, have p values <0.00013 and a posterior probability of differential expression value of 0.99. These 175 genes clustered into eight expression patterns and represent genes involved in a large number of cell processes, including small molecule biosynthesis, macromolecular synthesis, and aerobic/anaerobic respiration and fermentation. In addition, 119 of these 175 genes were also identified in our previous study of the fnr allele. A MEME/weight matrix method was used to identify a new putative ArcA-binding site for all genes of the E. coli genome. 16 new sites were identified upstream of genes in our gold standard set. The strict statistical analyses that we have performed on our data  allow us to predict that 1139 genes in the E. coli genome are regulated either directly or indirectly by the ArcA protein with a 99% confidence level. 
15736164	 We studied the physiological response of Escherichia coli central metabolism to the expression of heterologous pyruvate carboxylase (PYC) in the presence and absence of pyruvate oxidase (POX). These studies were complemented with expression analysis of central and intermediary metabolic genes and conventional  in vitro enzyme assays to evaluate glucose metabolism at steady-state growth conditions (chemostats). The absence of POX activity reduced nongrowth-related energy metabolism (maintenance coefficient) and increased the maximum specific rate of oxygen consumption. The presence of PYC activity (i.e., with POX activity) increased the biomass yield coefficient and reduced the maximum specific oxygen consumption rate compared to the wildtype. The presence of PYC in a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater biomass yield compared to the wildtype. Providing E. coli with PYC or removing POX increased the threshold specific growth rate at which acetate accumulation began, with an 80% reduction in acetate accumulation observed at a specific growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests utilization of energetically less favorable glucose metabolism via glucokinase and the Entner-Doudoroff pathway in the absence of functional POX, while the upregulation of the phosphotransferase glucose uptake system and several amino acid biosynthetic pathways occurs in the presence of PYC. The physiological and expression changes resulting from these genetic perturbations demonstrate the importance of the pyruvate node in respiration and its impact on acetate overflow during aerobic growth. 
15794748	 The Wnt pathway controls biological processes via the regulation of target gene expression. The expression of direct Wnt target genes, e.g. cyclin D1 and MYC, is activated by the transcription factor TCF, which binds to specific sequence motifs in the promoter. Indirect target genes are regulated via transcription regulators, which are targets of the Wnt pathway. As an example, MYC regulates the MYC interacting zinc finger protein-1 (MIZ-1), which is able to inhibit the expression of the indirect target p21WAF1. We intended to identify new Wnt target genes and to get a deeper insight into the regulatory mechanisms of Wnt target gene expression. For this we analyzed the differential expression pattern of Wnt-1 activated cells by microarray analysis. We identified 43 sequences including eight expressed sequence tags (ESTs), which showed increased transcript levels, and 104 sequences including 19 ESTs with decreased RNA levels. Northern blot and real-time quantitative PCR analysis of the differential expression levels of 15 genes confirmed the differential expression trends of eight candidate genes. When the Wnt pathway was regulated at the lower level of glycogen synthase kinase-3 beta (GSK-3 beta) or adenomatous polyposis coli (APC), we detected discrepant expression trends. We compared the number of binding sites of transcription factors in the genomic regions of all candidate target genes with the number of sites in control genes. We found that the genomic regions of the down-regulated genes include an increased number of putative MIZ-1 binding sites. Our study introduces several new Wnt target genes and provides indications that the specific gene expression pattern depends on the type of the activation trigger or the level of interference with the Wnt pathway. Furthermore, our data  indicate that a high proportion of Wnt target genes are regulated by indirect mechanisms. 
15817786	 The gene expression profile of Escherichia coli K-12 MG1655 grown in minimal medium supplemented with elevated copper concentrations (as copper-glycine) has been analysed using whole-genome oligonucleotide microarrays. At 750 muM copper-glycine, the expression of both the cue and cus copper-export systems is evident. At near-lethal copper concentrations (2 mM copper-glycine), the expression of these two regulons increases significantly. Other regulons with increased transcription in response to elevated concentrations of copper-glycine  include those for the superoxide stress response, iron homeostasis, and envelope  stress. Furthermore, a variety of ORFs with decreased expression in response to increased copper-glycine has been identified, including the zinc ABC transporter  and genes involved in the chemotactic response. 
15773991	 Transcriptional response of Escherichia coli upon exposure to external copper was studied using DNA microarray and in vivo and in vitro transcription assays. Transcription of three hitherto-identified copper-responsive genes, copA (copper  efflux transporter), cueO (multicopper oxidase) and cusC (tripartite copper pump  component) became maximum at 5 min after addition of copper sulphate, and thereafter decreased to the preshift levels within 30 min. Microarray analysis at 5 min after addition of copper indicated that a total of at least 29 genes including these three known genes were markedly and specifically affected (28 upregulated and one downregulated). Transcription of the divergent operons, cusCFB and cusRS, was found to be activated by CusR, which bound to a CusR box between the cusC and cusR promoters. Except for this site, the CusR box was not identified in the entire E. coli genome. On the other hand, transcription of copA and cueO was found to be activated by another copper-responsive factor CueR, which bound to a conserved inverted repeat sequence, CueR box. A total of 197 CueR boxes were identified on the E. coli genome, including the CueR box associated with the moa operon for molybdenum cofactor synthesis. At least 10 copper-induced genes were found to be under the control of CpxAR two-component system, indicating that copper is one of the signals for activation of the CpxAR  system. In addition, transcription of yedWV, a putative two-component system, was activated by copper in CusR-dependent manner. Taken together we conclude that the copper-responsive genes are organized into a hierarchy of the regulation network, forming at least four regulons, i.e. CueR, CusR, CpxR and YedW regulons. These copper-responsive regulons appear to sense and respond to different concentrations of external copper. 
15805776	 Eight nucleotide sequences containing a single rhodanese domain were found in the Acidithiobacillus ferrooxidans ATCC 23270 genome: p11, p14, p14.3, p15, p16, p16.2, p21, and p28. Amino acids sequence comparisons allowed us to identify the  potentially catalytic Cys residues and other highly conserved rhodanese family features in all eight proteins. The genomic contexts of some of the rhodanese-like genes and the determination of their expression at the mRNA level  by using macroarrays suggested their implication in sulfur oxidation and metabolism, formation of Fe-S clusters or detoxification mechanisms. Several of the putative rhodanese genes were successfully isolated, cloned and overexpressed in E. coli and their thiosulfate:cyanide sulfurtransferase (TST) and 3-mercaptopyruvate/cyanide sulfurtransferase (MST) activities were determined. Based on their sulfurtransferase activities and on structural comparisons of catalytic sites and electrostatic potentials between homology- modeled A. ferrooxidans rhodaneses and the reported crystal structures of E. coli GlpE (TST) and SseA (MST) proteins, two of the rhodanese-like proteins (P15 and P16.2) could clearly be defined as TSTs, and P14 and P16 could possibly correspond to MSTs. Nevertheless, several of the eight A. ferrooxidans rhodanese-like proteins may have some different functional activities yet to be discovered. 
15738384	 In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this  hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of  the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation. 
15728886	 We investigated the influence of a low concentration of the gyrase inhibitor norfloxacin on the transcriptome of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. For this purpose, we used a commercial DNA microarray containing oligonucleotides specific for E. coli O157:H7 strains EDL933 and RIMD0509952 and  E. coli K-12 strain MG1655. Under the conditions applied, 5,963 spots (94% of all spots) could be analyzed. Among these, 118 spots (P < 0.05) indicated transcriptional upregulation and 122 spots (P < 0.05) indicated transcriptional downregulation of the E. coli genes present on the array. Eighty-five upregulated EDL933 genes were phage borne. Fifty-two of them could be ascribed to the Shiga toxin-encoding phages (Stx phages) BP-933W and CP-933V; the other 33 genes belonged to non-Stx prophage elements in the EDL933 genome. Genes present in the  BP-933W prophage genome were induced most strongly up to 158-fold in the case of  stxA(2) upon induction with norfloxacin. Twenty-two additional upregulated genes  appeared to be E. coli O157:H7 strain RIMD0509952-specific phage elements, and the remaining 11 genes were related mainly to recombination and stress functions. Downregulation was indicated predominantly for genes responsible for bacterial primary metabolism, such as energy production, cell division, and amino acid biosynthesis. Interestingly, some genes present in the locus of enterocyte effacement appeared to be downregulated. The results of the study have shown that a low concentration of norfloxacin has profound effects on the transcriptome of E. coli O157:H7. 
15716448	 The BaeSR two-component regulatory system controls expression of exporter genes conferring drug resistance in Escherichia coli (S. Nagakubo, K. Nishino, T. Hirata, and A. Yamaguchi, J. Bacteriol. 184:4161-4167, 2002; N. Baranova and H. Nikaido, J. Bacteriol. 184:4168-4176, 2002). To understand the whole picture of BaeSR regulation, a DNA microarray analysis of the effect of BaeR overproduction  was performed. BaeR overproduction activated 59 genes related to two-component signal transduction, chemotactic responses, flagellar biosynthesis, maltose transport, and multidrug transport, and BaeR overproduction also repressed the expression of the ibpA and ibpB genes. All of the changes in the expression levels were also observed by quantitative real-time reverse transcription-PCR analysis. The expression levels of 15 of the 59 BaeR-activated genes were decreased by deletion of baeSR. Of 11 genes induced by indole (a putative inducer of the BaeSR system), 10 required the BaeSR system for induction. Combination of  the expression data sets revealed a BaeR-binding site sequence motif, 5'-TTTTTCTCCATDATTGGC-3' (where D is G, A, or T). Several genes up-regulated by BaeR overproduction, including genes for maltose transport, chemotactic responses, and flagellar biosynthesis, required an intact PhoBR or CreBC two-component regulatory system for up-regulation. These data indicate that there is cross-regulation among the BaeSR, PhoBR, and CreBC two-component regulatory systems. Such a global analysis should reveal the regulatory network of the BaeSR system. 
15626601	 A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between  microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could  be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA. 
15639085	 We investigated the global gene expression changes of Escherichia coli due to the presence of different concentrations of phenylalanine or shikimate in the growth  medium. The response to 0.5 g l(-1) phenylalanine primarily reflected a perturbed aromatic amino acid metabolism, in particular due to TyrR-mediated regulation. The addition of 5g l(-1) phenylalanine reduced the growth rate by half and elicited a great number of likely indirect effects on genes regulated in response to changed pH, nitrogen or carbon availability. Consistent with the observed gene expression changes, supplementation with shikimate, tyrosine and tryptophan relieved growth inhibition by phenylalanine. In contrast to the wild-type, a tyrR disruption strain showed increased expression of pckA and of tktB in the presence of phenylalanine, but its growth was not affected by phenylalanine at the concentrations tested. The absence of growth inhibition by phenylalanine suggested that at high phenylalanine concentrations TyrR-defective strains might  perform better in phenylalanine production. 
15701760	 We combine comparative genomic measures and the distance separating adjacent genes to predict operons in 124 completely sequenced prokaryotic genomes. Our method automatically tailors itself to each genome using sequence information alone, and thus can be applied to any prokaryote. For Escherichia coli K12 and Bacillus subtilis, our method is 85 and 83% accurate, respectively, which is similar to the accuracy of methods that use the same features but are trained on  experimentally characterized transcripts. In Halobacterium NRC-1 and in Helicobacter pylori, our method correctly infers that genes in operons are separated by shorter distances than they are in E.coli, and its predictions using distance alone are more accurate than distance-only predictions trained on a database of E.coli transcripts. We use microarray data from six phylogenetically  diverse prokaryotes to show that combining intergenic distance with comparative genomic measures further improves accuracy and that our method is broadly effective. Finally, we survey operon structure across 124 genomes, and find several surprises: H.pylori has many operons, contrary to previous reports; Bacillus anthracis has an unusual number of pseudogenes within conserved operons; and Synechocystis PCC 6803 has many operons even though it has unusually wide spacings between conserved adjacent genes. 
15677333	 The Wnt signaling pathway acts ubiquitously in metazoans to control various aspects of embryonic development. Wnt ligands bind their receptors Frizzled and low-density lipoprotein receptor-related protein 5/6 and function through Disheveled (Dvl), Axin, adenomatous polyposis coli, glycogen synthase kinase 3beta, and casein kinase (CK) 1 to stabilize beta-catenin and induce lymphocyte enhancer-binding factor (LEF)/T cell factor (TCF)-dependent transcriptional activities. To identify previously unrecognized Wnt signaling modulators, a genome-wide functional screen was performed using large-scale arrayed cDNA collections. From this screen, both known components and previously uncharacterized regulators of this pathway were identified, including beta-catenin, Dvl1, Dvl3, Fbxw-1, Cul1, CK1epsilon, CK1delta, and gamma-catenin.  In particular, a previously unrecognized activator, LRRFIP2 (leucine-rich repeat  in Flightless interaction protein 2), was found that interacts with Dvl to increase the cellular levels of beta-catenin and activate beta-catenin/LEF/TCF-dependent transcriptional activity. The function of LRRFIP2  is blocked when a dominant negative Dvl (Xdd1) is coexpressed. Expression of LRRFIP2 in Xenopus embryos induced double axis formation and Wnt target gene expression; a dominant negative form of LRRFIP2 suppresses ectopic Wnt signaling  in Xenopus embryos and partially inhibits endogenous dorsal axis formation. These data suggest that LRRFIP2 plays an important role in transducing Wnt signals. 
15691972	 Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes  that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance. 
15340867	 Hydrostatic pressure is a well-known physical stimulus, but its effects on cell physiology have not been clarified. To investigate pressure effects on Escherichia coli, we carried out DNA microarray analysis of the entire E. coli genome. The microarray results showed pleiotropic effects on gene expression. In  particular, heat- and cold-stress responses were induced simultaneously by the elevated pressure. Upon temperature stress (including both temperature up- and down-shifts) and other environmental stresses, gene expression adjusts to adapt to such environmental changes through regulations by several DNA-binding proteins. An E. coli mutant, which deleted the hns gene encoding one of the regulator proteins, exhibited great pressure sensitivity. The result suggested that the H-NS protein was a possible transcriptional regulator for adaptation of  the high-pressure stress. 
15687289	 To explore the role of transcriptome polymorphism in adaptation of organisms to their environment, we evaluated this parameter for the Escherichia coli/Shigella  bacterial species, which is composed of well-characterized phylogenetic groups that exhibit characteristic life styles ranging from commensalism to intracellular pathogenicity. Both the genomic content and the transcriptome of 10 strains representative of the major E. coli/Shigella phylogenetic groups were evaluated using macroarrays displaying the 4290 K12-MG1655 open reading frames (ORFs). Although Shigella and enteroinvasive E. coli (EIEC) are not monophyletic, phylogenetic analysis of the binary coded (presence/absence) gene content data showed that these organisms group together due to similar patterns of undetectable K12-MG1655 genes. The variation in transcript abundance was then analyzed using a core genome of 2880 genes present in all strains, after adjusting RNA hybridization signals for DNA hybridization signals. Nonrandom changes in gene expression during the evolution of the E. coli/Shigella species were evidenced. Phylogenetic analysis of transcriptome data again showed that Shigella and EIEC strains group together in terms of gene expression, and this convergence involved groups of genes displaying biologically coherent patterns of functional divergence. Unlike the other E. coli strains evaluated, Shigella and EIEC are intracellular pathogens, and therefore face similar selective pressures. Thus, within the E. coli/Shigella species, strains exhibiting a particular life style have converged toward a specific gene expression pattern in a subset of genes common to the species, revealing the role of selection in shaping transcriptome polymorphism. 
15659685	 We determined global transcriptional responses of Escherichia coli K-12 to sulfur (S)- or nitrogen (N)-limited growth in adapted batch cultures and cultures subjected to nutrient shifts. Using two limitations helped to distinguish between nutrient-specific changes in mRNA levels and common changes related to the growth rate. Both homeostatic and slow growth responses were amplified upon shifts. This made detection of these responses more reliable and increased the number of genes that were differentially expressed. We analyzed microarray data in several ways:  by determining expression changes after use of a statistical normalization algorithm, by hierarchical and k-means clustering, and by visual inspection of aligned genome images. Using these tools, we confirmed known homeostatic responses to global S limitation, which are controlled by the activators CysB and Cbl, and found that S limitation propagated into methionine metabolism, synthesis of FeS clusters, and oxidative stress. In addition, we identified several open reading frames likely to respond specifically to S availability. As predicted from the fact that the ddp operon is activated by NtrC, synthesis of cross-links  between diaminopimelate residues in the murein layer was increased under N-limiting conditions, as was the proportion of tripeptides. Both of these effects may allow increased scavenging of N from the dipeptide D-alanine-D-alanine, the substrate of the Ddp system. 
15660999	 In response to environmental signals in the host, bacterial pathogens express factors required during infection and repress those that interfere with specific  stages of this process. Signalling pathways controlling virulence factors of the  human respiratory pathogen, Haemophilus influenzae, are predominantly unknown. The lipooligosaccharide (LOS) outer core represents a prototypical virulence trait of H. influenzae that enhances virulence but also provides targets for innate and adaptive immunity. We report regulation of the display of the virulence-associated phosphorylcholine (PC) epitope on the LOS in response to environmental conditions. PC display is optimal under microaerobic conditions and markedly decreased under conditions of high culture aeration. Gene expression analysis using a DNA microarray was performed to begin to define the metabolic state of the cell under these conditions and to identify genes potentially involved in PC epitope modulation. Global gene expression profiling detected changes in redox responsive genes and in genes of carbohydrate metabolism. The effects on carbohydrate metabolism led us to examine the role of the putative H.  influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in Escherichia coli. A mutant containing an in-frame deletion of the H. influenzae csrA gene showed increased PC epitope levels under aerobic conditions. Furthermore, deletion of csrA elevated mRNA expression of galU, an essential virulence gene that is critical in generating sugar precursors needed for polysaccharide formation and LOS outer core synthesis. Growth conditions predicted to alter the redox state of the culture modulated the PC epitope and galU expression as well. The results are consistent with a multifactorial mechanism of control of LOS-PC epitope display involving csrA and environmental signals that coordinately regulate biosynthetic and metabolic genes controlling the LOS structure. 
15546870	 Nitric oxide produced by activated macrophages plays a key role as one of the immune system's weapons against pathogens. Because the lifetime of nitric oxide is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased as in the infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions. In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the  helix-turn-helix LysR-type (YidZ). The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide is shown to cause a significant alteration of the global E. coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulfur cluster assembly systems, DNA-repairing enzymes, and stress response regulators. 
15580578	 Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification.  However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain. The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression.  The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent. A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases. Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype. These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community. 
15640440	 ArrayOme is a new program that calculates the size of genomes represented by microarray-based probes and facilitates recognition of key bacterial strains carrying large numbers of novel genes. Protein-coding sequences (CDS) that are contiguous on annotated reference templates and classified as 'Present' in the test strain by hybridization to microarrays are merged into ICs (ICs). These ICs  are then extended to account for flanking intergenic sequences. Finally, the lengths of all extended ICs are summated to yield the 'microarray-visualized genome (MVG)' size. We tested and validated ArrayOme using both experimental and  in silico-generated genomic hybridization data. MVG sizing of five sequenced Escherichia coli and Shigella strains resulted in an accuracy of 97-99%, as compared to true genome sizes, when the comprehensive ShE.coli meta-array gene sequences (6239 CDS) were used for in silico hybridization analysis. However, the E.coli CFT073 genome size was underestimated by 14% as this meta-array lacked probes for many CFT073 CDS. ArrayOme permits rapid recognition of discordances between PFGE-measured genome and MVG sizes, thereby enabling high-throughput identification of strains rich in novel genes. Gene discovery studies focused on  these strains will greatly facilitate characterization of the global gene pool accessible to individual bacterial species. 
15782232	 To identify Escherichia coli genes potentially regulated by filamentous phage infection, we used oligonucleotide microarrays. Genome-wide comparison of phage M13-infected and uninfected E. coli, 2 and 20 min after infection, was performed. The analysis revealed altered transcription levels of 12 E. coli genes in response to phage infection, and the observed regulation of phage genes correlated with the known in vivo pattern of M13 mRNA species. Ten of the 12 host genes affected could be grouped into 3 different categories based on cellular function, suggesting a coordinated response. The significantly upregulated genes  encode proteins involved in reactions of the energy-generating phosphotransferase system and transcription processing, which could be related to phage transcription. No genes belonging to any known E. coli stress response pathways were scored as upregulated. Furthermore, phage infection led to significant downregulation of transcripts of the bacterial genes gadA, gadB, hdeA, gadE, slp, and crl. These downregulated genes are normally part of the host stress response  mechanisms that protect the bacterium during conditions of acid stress and stationary phase transition. The phage-infected cells demonstrated impaired function of the oxidative and the glutamate-dependent acid resistance systems. Thus, global transcriptional analysis and functional analysis revealed previously unknown host responses to filamentous phage infection. 
16233757	 A time-course gene-expression profile was generated for Escherichia coli TK31 when it was exposed to an organic solvent mixture, and classified by fuzzy adaptive resonance theory (Fuzzy ART). It was found that the purR regulon plays an important role in the organic solvent tolerance (OST) of E. coli. 
17150737	 We carried out an in vitro selection of RNA aptamers that bind to Escherichia coli release factor 1 (E. coli RF1). The selected aptamer (class II) showed an apparent dissociation constant of nM range. The binding of the class II aptamer with E. coli RF1 is highly specific (orthogonal), allowing selective inhibition of RF1 activity in the E. coli translation system. 
15485810	 The phage shock protein operon (pspABCDE) of Escherichia coli is strongly up-regulated in response to overexpression of the filamentous phage secretin protein IV (pIV) and by many other stress conditions including defects in protein export. PspA has an established role in maintenance of the proton-motive force of the cell under stress conditions. Here we present evidence for a new member of the phage shock response in E. coli. Using transcriptional profiling, we show that the synthesis of pIV in E. coli leads to a highly restricted response limited to the up-regulation of the psp operon genes and yjbO. The psp operon and yjbO are also up-regulated in response to pIV in Salmonella enterica serovar Typhimurium. yjbO is a highly conserved gene found exclusively in bacteria that contain a psp operon but is physically unlinked to the psp operon. yjbO encodes a putative inner membrane protein that is co-controlled with the psp operon genes and is predicted to be an effector of the psp response in E. coli. We present evidence that yjbO expression is driven by sigma(54)-RNA polymerase, activated by PspF and integration host factor, and negatively regulated by PspA. PspF specifically regulates only members of the PspF regulon: pspABCDE and yjbO. We found that increased expression of YjbO results in decreased motility of bacteria. Because yjbO is co-conserved and co-regulated with the psp operon and is a member of the phage shock protein F regulon, we propose that yjbO be renamed pspG. 
15616567	 Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias.  This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general. 
15558318	 The alternative sigma factor RpoS controls the expression of many stationary-phase genes in Escherichia coli and other bacteria. Though the RpoS regulon is a large, conserved system that is critical for adaptation to nutrient  deprivation and other stresses, it remains incompletely characterized. In this study, we have used oligonucleotide arrays to delineate the transcriptome that is controlled by RpoS during entry into stationary phase of cultures growing in rich medium. The expression of known RpoS-dependent genes was confirmed to be regulated by RpoS, thus validating the use of microarrays for expression analysis. The total number of positively regulated stationary-phase genes was found to be greater than 100. More than 45 new genes were identified as positively controlled by RpoS. Surprisingly, a similar number of genes were found to be negatively regulated by RpoS, and these included almost all genes required  for flagellum biosynthesis, genes encoding enzymes of the TCA cycle, and a physically contiguous group of genes located in the Rac prophage region. Negative regulation by RpoS is thus much more extensive than has previously been recognized, and is likely to be an important contributing factor to the competitive growth advantage of rpoS mutants reported in previous studies. 
15347649	 The Tat protein export system serves to export folded proteins harboring an N-terminal twin arginine signal peptide across the cytoplasmic membrane. In this  study, we have used gene expression profiling of Escherichia coli supported by phenotypic analysis to investigate how cells respond to a defect in the Tat pathway. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are defective in the integrity of their cell envelope because of the mislocalization of two amidases involved in cell wall metabolism (Ize, B., Stanley, N. R., Buchanan, G., and Palmer, T. (2003) Mol. Microbiol. 48, 1183-1193). To distinguish between genes that are differentially expressed specifically because of the cell envelope defect and those that result  from other effects of the tatC deletion, we also analyzed two different transposon mutants of the DeltatatC strain that have their outer membrane integrity restored. Approximately 50% of the genes that were differentially expressed in the tatC mutant are linked to the envelope defect, with the products of many of these genes involved in self-defense or protection mechanisms, including the production of exopolysaccharide. Among the changes that were not explicitly linked to envelope integrity, we characterized a role for the Tat system in iron acquisition and copper homeostasis. Finally, we have demonstrated  that overproduction of the Tat substrate SufI saturates the Tat translocon and produces effects on global gene expression that are similar to those resulting from the DeltatatC mutation. 
15528503	 We systematically investigated the physiological response as well as DNA damage repair and damage tolerance in Shewanella oneidensis MR-1 following UVC, UVB, UVA, and solar light exposure. MR-1 showed the highest UVC sensitivity among Shewanella strains examined, with D37 and D10 values of 5.6 and 16.5% of Escherichia coli K-12 values. Stationary cells did not show an increased UVA resistance compared to exponential-phase cells; instead, they were more sensitive at high UVA dose. UVA-irradiated MR-1 survived better on tryptic soy agar than Luria-Bertani plates regardless of the growth stage. A 20% survival rate of MR-1  was observed following doses of 3.3 J of UVC m(-2), 568 J of UVB m(-2), 25 kJ of  UVA m(-2), and 558 J of solar UVB m(-2), respectively. Photoreactivation conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11-  to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation, respectively. A significant UV mutability to rifampin resistance was detected in  both UVC- and UVB-treated samples, with the mutation frequency in the range of 10(-5) to 10(-6). Unlike in E. coli, the expression levels of the nucleotide excision repair (NER) component genes uvrA, uvrB, and uvrD were not damage inducible in MR-1. Complementation of Pseudomonas aeruginosa UA11079 (uvrA deficient) with uvrA of MR-1 increased the UVC survival of this strain by more than 3 orders of magnitude. Loss of damage inducibility of the NER system appears to contribute to the high sensitivity of this bacterium to UVR as well as to other DNA-damaging agents. 
15522587	 DNA microarrays that are used as end-point detectors for PCR assays are typically composed of short (15-25 mer) oligonucleotide probes bound to glass. When designing these detectors, we have frequently encountered situations where a probe would not hybridize to its complementary, terminally labeled PCR amplicon.  To determine if failures could be explained by general phenomenon such as secondary structure, we designed a microarray to detect eight regions of the Escherichia coli 16S rDNA gene. We then amplified eight amplicons of different lengths using a biotin conjugated, antisense primer. Amplicons were then hybridized to the microarray and detected using a combination of signal amplification and fluorescence. In most cases, probe sequences complementary to the 5' region of the amplified products (sense orientation) did not hybridize to  their respective amplicon. We tested for positional bias and showed that a biotin conjugated sense primer mirrored the same probe failures. Nick translated products, however, hybridized to all probes. Because nick translation generates many labeled fragments of random length, we concluded that this method disrupted  secondary structure that otherwise prevented the amplicons from hybridizing to their respective probes. We also show that nick translation does not compromise detector sensitivity even when used with long PCR amplicons (ca. 1.5 kbp). Despite the increased cost of the nick translation, we concluded that this labeling strategy will reduce the time needed to design new assays as well as avoid possible false negatives during field applications. Alternative labeling strategies are also discussed. 
15516594	 Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. However, the genetic basis and regulatory mechanisms underlying the ability of S. oneidensis to survive and adapt to various environmentally relevant stresses is poorly understood. To define this organism's molecular response to elevated growth temperatures, temporal gene expression profiles were examined in cells subjected to heat stress by using whole-genome DNA microarrays for S. oneidensis. Approximately 15% (n = 711) of the total predicted S. oneidensis genes (n = 4,648) represented on the microarray were significantly up- or downregulated (P < 0.05) over a 25-min period after shift to the heat shock temperature. As expected, the majority of the genes that showed homology to known chaperones and heat shock proteins in other organisms were highly induced. In addition, a number of predicted genes, including those encoding enzymes in glycolysis and the pentose cycle, serine proteases, transcriptional regulators (MerR, LysR, and TetR families), histidine  kinases, and hypothetical proteins were induced. Genes encoding membrane proteins were differentially expressed, suggesting that cells possibly alter their membrane composition or structure in response to variations in growth temperature. A substantial number of the genes encoding ribosomal proteins displayed downregulated coexpression patterns in response to heat stress, as did  genes encoding prophage and flagellar proteins. Finally, a putative regulatory site with high conservation to the Escherichia coli sigma32-binding consensus sequence was identified upstream of a number of heat-inducible genes. 
15489430	 Treatment of Escherichia coli with p-hydroxybenzoic acid (pHBA) resulted in upregulation of yhcP, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes yhcQ, encoding a protein of the membrane fusion protein family, and yhcR, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene yhcS, encoding a regulatory protein of the LysR family, in regulating expression  of yhcRQP was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of yhcRQP expression. The efflux function encoded by yhcP was proven by the hypersensitivity to pHBA of a yhcP mutant strain. A yhcS mutant strain was also hypersensitive to pHBA. Expression of yhcQ and yhcP was necessary and sufficient for suppression of the pHBA hypersensitivity of the yhcS mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming yhcS, yhcR, yhcQ, and yhcP, to reflect their role in aromatic carboxylic acid efflux, to aaeR, aaeX, aaeA, and aaeB, respectively. The role of pHBA in normal E. coli metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a "metabolic relief valve" to alleviate toxic effects of imbalanced metabolism. 
15489429	 The sigma(S) subunit of RNA polymerase, the product of the rpoS gene, controls the expression of genes responding to starvation and cellular stresses. Using gene array technology, we investigated rpoS-dependent expression at the onset of  stationary phase in Escherichia coli grown in rich medium. Forty-one genes were expressed at significantly lower levels in an rpoS mutant derived from the MG1655 strain; for 10 of these, we also confirmed rpoS and stationary-phase dependence by reverse transcription-PCR. Only seven genes (dps, osmE, osmY, sodC, rpsV, wrbA, and yahO) had previously been recognized as rpoS dependent. Several newly identified rpoS-dependent genes are involved in the uptake and metabolism of amino acids, sugars, and iron. Indeed, the rpoS mutant strain shows severely impaired growth on some sugars such as fructose and N-acetylglucosamine. The rpoS gene controls the production of indole, which acts as a signal molecule in stationary-phase cells, via regulation of the tnaA-encoded tryptophanase enzyme.  Genes involved in protein biosynthesis, encoding the ribosome-associated protein  RpsV (sra) and the initiation factor IF-1 (infA), were also induced in an rpoS-dependent fashion. Using primer extension, we determined the promoter sequences of a selection of rpoS-regulated genes representative of different functional classes. Significant fractions of these promoters carry sequence features specific for Esigma(S) recognition of the -10 region, such as cytosines  at positions -13 (70%) and -12 (30%) as well as a TG motif located upstream of the -10 region (50%), thus supporting the TGN(0-2)C(C/T)ATA(C/A)T consensus sequence recently proposed for sigma(S). 
15489422	 When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300- to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the  red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by  testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes. 
15520470	 The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that controls transcription initiation from more than 100 promoters by binding to a specific DNA sequence within cognate promoters. Many genes in the CRP regulon have been predicted simply based on the presence of DNA-binding sites within gene promoters. In this study, we have exploited a newly developed technique, run-off  transcription/microarray analysis (ROMA) to define CRP-regulated promoters. Using ROMA, we identified 176 operons that were activated by CRP in vitro and 16 operons that were repressed. Using positive control mutants in different regions  of CRP, we were able to classify the different promoters into class I or class II/III. A total of 104 operons were predicted to contain Class II CRP-binding sites. Sequence analysis of the operons that were repressed by CRP revealed different mechanisms for CRP inhibition. In contrast, the in vivo transcriptional profiles failed to identify most CRP-dependent regulation because of the complexity of the regulatory network. Analysis of these operons supports the hypothesis that CRP is not only a regulator of genes required for catabolism of sugars other than glucose, but also regulates the expression of a large number of other genes in E.coli. ROMA has revealed 152 hitherto unknown CRP regulons. 
15464604	 In a recent investigation, expression of a double Vitreoscilla haemoglobin (two fused VHb molecules) in Escherichia coli grown in shake flasks resulted in higher final cell density and considerably higher levels of ribosomes and tRNA. In this  study, we have investigated the E. coli transcriptome in cells expressing native  VHb, double VHb and control cells lacking VHb by hybridising mRNA from the different constructs to high-density oligonucleotide arrays. Within the 95% confidence interval, 4 and 5% of all detected genes in native VHb cells were up-  and down-regulated, respectively; in double VHb cells the corresponding numbers were 6 and 10%, respectively. Dividing the data into different functional groups  revealed that genes involved in energy metabolism, central intermediary metabolism and cell processes were the most affected at the mRNA level. Particularly, the up-regulation of genes involved in translation and posttranslational modification observed in double VHb cells demonstrates a strong relationship between the regulation of ribosomal genes and the actual number of ribosomes. 
15476863	 To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h  after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of  bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus,  these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the  cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment. 
15466556	 Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was  found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters  during chemostat cultivation on maltose-based media at 80 degrees C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in  sessile cells (6% of the genome), over a third of which were initially annotated  as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several beta-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased beta-glycosidase levels are unclear but are  likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments. 
15466053	 A DNA microarray-based global transcript profiling of Escherichia coli in response to cold shock showed that in addition to the known cold shock-inducible  genes, new genes such as the flagellar operon, those encoding proteins involved in sugar transport and metabolism, and remarkably, genes encoding certain heat shock proteins are induced by cold shock. In the light of strong reduction in metabolic activity of the cell after temperature downshift, the induction of sugar metabolism machinery is unexpected. The deletion of four csps (cspA, cspB,  cspG, and cspE) affected cold shock induction of mostly those genes that are transiently induced in the acclimation phase, emphasizing that CspA homologues are essential in the acclimation phase. Relevance of these findings with respect  to the known RNA chaperone function of CspA homologues is discussed. 
15466047	 Escherichia coli MelR protein is a transcription activator that is essential for  melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB  promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR. 
15466022	 Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF  might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at  the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes  have been renamed frmR, frmA, and frmB, respectively. 
15469514	 The MtrAB two-component signal transduction system is highly conserved in sequence and genomic organization in Mycobacterium and Corynebacterium species, but its function is completely unknown. Here, the role of MtrAB was studied with  C. glutamicum as model organism. In contrast to M. tuberculosis, it was possible  to delete the mtrAB genes in C. glutamicum. The mutant cells showed a radically different cell morphology and were more sensitive to penicillin, vancomycin and lysozyme but more resistant to ethambutol. In order to identify the molecular basis for this pleiotropic phenotype, the mRNA profiles of mutant and wild type were compared with DNA microarrays. Three genes showed a more than threefold increased RNA level in the mutant, i.e. mepA (NCgl2411) encoding a putative secreted metalloprotease, ppmA (NCgl2737 ) encoding a putative membrane-bound protease modulator, and lpqB encoding a putative lipoprotein of unknown function. Expression of plasmid-encoded mepA in Escherichia coli led to elongated cells that were hypersensitive to an osmotic downshift, supporting the idea that peptidoglycan is the target of MepA. The mRNA level of two genes was more than fivefold decreased in the mutant, i.e. betP and proP which encode transporters for the uptake of betaine and proline respectively. The microarray results were confirmed by primer extension and RNA dot blot experiments. In the latter, the transcript level of genes involved in osmoprotection was tested before and after  an osmotic upshift. The mRNA level of betP, proP and lcoP was strongly reduced or undetectable in the mutant, whereas that of mscL (mechanosensitive channel) was increased. The changes in cell morphology, antibiotics susceptibility and the mRNA levels of betP, proP, lcoP, mscL and mepA could be reversed by expression of plasmid-encoded copies of mtrAB in the DeltamtrAB mutant, confirming that these changes occurred as a consequence of the mtrAB deletion. 
15313213	 Identifying the genes required for the growth or viability of an organism under a given condition is an important step toward understanding the roles these genes play in the physiology of the organism. Currently, the combination of global transposon mutagenesis with PCR-based mapping of transposon insertion sites is the most common method for determining conditional gene essentiality. In order to accelerate the detection of essential gene products, here we test the utility and reliability of a DNA microarray technology-based method for the identification of conditionally essential genes of the bacterium, Escherichia coli, grown in rich medium under aerobic or anaerobic growth conditions using two different DNA microarray platforms. Identification and experimental verification of five hypothetical E. coli genes essential for anaerobic growth directly demonstrated the utility of the method. However, the two different DNA microarray platforms yielded largely non-overlapping results after a two standard deviations cutoff and were subjected to high false positive background levels. Thus, further methodological improvements are needed prior to the use of DNA microarrays to reliably identify conditionally essential genes on genome-scale. 
15328082	 Most antimicrobial peptides (AMPs) impair the viability of target bacteria by permeabilizing bacterial membranes. However, the proline-rich AMPs have been shown to kill susceptible organisms without causing significant membrane perturbation and may act by inhibiting the activity of bacterial targets. To gain initial insight into the events that follow interaction of a proline-rich peptide with bacterial cells, we used DNA macroarray technology to monitor transcriptional alterations of Escherichia coli in response to challenge with a subinhibitory concentration of the proline-rich Bac7(1-35). Substantial changes in the expression levels of 70 bacterial genes from various functional categories were detected. Among these, 26 genes showed decreased expression, while 44 genes, including genes that are potentially involved in bacterial resistance to antimicrobials, showed increased expression. The generation of a transcriptional  response under the experimental conditions used is consistent with the ability of Bac7(1-35) to interact with bacterial components and affect biological processes  in this organism. 
15322044	 Using a DNA microarray, we determined changes in enterohemorrhagic Escherichia coli O157:H7 gene expression during binding to plasma membranes. Analysis of the  complete transcriptomes of the bound bacteria revealed increased levels of stress-associated mRNAs and decreased levels of mRNA encoding proteins involved in translation and type III secretion. 
15342588	 DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts  by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (sigmas), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction,  including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in  the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic  E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli. 
15364994	 The incidence of resistance against fluoroquinolones among pathogenic bacteria has been increasing in accordance with the worldwide use of this drug. Escherichia coli is one of the most relevant species for quinolone resistance. In this study, a diagnostic microarray for single-base-mutation detection was developed, which can readily identify the most prevalent E. coli genotypes leading to quinolone resistance. Based on genomic sequence analysis using public  databases and our own DNA sequencing results, two amino acid positions (83 and 87) on the A subunit of the DNA gyrase, encoded by the gyrA gene, have been identified as mutation hot spots and were selected for DNA microarray detection.  Oligonucleotide probes directed against these two positions were designed so that they could cover the most important resistance-causing and silent mutations. The  performance of the array was validated with 30 clinical isolates of E. coli from  four different hospitals in Germany. The microarray results were confirmed by standard DNA sequencing and were in full agreement with phenotypic antimicrobial  susceptibility testing. 
15364980	 A DNA microarray for detection of Campylobacter spp. was recently developed and applied to detect Campylobacter spp. directly from chicken feces. Sixty-five pooled chicken cloacal swab samples from 650 individual broiler chickens were included in the study. The results of Campylobacter sp. detection obtained with DNA microarrays were compared to those obtained by conventional culture and gel electrophoresis. By conventional culture, 60% of the samples were positive for either Campylobacter jejuni or Campylobacter coli. By PCR and capillary electrophoresis, 95% of the samples were positive for Campylobacter spp., whereas with DNA microarrays all samples were positive for Campylobacter spp. By application of DNA microarray analysis, the isolates in 4 samples (6%) could not  be identified to the species level, whereas by PCR-capillary electrophoresis, the isolates in 12 samples (19%) remained unidentified. Interestingly, PCR-capillary  electrophoresis analysis revealed that two (3%) of the samples were positive for  both C. jejuni and C. coli, while DNA microarray analysis revealed that nine (14%) of the samples were positive for both species. Of 65 samples, 2 samples were identified to contain C. coli by conventional culture but were positive for  C. jejuni by both PCR-capillary electrophoresis and DNA microarray analysis. The  discrepancy between the methods is discussed. 
15044239	 MOTIVATION: A major issue in computational biology is the reconstruction of functional relationships among genes, for example the definition of regulatory or biochemical pathways. One step towards this aim is the elucidation of transcriptional units, which are characterized by co-responding changes in mRNA expression levels. These units of genes will allow the generation of hypotheses about respective functional interrelationships. Thus, the focus of analysis currently moves from well-established functional assignment through comparison of protein and DNA sequences towards analysis of transcriptional co-response. Tools  that allow deducing common control of gene expression have the potential to complement and extend routine BLAST comparisons, because gene function may be inferred from common transcriptional control. RESULTS: We present a co-clustering strategy of genome sequence information and gene expression data, which was applied to identify transcriptional units within  diverse compendia of expression profiles. The phenomenon of prokaryotic operons was selected as an ideal test case to generate well-founded hypotheses about transcriptional units. The existence of overlapping and ambiguous operon definitions allowed the investigation of constitutive and conditional expression  of transcriptional units in independent gene expression experiments of Escherichia coli. Our approach allowed identification of operons with high accuracy. Furthermore, both constitutive mRNA co-response as well as conditional  differences became apparent. Thus, we were able to generate insight into the possible biological relevance of gene co-response. We conclude that the suggested strategy will be amenable in general to the identification of transcriptional units beyond the chosen example of E.coli operons. AVAILABILITY: The analyses of E.coli transcript data presented here are available upon request or at http://csbdb.mpimp-golm.mpg.de/ 
15115803	 Horizontally transferred genes are believed to play a critical role in the divergence of bacterial strains from a common ancestor, but whether all of these  genes express functional proteins in the cell remains unknown. Here, we used an integrated LC-based protein identification technology to analyze the proteome of  Escherichia coli strain K12 (JM109) and identified 1,480 expressed proteins, which are equivalent to approximately 35% of the total open reading frames predicted in the genome. This subset contained proteins with cellular abundance of several dozens to hundreds of thousands of copies, and included nearly all types of proteins in terms of chemical characteristics, subcellular distribution, and function. Interestingly, the subset also contained 138 of 164 gene products that are currently known to be essential for bacterial viability (84% coverage).  However, the subset contained only a very small population (10%) of protein products from genes mapped within K-loops, which are "hot spots" for the integration of foreign DNAs within the K12 genome. On the other hand, these genes in K-loops appeared to be transcribed to RNAs almost as efficiently as the native genes in the bacterial cell as monitored by DNA microarray analysis, raising the  possibility that most of the recently acquired foreign genes are inadequate for the translational machinery for the native genes and do not generate functional proteins within the cell. 
15101820	 Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a  small plant-specific protein whose cellular mode of action defies deduction based on its sequence or homology analyses, is one of numerous plant gene products with unknown biological roles that become over-expressed under water- and salt-stress  conditions. Steady-state cellular levels of tomato ASR1 mRNA and protein are transiently increased following exposure of plants to poly(ethylene glycol), NaCl or abscisic acid. Western blot and indirect immunofluorescence analysis with anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic and nuclear subcellular compartments; approx. one-third of the total ASR1 protein could be detected in the nucleus. Nuclear ASR1 is a chromatin-bound protein, and  can be extracted with 1 M NaCl, but not with 0.5% Triton X-100. ASR1, overexpressed in Escherichia coli and purified to homogeneity, possesses zinc-dependent DNA-binding activity. Competitive-binding experiments and SELEX (systematic evolution of ligands by exponential enrichment) analysis suggest that ASR1 binds at a preferred DNA sequence. 
15256503	 The circular chromosome of Escherichia coli is organized into independently supercoiled loops, or topological domains. We investigated the organization and size of these domains in vivo and in vitro. Using the expression of >300 supercoiling-sensitive genes to gauge local chromosomal supercoiling, we quantitatively measured the spread of relaxation from double-strand breaks generated in vivo and thereby calculated the distance to the nearest domain boundary. In a complementary approach, we gently isolated chromosomes and examined the lengths of individual supercoiled loops by electron microscopy. The  results from these two very different methods agree remarkably well. By comparing our results to Monte Carlo simulations of domain organization models, we conclude that domain barriers are not placed stably at fixed sites on the chromosome but instead are effectively randomly distributed. We find that domains are much smaller than previously reported, approximately 10 kb on average. We discuss the  implications of these findings and present models for how domain barriers may be  generated and displaced during the cell cycle in a stochastic fashion. 
15231542	 MOTIVATION: Microarray technology allows us to profile the expression of a large  subset or all genes of a cell. Biochemical research over the last three decades has elucidated an increasingly complete image of the metabolic architecture. For  less complex organisms, such as Escherichia coli, the biochemical network has been described in much detail. Here, we investigate the clustering of such networks by applying gene expression data that define edge lengths in the network. RESULTS: The Potts spin model is used as a nearest neighbour based clustering algorithm to discover fragmentation of the network in mutants or in biological samples when treated with drugs. As an example, we tested our method with gene expression data from E.coli treated with tryptophan excess, starvation and trpyptophan repressor mutants. We observed fragmentation of the tryptophan biosynthesis pathway, which corresponds well to the commonly known regulatory response of the cells. 
15256571	 This paper focuses on global gene regulation by FlhD/FlhC in enteric bacteria. Even though Yersinia enterocolitica FlhD/FlhC can complement an Escherichia coli  flhDC mutant for motility, it is not known if the Y. enterocolitica FlhD/FlhC complex has an effect on metabolism similar to E. coli. To study metabolic gene regulation, a partial Yersinia enterocolitica 8081c microarray was constructed and the expression patterns of wild-type cells were compared to an flhDC mutant strain at 25 and 37 degrees C. The overlap between the E. coli and Y. enterocolitica FlhD/FlhC regulated genes was 25 %. Genes that were regulated at least fivefold by FlhD/FlhC in Y. enterocolitica are genes encoding urocanate hydratase (hutU), imidazolone propionase (hutI), carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a pathway that is involved in the degradation of L-histidine to L-glutamate and eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and carbamoylaspartate. A number of other genes were regulated at a lower rate. In two additional experiments, the expression of wild-type cells grown at 4 or 25 degrees C was compared to the same strain grown at 37 degrees C. The expression of the flagella master operon flhD was not affected by temperature, whereas the flagella-specific sigma factor fliA was highly expressed at 25 degrees C and reduced at 4 and 37 degrees C. Several other flagella genes, all of which are under the control of FliA, exhibited a similar temperature profile. These data are consistent with the hypothesis that temperature regulation of flagella genes  might be mediated by the flagella-specific sigma factor FliA and not the flagella master regulator FlhD/FlhC. 
15210965	 Transposable elements have influenced the genetic and physical composition of all modern organisms. Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function. Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction. Replicated copies are excised from the chromosome and packaged into virus particles. Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion. To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA. Insertion hot- and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency. A moderate preference was observed for genes near the origin compared to terminus of replication. Large biases were found at hot and cold spots, which often include several consecutive genes. Efficient transcription of genes had a strong  negative influence on transposition. Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection. 
15167797	 An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for  methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance. 
14991425	 The thermal stress response of the hyperthermophilic bacterium Thermotoga maritima was characterized using a 407-open reading frame-targeted cDNA microarray. Transient gene expression was followed for 90 min, following a shift  from 80 degrees C to 90 degrees C. While some aspects of mesophilic heat-shock response were conserved in T. maritima, genome content suggested differentiating  features that were borne out by transcriptional analysis. Early induction of predicted heat-shock operons hrcA-grpE-dnaJ (TM0851-TM0850-TM0849), groES-groEL (TM0505-TM0506), and dnaK-sHSP (TM0373-TM0374) was consistent with conserved CIRCE elements upstream of hrcA and groES. Induction of the T. maritima rpoE/ sigW and rpoD/ sigA homologs suggests a mechanism for global heat-shock response  in the absence of an identifiable ortholog to a major heat-shock sigma factor. In contrast to heat-shock response in Escherichia coli, the majority of genes encoding ATP-dependent proteases were downregulated, including clpP (TM0695), clpQ (TM0521), clpY (TM0522), lonA (TM1633), and lonB (TM1869). Notably, T. maritima showed indications of a late heat-shock response with the induction of a marR homolog (TM0816), several other putative transcriptional regulators (TM1023, TM1069), and two alpha-glucosidases (TM0434 and TM1068). Taken together, the results reported here indicate that, while T. maritima shares core elements of the bacterial heat-shock response with mesophiles, the thermal stress regulatory  strategies of this organism differ significantly. However, it remains to be elucidated whether these differences are related to thermophilicity or phylogenetic placement. 
15177116	 The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St. Louis, MO). Of these target sequences, 44 were differentially expressed upon exposure to LPS, including 18 genes not previously reported to exist in cattle. These included a pentaxin-related gene, CASP8, TNF-induced genes, interferon-induced genes, and inhibitors of apoptosis. Using the human microarray, cDNA from bovine LPS-treated and control macrophages consistently hybridized to targets known to be expressed  constitutively by macrophages, as expected given the predicted cDNA sequence homology. That this human system was accurately estimating levels of bovine transcripts was further verified by real-time quantitative reverse transcriptase  polymerase chain reaction (RTQ-PCR) using bovine-specific primers. This first report of bovine-human cross-species expression profiling by microarray hybridization demonstrates the utility of this technique in bovine gene expression and discovery. 
15107501	 Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using microarray-based marker frequency analysis. Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms. Two of the three replication origins in each species were located in the vicinity of a cdc6/orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism. In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated. In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic  elongation rates. In S. acidocaldarius, a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the  chromosome, raising the possibility of a centromere-like function. 
14727089	 DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010  plates in continuous reactors were used to create the biofilms. For the biofilms  grown on glass wool, 22 genes were induced significantly (p< or =0.05) compared to suspension cells, including several genes for the stress response ( hslS, hslT, hha, and soxS), type I fimbriae ( fimG), metabolism ( metK), and 11 genes of unknown function ( ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373,  b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared  to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation. 
15052357	 A hybrid multiplex microarray microsystem has been developed that consists of 32  individually addressable array reaction chambers, supporting the use of multichannel pipettes for addition of up to 8 samples simultaneously. Discrimination between Campylobacter jejuni and Campylobacter coli bacteria was observed in DNA samples containing Campylobacter spp., with the same specificity  and sensitivity as when compared to a full-size microarray. The spinloaded multiplex microarray microsystem described provides a novel and convenient test format for simultaneous low-density microarray analysis and is universally adaptable to other DNA, protein or small molecule microarray based applications. 
14711822	 The ArcB/ArcA two-component signal transduction system of Escherichia coli regulates gene expression in response to the redox conditions of growth. Over the years, genetic screens have lead to the identification of about 30 ArcA-P-controlled operons that are involved in redox metabolism. However, the discovery of 3 targets that are not implicated in respiratory metabolism (the tra operon for plasmid conjugation, psi site for Xer-based recombination, and oriC site for chromosome replication) suggests that the Arc modulon may comprise additional operons that are involved in a myriad of functions. To identify these  operons, we derived the ArcA-P-dependent transcription profile of E. coli using oligonucleotide-based microarray analysis. The findings indicated that 9% of all  open reading frames in E. coli are affected either directly or indirectly by ArcA-P. To identify which operons are under the direct control of ArcA-P, we developed the ArcA-P recognition weight matrix from footprinting data and used it to scan the genome, yielding an ArcA-P sequence affinity map. By overlaying both  methods, we identified 55 new Arc-regulated operons that are implicated in energy metabolism, transport, survival, catabolism, and transcriptional regulation. The  data also suggest that the Arc response pathway, which translates into a net global downscaling of gene expression, overlaps partly with the FNR regulatory network. A conservative but reasonable assessment is that the Arc pathway recruits 100-150 operons to mediate a role in cellular adaptation that is more extensive than hitherto anticipated. 
15035675	 BACKGROUND: We describe a novel application of microarray technology for comparative genomics of bacteria in which libraries of entire genomes rather than the sequence of a single genome or sets of genes are arrayed on the slide and then probed for the presence or absence of specific genes and/or gene alleles. RESULTS: We first adopted a 96-well high throughput working protocol to efficiently isolate high quality genomic DNA. We then optimized conditions to print genomic DNA onto a glass slide with high density (up to 15000 spots) and to sensitively detect gene targets in each genome spot using fluorescently labeled DNA probe. Finally, we created an E. coli reference collection array and probed it for the presence or absence of the hemolysin (hly) gene using a dual channel non-competing hybridization strategy. Results from the array hybridization matched perfectly with previous tests. CONCLUSIONS: This new form of microarray technology, Library on a Slide, is an efficient way for sharing and utilizing large strain collections in comparative genomic analyses. 
14981237	 RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs  for decay by degradosomes. 
14645248	 2-O-alpha-mannosyl-D-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100. Uptake  is mediated by the P-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent Km of MG uptake in induced cells was 10 microm, and the Vmax was 0.65 nmol/min/10(9) cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with alpha-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR  is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system. 
15163562	 The mechanisms underlying alcoholic liver disease are not fully understood. It has been established that alcohol interferes with transcriptional and translational regulatory steps of cell function. To understand such an effect, assessment of alcohol-induced changes in the simultaneous expression of a large number of genes may prove very useful. The purpose of the current study was to test a large number of genes ( approximately 8700) for possible changes in expression induced by alcohol alone or in addition to treatment with lipopolysaccharide (LPS), a putative mediator of alcohol effects on the liver. Male rats were fed an alcohol-containing liquid diet (Lieber-DeCarli) for 14-15 weeks, injected with Escherichia coli LPS (0.8 mg x kg(-1)), and killed 24 h later. Blood samples were taken for determination of plasma liver enzyme activity, and liver samples were obtained for histologic evaluation and total RNA extraction. Total RNA was analyzed for gene expression (Rat Toxicology U34 Array; Affymetrix, Santa Clara, CA). Of 8740 genes on the microchip, 2259 were expressed in the liver. Seven hundred ninety-eight genes underwent significant changes induced by either alcohol or LPS, but listed in this article are only those that  significantly increased or decreased expression twofold or more. The genes were assigned to functional groups and reviewed. Gene changes were discussed from two  viewpoints: relevance to established hypotheses of alcohol and LPS mechanisms of  action and revealing of novel mechanisms of alcohol-induced liver injury. Application of DNA microarray technology to the study of alcohol-induced liver injury generated novel theoretical and experimental approaches to alcohol-induced liver injury. 
14718666	 We examined the genomewide transcriptional responses of Escherichia coli treated  with nitrosylated glutathione or the nitric oxide (NO)-generator acidified sodium nitrite (NaNO(2)) during aerobic growth. These assays showed that NorR, a homolog of NO-responsive transcription factors in Ralstonia eutrophus, and Fur, the global repressor of ferric ion uptake, are major regulators of the response to reactive nitrogen species. In contrast, SoxR and OxyR, regulators of the E. coli  defenses against superoxide-generating compounds and hydrogen peroxide, respectively, have minor roles. Moreover, additional regulators of the E. coli response to reactive nitrogen species remain to be identified because several of  the induced genes were regulated normally in norR, fur, soxRS, and oxyR mutant strains. We propose that the E. coli transcriptional response to reactive nitrogen species is a composite response mediated by the modification of multiple transcription factors containing iron or redox-active cysteines, some specifically designed to sense NO and its derivatives and others that are collaterally activated by the reactive nitrogen species. 
14719884	 A nucleic acid hybridization assay was assembled onto a robust and readily addressable silicon-based chip using polysaccharide chitosan as a scaffold for the covalent coupling of probe DNA to the chip's surface. Chitosan is a unique polymer, ideally suited for this application because its net charge and solubility are pH dependent. Specifically in this work, gold-patterned electrodes were created using standard photolithographic techniques, chitosan was electrodeposited in a spatially resolved manner onto the polarized electrodes, probe DNA was covalently assembled onto the chitosan, and both DNA:DNA and DNA:mRNA hybridization detection schemes were evaluated. Hybridization of target  nucleic acid was quantifiable, reproducible, and robust; the surface was regenerated and rehybridized up to eight times without loss of signal. Finally, transcriptional upregulation of the Escherichia coli chaperone, DnaK, which is an indicator of cellular stress, was observed using the hybridization chip sandwich  assay. Thus, this method enables rapid and facile monitoring of gene expression in a format that is reusable and requires minimal reagent quantities. 
14694202	 Cells adjust gene expression profiles in response to environmental and physiological changes through a series of signal transduction pathways. Upon activation or deactivation, the terminal regulators bind to or dissociate from DNA, respectively, and modulate transcriptional activities on particular promoters. Traditionally, individual reporter genes have been used to detect the  activity of the transcription factors. This approach works well for simple, non-overlapping transcription pathways. For complex transcriptional networks, more sophisticated tools are required to deconvolute the contribution of each regulator. Here, we demonstrate the utility of network component analysis in determining multiple transcription factor activities based on transcriptome profiles and available connectivity information regarding network connectivity. We used Escherichia coli carbon source transition from glucose to acetate as a model system. Key results from this analysis were either consistent with physiology or verified by using independent measurements. 
15535863	 BACKGROUND: The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure. RESULTS: We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions. CONCLUSIONS: The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as  a second messenger that transmits information about the environment to many regulatory networks in the cell. 
14585931	 The rnpA mutation, A49, in Escherichia coli reduces the level of RNase P at 43 degrees C because of a temperature-sensitive mutation in C5 protein, the protein  subunit of the enzyme. Microarray analysis reveals the expression of several noncoding intergenic regions that are increased at 43 degrees C compared with 30  degrees C. These regions are substrates for RNase P, and they are cleaved less efficiently than, for example, tRNA precursors. An analysis of the tna, secG, rbs, and his operons, all of which contain RNase P cleavage sites, indicates that RNase P affects gene expression for regions downstream of its cleavage sites. 
14597655	 The transcriptional network of Escherichia coli may well be the most complete experimentally characterized network of a single cell. A rule-based approach was  built to assess the degree of consistency between whole-genome microarray experiments in different experimental conditions and the accumulated knowledge in the literature compiled in RegulonDB, a data base of transcriptional regulation and operon organization in E. coli. We observed a high and statistical significant level of consistency, ranging from 70%-87%. When effector metabolites of regulatory proteins are not considered in the prediction of the active or inactive state of the regulators, consistency falls by up to 40%. Similarly, consistency decreases when rules for multiple regulatory interactions are altered or when "on" and "off" entries were assigned randomly. We modified the initial state of regulators and evaluated the propagation of errors in the network that do not correlate linearly with the connectivity of regulators. We interpret this  deviation mainly as a result of the existence of redundant regulatory interactions. Consistency evaluation opens a new space of dialogue between theory and experiment, as the consequences of different assumptions can be evaluated and compared. 
12777642	 OBJECTIVE: To identify disease-related genes and immune-regulatory pathways in the pathogenesis of systemic lupus erythematosus (SLE) by using gene expression profiling and protein-protein interaction analysis. METHODS: Peripheral white blood cell gene expression profiles of 10 SLE patients  were determined by oligonucleotide microarray analysis. Clustering of the gene expression profile was compared with the clinical immune phenotype. SLE-induced genes that were over- or under-expressed were determined and independently validated using a real-time polymerase chain reaction (PCR) method. To study their potential function and the possible pathways involved, a candidate gene was cloned and a GST (glutathione S-transferase) fusion protein was expressed in Escherichia coli. The fusion protein was further purified using the glutathione Sepharose 4B system, and was treated as bait to capture prey from SLE peripheral  white blood cell lysate. MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry was then performed to determine the prey protein. RESULTS: Similarity was found between the gene expression profile and the immune  phenotype clusters of the SLE patients. More than 20 disease-associated genes were identified, some of which have not been related to SLE previously. Of these  genes, a cluster of interferon-induced genes were highly correlated. IFIT1 (interferon-induced with tetratricopeptide repeats 1) was one of these genes, and overexpression of its mRNA was confirmed independently by real-time PCR in a larger population (40 SLE patients and 29 normal controls). An IFIT1 protein- protein interaction study showed that IFIT1 may interact with Rho/Rac guanine nucleotide exchange factor. CONCLUSION: The gene expression profile seems to be the molecular basis of the diverse immune phenotype of SLE. On the basis of the SLE-related genes found in this study, we suggest that the interferon-related immune pathway is important in the pathogenesis of SLE. IFIT1 is the first gene described as a candidate gene for SLE, and may function by activating Rho proteins through interaction with Rho/Rac guanine nucleotide exchange factor. IFIT1 and the interferon-related pathway may provide potential targets for novel interventions in the treatment of SLE. 
12889036	 Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes. 
12754220	 The work presented here is a first step toward a long term goal of systems biology, the complete elucidation of the gene regulatory networks of a living organism. To this end, we have employed DNA microarray technology to identify genes involved in the regulatory networks that facilitate the transition of Escherichia coli cells from an aerobic to an anaerobic growth state. We also report the identification of a subset of these genes that are regulated by a global regulatory protein for anaerobic metabolism, FNR. Analysis of these data demonstrated that the expression of over one-third of the genes expressed during  growth under aerobic conditions are altered when E. coli cells transition to an anaerobic growth state, and that the expression of 712 (49%) of these genes are either directly or indirectly modulated by FNR. The results presented here also suggest interactions between the FNR and the leucine-responsive regulatory protein (Lrp) regulatory networks. Because computational methods to analyze and interpret high dimensional DNA microarray data are still at an early stage, and because basic issues of data analysis are still being sorted out, much of the emphasis of this work is directed toward the development of methods to identify differentially expressed genes with a high level of confidence. In particular, we describe an approach for identifying gene expression patterns (clusters) obtained from multiple perturbation experiments based on a subset of genes that exhibit high probability for differential expression values. 
12878731	 High-pressure liquid chromatography-tandem mass spectrometry was used to obtain a protein profile of Escherichia coli strain MG1655 grown in minimal medium with glycerol as the carbon source. By using cell lysate from only 3 x 108 cells, at least four different tryptic peptides were detected for each of 404 proteins in a short 4-h experiment. At least one peptide with a high reliability score was detected for 986 proteins. Because membrane proteins were underrepresented, a second experiment was performed with a preparation enriched in membranes. An additional 161 proteins were detected, of which from half to two-thirds were membrane proteins. Overall, 1,147 different E. coli proteins were identified, almost 4 times as many as had been identified previously by using other tools. The protein list was compared with the transcription profile obtained on Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein products were found was detected at the mRNA level. The arithmetic mean mRNA signal intensity for these genes was 3-fold higher than that for all 4,300 protein-coding genes of E. coli. Thus, GeneChip data confirmed the high reliability of the protein list, which contains about one-fourth of the proteins  of E. coli. Detection of even those membrane proteins and proteins of undefined function that are encoded by the same operons (transcriptional units) encoding proteins on the list remained low. 
12672900	 The involvement of heat-inducible genes, including the heat-shock genes, in the acute response to temperature stress is well established. However, their importance in genetic adaptation to long-term temperature stress is less clear. Here we use high-density arrays to examine changes in expression for 35 heat-inducible genes in three independent lines of Escherichia coli that evolved  at high temperature (41.5 degrees C) for 2,000 generations. These lines exhibited significant changes in heat-inducible gene expression relative to their ancestor, including parallel changes in fkpA, gapA, and hslT. As a group, the heat-inducible genes were significantly more likely than noncandidate genes to have evolved changes in expression. Genes encoding molecular chaperones and ATP-dependent proteases, key components of the cytoplasmic stress response, exhibit relatively little expression change; whereas genes with periplasmic functions exhibit significant expression changes suggesting a key role for the extracytoplasmic stress response in the adaptation to high temperature. Following acclimation at 41.5 degrees C, two of the three lines exhibited significantly improved survival at 50 degrees C, indicating changes in inducible thermotolerance. Thus evolution at high temperature led to significant changes at the molecular level in heat-inducible gene expression and at the organismal level in inducible thermotolerance and fitness. 
12682301	 High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells  lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon. Bacteria devoid of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase. The latter two strains differed from WT in two ways. First, the origin proximal genes were transcribed with increased frequency due to increased gene dosage. Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction. We suggest that the methylation status of the cell is an important factor in forming and/or  maintaining chromosome structure. 
12700153	 The vast majority of microorganisms in the environment remain uncultured, and their existence is known only from sequences retrieved by PCR. As a consequence,  our understanding of the ecological function of dominant microbial populations in the environment is limited. We will review microbial diversity studies and show that these may have moved from an extreme underestimation to a potentially severe overestimation of diversity. The latter results from a simple PCR-generated artifact: the cloning of heteroduplex molecules followed by Escherichia coli mismatch repair, which may generate an exponential increase in observed sequence  diversity. However, simple modifications to current PCR amplification protocols minimize such artifactual sequences and may bring within our reach estimation of  bacterial diversity in environmental samples. Such estimates may spur new culture-independent approaches based on genomic and microarray technology, allowing correlation of phylogenetic identity with the ecological function of unculturable organisms. In particular, we are developing a DNA microarray that enables identification of individual populations active in utilization of specific organic substrates. The array consists of 16S and 23S rDNA-targeted oligonucleotides and is hybridized to RNA extracted from samples incubated with (14)C-labeled organic substrates. Populations that metabolize the substrate can be identified by the radiolabel incorporated in their rRNA after only one to two  cell doublings, ensuring realistic preservation of community structure. Thus, the microarray approach may provide a powerful means to link microbial community structure with in situ function of individual populations. 
12654720	 In this communication, we describe the use of specialized transposons (Tn5 derivatives) to create deletions in the Escherichia coli K-12 chromosome. These transposons are essentially rearranged composite transposons that have been assembled to promote the use of the internal transposon ends, resulting in intramolecular transposition events. Two similar transposons were developed. The  first deletion transposon was utilized to create a consecutive set of deletions in the E. coli chromosome. The deletion procedure has been repeated 20 serial times to reduce the genome an average of 200 kb (averaging 10 kb per deletion). The second deletion transposon contains a conditional origin of replication that  allows deleted chromosomal DNA to be captured as a complementary plasmid. By plating cells on media that do not support plasmid replication, the deleted chromosomal material is lost and if it is essential, the cells do not survive. This methodology was used to analyze 15 chromosomal regions and more than 100 open reading frames (ORFs). This provides a robust technology for identifying essential and dispensable genes. 
12514191	 In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM)  lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns. Such deficiencies can result from inborn errors in the MCM structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin adenosyltransferase (ATR) catalyzes the terminal step in the conversion of cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has not been identified. Here we report the identification of the bovine and human ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression library was screened for clones that complemented an ATR-deficient bacterial strain for color formation on aldehyde indicator medium, and four positive clones were isolated. The DNA sequences of two clones were determined and found to be identical. Sequence similarity searching was then used to identify a homologous human cDNA (89% identity) and its corresponding gene that is located on chromosome XII. The bovine and human cDNAs were independently cloned and expressed in Escherichia coli. Enzyme assays showed that expression strains produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific activities are in line with values reported previously for bacterial ATR enzymes. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This demonstrated that the human ATR is active under physiological conditions albeit in a heterologous host. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonyl aciduria patients compared with cell lines from normal individuals. We propose that inborn errors in the human ATR gene identified here result in methylmalonyl aciduria. The identification of genes involved in this disorder will allow improvements in  the diagnosis and treatment of this serious disease. 
12675606	 Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the production of ethanol. Gene arrays were used to identify expression changes that  occurred in the mutant, LY01, during directed evolution to improve ethanol tolerance (defined as extent of growth in the presence of added ethanol). Expression levels for 205 (5%) of the ORFs were found to differ significantly (p  < 0.10) between KO11 and LY01 under each of six different growth conditions (p <  0.000001). Statistical evaluation of differentially expressed genes according to  various classification schemes identified physiological areas of importance. A large fraction of differentially expressed ORFs were globally regulated, leading  to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a  putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+)  in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the absence of ethanol. Other differences in gene expression provided additional clues that permitted experimentation. Tolerance appears to involve increased metabolism of glycine (higher expression of gcv genes) and increased production of betaine (higher expression of betIBA and betT encoding betaine synthesis from  choline and choline uptake, respectively). Addition of glycine (10 mM) increased  ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and KO11 but had no effect on growth in the absence of ethanol. Both glycine and betaine can serve as protective osmolytes, and this may be the basis of their beneficial action. In addition, the marAB genes encoding multiple antibiotic resistance proteins were expressed at higher levels in LY01 as compared to KO11.  Interestingly, overexpression of marAB in KO11 made this strain more ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine deamination (sdaB) also appear beneficial for LY01. Addition of serine increased  the growth of LY01 in the presence and absence of ethanol but had no effect on KO11. Changes in the expression of several genes concerned with the synthesis of  the cell envelope components were also noted, which may contribute to increased ethanol tolerance. 
12566398	 Changes in DNA supercoiling are induced by a wide range of environmental stresses in Escherichia coli, but the physiological significance of these responses remains unclear. We now demonstrate that an increase in negative supercoiling is  necessary for transcriptional activation of a large subset of osmotic stress-response genes. Using a microarray-based approach, we have characterized supercoiling-dependent gene transcription by expression profiling under conditions of high salt, in conjunction with the microbial antibiotics novobiocin, pefloxacin, and chloramphenicol. Algorithmic clustering and statistical measures for gauging cellular function show that this subset is enriched for genes critical in osmoprotectant transport/synthesis and rpoS-driven stationary phase adaptation. Transcription factor binding site analysis also supports regulation by the global stress sigma factor rpoS. In addition, these studies implicate 60 uncharacterized genes in the osmotic stress regulon, and offer evidence for a broader role for supercoiling in the control of stress-induced transcription. 
12736533	 DNA microarrays provide a global view of the physiological state of the cell by parallel analysis of the expression levels of all the genes in an organism. The effects of four bactericidal agents on the expression pattern of Escherichia coli MG1655 were assessed. Compounds were chosen on the basis of their different mechanisms of action and included inhibitors of DNA replication and recombination, translation, transcription and cell wall biosynthesis. The addition of rifampin resulted in increased expression of the target, rpoB, as well as several genes involved in nucleotide salvage and purine biosynthesis. The addition of ampicillin resulted in overall changes in gene expression that showed some similarity to changes induced by rifampin. The addition of the antibiotics kanamycin or norfloxacin resulted in the induction of unique gene expression signatures: a heat shock response to kanamycin and an SOS response to norfloxacin. Several genes of unknown function showed expression profiles similar to the genes associated with the SOS or the heat shock response. Thus, these profiles define families of genes with similar expression phenotypes that can be  tested for related function. 
12673060	 The addition of exogenous pyrophosphate increases the growth yield and cAMP synthesis in stationary phase when Escherichia coli is grown in minimal medium. Pyrophosphate increases the yield by altering the enterobactin uptake system. We  studied the physiological effects and examined how the E. coli transcriptome was  modified when two structural analogs of pyrophosphate were added to the growth medium. Methylenediphosphonic acid or a high concentration of iron had the same positive effects as pyrophosphate on growth yield, cAMP synthesis and the repression of Fur-regulated genes. In contrast, imidodiphosphate did not affect these cellular processes significantly. The transcriptome modifications generated by pyrophosphate or methylenediphosphonic acid were more similar than those generated by imidodiphosphate or excess iron. The transcriptome data also indicated that processes other than iron uptake might be involved in the cellular response to exogenous pyrophosphate or methylenediphosphonic acid. 
12710666	 Author information:  (1)Department of Biochemistry, Biophysics and Molecular Biology, University of Minnesota, St. Paul, USA. 
12324455	 Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys(3)His motif proteins required for proper regulation of tumor necrosis factor  mRNA stability in macrophages. We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift. This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs. Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by site-directed  mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-gamma, and interferon-beta ARE fragments. A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex. 
16013212	 BACKGROUND: Necrotizing ulcerative periodontitis (NUP) is a painful and potentially debilitating affliction that affects about 2% to 6% of HIV-positive subjects. NUP may be caused by specific microorganisms that are presently unknown or by microbial species not usually thought to cause periodontal infections. The  purpose of this study was to define the bacterial species associated with NUP in  HIV-positive patients. METHODS: 16S rRNA bacterial genes of DNA isolated from subgingival plaque of 8 HIV-positive subjects with NUP were amplified by polymerase chain reaction (PCR)  and cloned into Escherichia coli. The sequences of cloned inserts were used to determine species identity or closest relatives by comparison with known sequences. The microbial profiles in subgingival plaque of subjects with NUP, chronic periodontitis, and periodontal health were compared using a battery of over 200 oligonucleotide probes in a PCR-based, reverse-capture, checkerboard DNA-DNA hybridization assay. RESULTS: Sequence analysis of over 400 clones revealed 108 species; 65 were "uncultivable" phylotypes, of which 26 were novel to NUP subjects. Species or phylotypes most commonly detected were Bulleidia extructa, Dialister, Fusobacterium, Selenomonas, Peptostreptococcus, Veillonella, and the phylum TM7.  Based on sequence analysis and checkerboard analysis, NUP did not possess the classical periodontal pathogens such as Porphyromonas gingivalis. Otherwise, the  microbial profiles of NUP and periodontitis had many similarities. The microbial  profiles of subgingival plaque from periodontally healthy subjects were different and less complex in comparison to the profiles of both disease groups. CONCLUSIONS: Certain species appear to be associated with health and periodontal  diseases. The putative pathogens associated with periodontal disease in HIV-negative subjects are not associated with NUP in HIV-positive subjects. 
12130640	 Leucine-responsive regulatory protein (Lrp) is a global regulatory protein that affects the expression of multiple genes and operons in bacteria. Although the physiological purpose of Lrp-mediated gene regulation remains unclear, it has been suggested that it functions to coordinate cellular metabolism with the nutritional state of the environment. The results of gene expression profiles between otherwise isogenic lrp(+) and lrp(-) strains of Escherichia coli support  this suggestion. The newly discovered Lrp-regulated genes reported here are involved either in small molecule or macromolecule synthesis or degradation, or in small molecule transport and environmental stress responses. Although many of  these regulatory effects are direct, others are indirect consequences of Lrp-mediated changes in the expression levels of other global regulatory proteins. Because computational methods to analyze and interpret high dimensional DNA microarray data are still an early stage, much of the emphasis of this work is directed toward the development of methods to identify differentially expressed genes with a high level of confidence. In particular, we describe a Bayesian statistical framework for a posterior estimate of the standard deviation of gene measurements based on a limited number of replications. We also describe  an algorithm to compute a posterior estimate of differential expression for each  gene based on the experiment-wide global false positive and false negative level  for a DNA microarray data set. This allows the experimenter to compute posterior  probabilities of differential expression for each individual differential gene expression measurement. 
12374860	 Bacterial adaptation to nutrient limitation and increased population densities is central to survival and virulence. Surprisingly, <3% of Escherichia coli genes are known to play roles specific to the stationary phase. There is evidence that  the leucine-responsive regulatory protein (Lrp) may play an important role in stationary phase, so this study used microarrays representing >98% of E. coli genes to more comprehensively identify those controlled by Lrp. The primary analysis compared isogenic Lrp(+) and Lrp(-) strains in cells growing in steady state in glucose minimal medium, either in the presence or absence of leucine. More than 400 genes were significantly Lrp-responsive under the conditions used.  Transcription of 147 genes was lower in Lrp(+) than in Lrp(-) cells whether or not leucine was present; most of these genes were tightly coregulated under several conditions, including a burst of synthesis on transition to stationary phase. This cluster includes 56 of 115 genes already known to play roles in stationary phase. Our results suggest that the actual number of genes induced on  entrance into stationary phase is closer to 200 and that Lrp affects nearly three-quarters of them, including genes involved in response to nutrient limitation, high concentrations of organic acids, and osmotic stress. 
12207220	 Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type 1 fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated with virulence in the urinary tract. A typical type 1 fimbriated bacterium has up to 500 fimbriae on its surface, with each fimbria consisting of approximately 1000 individual subunits. This equates to approximately 8% of the total cellular protein and is potentially a significant resource drain for the cell. Here we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type 1 fimbriae. Changes in fim expression correlated directly with alterations in colony morphology. Deletion of the entire fim gene cluster resulted in the converse expression of another surface protein Antigen 43 (Ag43). Specific deletion of the fimH gene did not affect expression of other fim genes or Ag43, but did dramatically reduce the number of fimbriae expressed on the cell surface. The use of high-resolution oligonucleotide arrays for defining points of transcription initiation and termination is also demonstrated. 
12119387	 Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the approximately 4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although approximately 80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5' or 3' untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at  a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability. 
12118877	 Microarray technology was used to identify genes in Erwinia chrysanthemi 3937 that are specifically up- or down-regulated in a plant host compared with growth  in laboratory culture medium. Several genes were plant down-regulated, and almost all of them were homologues of well-known housekeeping genes, such as those encoding metabolic functions, oxidative phosphorylation components, and transcription or translation processes. On the other hand, almost all of the plant up-regulated genes were involved with specialized functions, including already known or new putative virulence factors, anaerobiosis, iron uptake, transporters or permeases, xenobiotic resistance, chemotaxis, and stress responses to reactive oxygen species and heat. A substantial number of the plant  up-regulated genes do not appear to be directly involved in damaging the host, but are probably important in adapting the pathogen to the host environment. We constructed insertion mutations in several of the plant up-regulated E. chrysanthemi 3937 genes. Among these, mutations of Bacillus subtilis pps1, Escherichia coli purU, and Pseudomonas aeruginosa pheC homologues reduced virulence on African violet leaves. Thus, new insights were obtained into genes important in bacterial virulence. 
11997466	 We report a DNA microarray-based method for genome-wide monitoring of competitively grown transformants to identify genes whose overexpression confers  a specific cellular phenotype. Whereas transcriptional profiling identifies differentially expressed genes that are correlated with particular aspects of the cellular phenotype, this functional genomics approach determines genes that result in a specific physiology. This parallel gene-trait mapping method consists of transforming a strain with a genomic library, enriching the cell population in transformants containing the trait conferring gene(s), and finally using DNA microarrays to simultaneously isolate and identify the enriched gene inserts. Various methods of enrichment can be used; here, genes conferring low-level antibiotic resistance were identified by growth in selective media. We demonstrated the method by transforming Escherichia coli cells with a genomic E.  coli library and selecting for transformants exhibiting a growth advantage in the presence of the anti-microbial agent Pine-Sol. Genes conferring Pine-Sol tolerance (19 genes) or sensitivity (27 genes) were identified by hybridizing, on DNA microarrays containing 1,160 E. coli gene probes, extra-chromosomal DNA isolated from transformed cells grown in the presence of various levels of Pine-Sol. Results were further validated by plating and sequencing of individual  colonies, and also by assessing the Pine-Sol resistance of cells transformed with enriched plasmid library or individual resistance genes identified by the microarrays. Applications of this method beyond antibiotic resistance include identification of genes resulting in resistance to chemotherapeutic agents, genes yielding resistance to toxic products (recombinant proteins, chemical feedstocks) in industrial fermentations, genes providing enhanced growth in cell culture or high cell density fermentations, genes facilitating growth on unconventional substrates, and others. 
11815613	 This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA microarray on glass slides. Glucose-grown cultures were used as a reference. At the 95% confidence level, 354 genes were up-regulated in acetate, while 370 genes were down-regulated compared with the glucose-grown culture. Generally, more metabolic genes were up-regulated in acetate than other  gene groups, while genes involved in cell replication, transcription, and translation machinery tended to be down-regulated. It appears that E. coli commits more resources to metabolism at the expense of growth when cultured in the poor carbon source. The expression profile confirms many known features in acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic acid cycle, and gluconeogenic genes. It also provided many previously unknown features, including induction of malic enzymes, ppsA, and the glycolate pathway and repression of glycolytic and glucose phosphotransferase genes in acetate. The carbon flux delivered from the malic enzymes and PpsA in acetate was further confirmed by deletion mutations. In general, the gene expression profiles qualitatively agree with the metabolic flux changes and may serve as a predictor  for gene function and metabolic flux distribution. 
11752446	 Genetic studies have identified mutations in key regulators of the Wnt/beta-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is  not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt/beta-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which beta-catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with beta-catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these  mRNAs are known inhibitors of the beta-catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt/beta-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon  tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with  truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers. 
11689852	 We describe a method of genome-wide analysis of quantitative growth phenotypes using insertional mutagenesis and DNA microarrays. We applied the method to assess the fitness contributions of Escherichia coli gene domains under specific  growth conditions. A transposon library was subjected to competitive growth selection in Luria-Bertani (LB) and in glucose minimal media. Transposon-containing genomic DNA fragments from the selected libraries were compared with the initial unselected transposon insertion library on DNA microarrays to identify insertions that affect fitness. Genes involved in the biosynthesis of nutrients not provided in the growth medium were found to be significantly enriched in the set of genes containing negatively selected insertions. The data also identify fitness contributions of several uncharacterized genes, including putative transcriptional regulators and enzymes. The applicability of this high-resolution array selection in other species is discussed. 
11679675	 Dendritic cells are involved in the initiation of both innate and adaptive immunity. To systematically explore how dendritic cells modulate the immune system in response to different pathogens, we used oligonucleotide microarrays to measure gene expression profiles of dendritic cells in response to Escherichia coli, Candida albicans, and influenza virus as well as to their molecular components. Both a shared core response and pathogen-specific programs of gene expression were observed upon exposure to each of these pathogens. These results  reveal that dendritic cells sense diverse pathogens and elicit tailored pathogen-specific immune responses. 
11553553	 Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection. 
11544210	 A sequenced collection of plasmid-borne random fusions of Escherichia coli DNA to a Photorhabdus luminescens luxCDABE reporter was used as a starting point to select a set of 689 nonredundant functional gene fusions. This group, called LuxArray 1.0, represented 27% of the predicted transcriptional units in E. coli.  High-density printing of the LuxArray 1.0 reporter strains to membranes on agar plates was used for simultaneous reporter gene assays of gene expression. The cellular response to nalidixic acid perturbation was analyzed using this format.  As expected, fusions to promoters of LexA-controlled SOS-responsive genes dinG, dinB, uvrA, and ydjM were found to be upregulated in the presence of nalidixic acid. In addition, six fusions to genes not previously known to be induced by nalidixic acid were also reproducibly upregulated. The responses of two of these, fusions to oraA and yigN, were induced in a LexA-dependent manner by both nalidixic acid and mitomycin C, identifying these as members of the LexA regulon. The responses of the other four were neither induced by mitomycin C nor dependent on lexA function. Thus, the promoters of ycgH, intG, rihC, and a putative operon  consisting of lpxA, lpxB, rnhB, and dnaE were not generally DNA damage responsive and represent a more specific response to nalidixic acid. These results demonstrate that cellular arrays of reporter gene fusions are an important alternative to DNA arrays for genomewide transcriptional analyses. 
11568803	 OBJECTIVE: We propose that elucidation of the pathophysiology of preterm labor can be achieved with genome-scale analyses of differential gene expression. STUDY DESIGN: CD-1 mice on day 14.5 of a 19- to 20-day gestation were assigned to one of 4 treatment groups modeling different clinical conditions (n = 5 per group): group A, infection with labor (intrauterine injection of 10(10) heat-killed Escherichia coli, which causes delivery within an average of 20 hours); group B, infection without labor (intrauterine injection of 10(7) heat-killed E coli, which leads to normal delivery at term); group C, labor without infection (ovariectomy, which causes delivery within an average of 27 hours); and group D, no infection and no labor (intrauterine injection of vehicle). Total pooled myometrial RNA was prepared 3.5 hours after surgery for groups A, B, and D and 5 hours after surgery for group C. The relative expression of 4963 genes was assayed in these pools by using DNA microarrays. Transcripts specifically involved in infection-induced labor were identified by subtracting from the list of differentially regulated genes in group A those with common expression in groups B and C. RESULTS: In group A 68 differentially expressed transcripts (>or=2-fold upregulation or downregulation) were identified. Among these are 39 characterized genes. Fourteen (45%) are involved in inflammatory responses, 7 (18%) are involved in growth-differentiation-oncogenesis, and 3 (8%) are involved in apoptosis. Subtraction identified 13 gene products most likely to be important for bacterially induced labor, as opposed to labor without infection or bacterial exposure without labor. CONCLUSION: This study demonstrates the potential of the subtractive DNA microarray technique to identify transcripts important specifically for bacterially induced preterm labor. 
11493693	 DNA microarrays constructed with full length ORFs from Shewanella oneidensis, MR-1, were hybridized with genomic DNA from nine other Shewanella species and Escherichia coli K-12. This approach enabled visualization of relationships between organisms by comparing individual ORF hybridizations to 164 genes and is  further amenable to high-density high-throughput analyses of complete microbial genomes. Conserved genes (arcA and ATP synthase) were identified among all species investigated. The mtr operon, which is involved in iron reduction, was poorly conserved among other known metal-reducing Shewanella species. Results were most informative for closely related organisms with small subunit rRNA sequence similarities greater than 93% and gyrB sequence similarities greater than 80%. At this level of relatedness, the similarity between hybridization profiles was strongly correlated with sequence divergence in the gyrB gene. Results revealed that two strains of S. oneidensis (MR-1 and DLM7) were nearly identical, with only 3% of the ORFs hybridizing poorly, in contrast to hybridizations with Shewanella putrefaciens, formerly considered to be the same species as MR-1, in which 63% of the ORFs hybridized poorly (log ratios below -0.75). Genomic hybridizations showed that genes in operons had consistent levels of hybridization across an operon in comparison to a randomly sampled data set, suggesting that similar applications will be informative for identification of horizontally acquired genes. The full value of microbial genomic hybridizations lies in providing the ability to understand and display specific differences between closely related organisms providing a window into understanding microheterogeneity, bacterial speciation, and taxonomic relationships. 
11443091	 The genome-wide transcription profile of Escherichia coli cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169 E. coli open reading frames. By measuring gene expression in isogenic wild-type and oxyR  deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes. The DNA microarray measurements allowed the identification of several new OxyR-activated  genes, including the hemH heme biosynthetic gene; the six-gene suf operon, which  may participate in Fe-S cluster assembly or repair; and four genes of unknown function. We also identified several genes, including uxuA, encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the DeltaoxyR mutant strain. In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in E. coli. For example, the isc operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS. These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide. 
11425749	 Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip  analysis were confirmed by hybridization of radiolabeled gene-specific probes to  genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results  suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens. 
11445539	 A burgeoning list of small RNAs with a variety of regulatory functions has been identified in both prokaryotic and eukaryotic cells. However, it remains difficult to identify small RNAs by sequence inspection. We used the high conservation of small RNAs among closely related bacterial species, as well as analysis of transcripts detected by high-density oligonucleotide probe arrays, to predict the presence of novel small RNA genes in the intergenic regions of the Escherichia coli genome. The existence of 23 distinct new RNA species was confirmed by Northern analysis. Of these, six are predicted to encode short ORFs, whereas 17 are likely to be novel functional small RNAs. We discovered that many  of these small RNAs interact with the RNA-binding protein Hfq, pointing to a global role of the Hfq protein in facilitating small RNA function. The approaches used here should allow identification of small RNAs in other organisms. 
11404467	 Symbiotic associations with microorganisms are pivotal in many insects. Yet, the  functional roles of obligate symbionts have been difficult to study because it has not been possible to cultivate these organisms in vitro. The medically important tsetse fly (Diptera: Glossinidae) relies on its obligate endosymbiont,  Wigglesworthia glossinidia, a member of the Enterobacteriaceae, closely related to Escherichia coli, for fertility and possibly nutrition. We show here that the  intracellular Wigglesworthia has a reduced genome size smaller than 770 kb. In an attempt to understand the composition of its genome, we used the gene arrays developed for E. coli. We were able to identify 650 orthologous genes in Wigglesworthia corresponding to approximately 85% of its genome. The arrays were  also applied for expression analysis using Wigglesworthia cDNA and 61 gene products were detected, presumably coding for some of its most abundant products. Overall, genes involved in cell processes, DNA replication, transcription, and translation were found largely retained in the small genome of Wigglesworthia. In addition, genes coding for transport proteins, chaperones, biosynthesis of cofactors, and some amino acids were found to comprise a significant portion, suggesting an important role for these proteins in its symbiotic life. Based on its expression profile, we predict that Wigglesworthia may be a facultative anaerobic organism that utilizes ammonia as its major source of nitrogen. We present an application of E. coli gene arrays to obtain broad genome information  for a closely related organism in the absence of complete genome sequence data. 
11259426	 We describe statistical methods based on the t test that can be conveniently used on high density array data to test for statistically significant differences between treatments. These t tests employ either the observed variance among replicates within treatments or a Bayesian estimate of the variance among replicates within treatments based on a prior estimate obtained from a local estimate of the standard deviation. The Bayesian prior allows statistical inference to be made from microarray data even when experiments are only replicated at nominal levels. We apply these new statistical tests to a data set  that examined differential gene expression patterns in IHF(+) and IHF(-) Escherichia coli cells (Arfin, S. M., Long, A. D., Ito, E. T., Tolleri, L., Riehle, M. M., Paegle, E. S., and Hatfield, G. W. (2000) J. Biol. Chem. 275, 29672-29684). These analyses identify a more biologically reasonable set of candidate genes than those identified using statistical tests not incorporating a Bayesian prior. We also show that statistical tests based on analysis of variance and a Bayesian prior identify genes that are up- or down-regulated following an experimental manipulation more reliably than approaches based only on a t test or fold change. All the described tests are implemented in a simple-to-use web interface called Cyber-T that is located on the University of California at Irvine genomics web site. 
11325924	 Replacement of the native fermentation pathway in Escherichia coli B with a homo-ethanol pathway from Zymomonas mobilis (pdc and adhB genes) resulted in a 30 to 50% increase in growth rate and glycolytic flux during the anaerobic fermentation of xylose. Gene array analysis was used as a tool to investigate differences in expression levels for the 30 genes involved in xylose catabolism in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total open reading frames, only 8% were expressed at a significantly higher level in KO11 (P < 0.05). In contrast, over half of the 30 genes involved in the catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher levels in KO11. For 14 of the 30 genes, higher expression was statistically significant at the 95% confidence level (xylAB, xylE, xylFG, xylR, rpiA, rpiB, pfkA, fbaA, tpiA, gapA, pgk, and pykA) during active fermentation (6, 12, and 24  h). Values at single time points for only four of these genes (eno, fbaA, fbaB, and talA) were higher in strain B than in KO11. The relationship between changes  in mRNA (cDNA) levels and changes in specific activities was verified for two genes (xylA and xylB) with good agreement. In KO11, expression levels and activities were threefold higher than in strain B for xylose isomerase (xylA) and twofold higher for xylulokinase (xylB). Increased expression of genes involved in xylose catabolism is proposed as the basis for the increase in growth rate and glycolytic flux in ethanologenic KO11. 
11282649	 In an effort to efficiently discover genes in the diazotrophic endophyte of maize, Klebsiella pneumoniae 342, DNA from strain 342 was hybridized to a microarray containing 96% (n = 4,098) of the annotated open reading frames from Escherichia coli K-12. Using a criterion of 55% identity or greater, 3,000 (70%)  of the E. coli K-12 open reading frames were also found to be present in strain 342. Approximately 24% (n = 1,030) of the E. coli K-12 open reading frames are absent in strain 342. For 1.6% (n = 68) of the open reading frames, the signal was too low to make a determination regarding the presence or absence of the gene. Genes with high identity between the two organisms are those involved in energy metabolism, amino acid metabolism, fatty acid metabolism, cofactor synthesis, cell division, DNA replication, transcription, translation, transport, and regulatory proteins. Functions that were less highly conserved included carbon compound metabolism, membrane proteins, structural proteins, putative transport proteins, cell processes such as adaptation and protection, and central intermediary metabolism. Open reading frames of E. coli K-12 with little or no identity in strain 342 included putative regulatory proteins, putative chaperones, surface structure proteins, mobility proteins, putative enzymes, hypothetical proteins, and proteins of unknown function, as well as genes presumed to have been acquired by lateral transfer from sources such as phage, plasmids, or transposons. The results were in agreement with the physiological properties of the two strains. Whole genome comparisons by genomic interspecies microarray hybridization are shown to rapidly identify thousands of genes in a previously uncharacterized bacterial genome provided that the genome of a close relative has been fully sequenced. This approach will become increasingly more useful as more full genome sequences become available. 
11283591	 A prototype biosensor array has been assembled from engineered RNA molecular switches that undergo ribozyme-mediated self-cleavage when triggered by specific  effectors. Each type of switch is prepared with a 5'-thiotriphosphate moiety that permits immobilization on gold to form individually addressable pixels. The ribozymes comprising each pixel become active only when presented with their corresponding effector, such that each type of switch serves as a specific analyte sensor. An addressed array created with seven different RNA switches was  used to report the status of targets in complex mixtures containing metal ion, enzyme cofactor, metabolite, and drug analytes. The RNA switch array also was used to determine the phenotypes of Escherichia coli strains for adenylate cyclase function by detecting naturally produced 3',5'- cyclic adenosine monophosphate (cAMP) in bacterial culture media. 
11237321	 Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of  Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears  to be selective for mRNA. These and additional studies of genome-wide expression  with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs. 
11226277	 Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E.  coli indicated elevated expression of expected and unanticipated genes. Selected  luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon. 
11157263	 We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager. 
11149947	 Elucidating the genetic basis of adaptation on a genomewide scale has evaded biologists, but complete genome sequences and DNA high-density array technology make genomewide surveys more tractable. Six lines of Escherichia coli adapted for 2,000 generations to a stressful high temperature of 41.5 degrees C were examined on a genomewide scale for duplication/deletion events by using DNA high-density arrays. A total of five duplication and deletion events were detected. These five events occurred in three of the six lines, whereas the remaining three lines contained no detectable events. Three of the duplications were at 2.85 Mb of the  E. coli chromosome, providing evidence for the replicability of the adaptation to high temperature. Four candidate genes previously shown to play roles in stress and starvation survival were identified in the region of common duplication. Expression of the two candidate genes examined is elevated over expression levels in the ancestral lines or the lines without the duplication. In the two cases where the duplication at 2.85 Mb has been further characterized, the timing of the genome reorganization is coincident with significant increases in relative fitness. In both of these cases, the model for the origin of the duplication is a complex recombination event involving insertion sequences and repeat sequences. These results provide additional evidence for the idea that gene duplication plays an integral role in adaptation, specifically as a means for gene amplification. 
11339905	 We consider the problem of inferring fold changes in gene expression from cDNA microarray data. Standard procedures focus on the ratio of measured fluorescent intensities at each spot on the microarray, but to do so is to ignore the fact that the variation of such ratios is not constant. Estimates of gene expression changes are derived within a simple hierarchical model that accounts for measurement error and fluctuations in absolute gene expression levels. Significant gene expression changes are identified by deriving the posterior odds of change within a similar model. The methods are tested via simulation and are applied to a panel of Escherichia coli microarrays. 
11121068	 Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription  of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons  encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends  on the nitrogen assimilation control protein, which serves as an adapter between  NtrC and final sigma(70)-dependent promoters. 
11101804	 We have developed a high-resolution "genome array" for the study of gene expression and regulation in Escherichia coli. This array contains on average one 25-mer oligonucleotide probe per 30 base pairs over the entire genome, with one every 6 bases for the intergenic regions and every 60 bases for the 4,290 open reading frames (ORFs). Twofold concentration differences can be detected at levels as low as 0.2 messenger RNA (mRNA) copies per cell, and differences can be seen over a dynamic range of three orders of magnitude. In rich medium we detected transcripts for 97% and 87% of the ORFs in stationary and log phases, respectively. We found that 1, 529 transcripts were differentially expressed under these conditions. As expected, genes involved in translation were expressed at higher levels in log phase, whereas many genes known to be involved in the starvation response were expressed at higher levels in stationary phase. Many previously unrecognized growth phase-regulated genes were identified, such as a putative receptor (b0836) and a 30S ribosomal protein subunit (S22), both of which are highly upregulated in stationary phase. Transcription of between 3,000  and 4,000 predicted ORFs was observed from the antisense strand, indicating that  most of the genome is transcribed at a detectable level. Examples are also presented for high-resolution array analysis of transcript start and stop sites and RNA secondary structure. 
10974115	 Escherichia coli was adapted to grow in medium containing substantially elevated  concentrations of either Zn(II), Cd(II), Co(II) or Ni(II). Whole-genome transcriptional profiles were generated from adapted strains and analysed for significant alteration in transcript abundance with reference to a wild-type strain. Similar alterations in specific message levels were observed for strains  adapted to the four metal ions. One unexpected trend was the increase in transcript level of genes involved in transposition of IS elements, particularly  insA. Subsequent expression of insA-7 from a heterologous promoter in E. coli conferred tolerance to Zn(II). 
11056062	 It has been commonly observed that gratuitous overexpression of proteins in Escherichia coli causes growth retardation. However, the molecular events involved in the metabolic response to the over-expression of proteins are still unclear. Here we used DNA microarray technology to characterize the changes in transcriptional patterns of selected host genes during protein overexpression. A  nontoxic, soluble protein, LuxA (coded by luxA), which is the alpha-subunit of the luciferase heterodimer, was overexpressed for this purpose. A total of 132 E. coli genes, including those in the central metabolism, key biosynthetic pathways, and selected regulatory functions, were used as probes for detecting the level of mRNA transcripts in E. coli strains JM109, MC4100, and VJS676A during protein overexpression. Upon induction, these strains shared several common responses, such as the upregulation of glk and the heat shock genes as well as the downregulation of fba, ppc, atpA, and gnd. In addition, the biosynthesis genes glnA, glyA, and leuA were downregulated in all three strains. Media-dependent responses were also observed in our study. For example, many respiratory genes that were upregulated in defined media showed an opposite effect in complex media under protein-overproducing conditions. These results demonstrate that gratuitous overexpression of proteins triggers a complex global response that involves several metabolic and regulatory systems. Explanations based on either existing knowledge of global regulations such as the heat shock response and the stringent response or stoichiometric analysis without regulatory considerations cannot account for the response induced by protein overexpression. 
10753455	 DNA microarray technology was applied to detect differential transcription profiles of a subset of the Escherichia coli genome. A total of 111 E. coli genes, including those in central metabolism, key biosyntheses, and some regulatory functions, were cloned, amplified, and used as probes for detecting the level of transcripts. An E. coli strain was grown in glucose, acetate, and glycerol media, and the transcript levels of the selected genes were detected. Despite extensive studies on E. coli physiology, many new features were found in  the regulation of these genes. For example, several genes were unexpectedly up-regulated, such as pps, ilvG, aroF, secA, and dsbA in acetate and asnA and asnB in glycerol, or down-regulated, such as ackA, pta, and adhE in acetate. These genes were regulated with no apparent reasons by unknown mechanisms. Meanwhile, many genes were regulated for apparent purposes but by unknown mechanisms. For example, the glucose transport genes (ptsHI, ptsG, crr) in both acetate and glycerol media were down-regulated, and the ppc, glycolytic, and biosynthetic genes in acetate were also down-regulated because of the reduced fluxes. However, their molecular mechanisms remain to be elucidated. Furthermore, a group of genes were regulated by known mechanisms, but the physiological roles  of such regulation remain unclear. This group includes pckA and aspA, which are up-regulated in glycerol, and gnd and aspA, which are down- and up-regulated, respectively, in acetate. The DNA microarray technology demonstrated here is a powerful yet economical tool for characterizing gene regulation and will prove to be useful for strain improvement and bioprocess development. 
10718123	 The human genome contains an estimated 3 billion bases of DNA making up some 100000 genes, and the variation within this genome accounts for human diversity and, in many cases, disease. Defining and understanding the expression profile of given genotypes is essential to understanding adverse effects from acute or chronic exposure to environmental toxicants or other stimuli. DNA array technology could help researchers understand how organisms function in response to exposure by elucidating the molecular mechanisms that underlie them. DNA arrays have been developed and refined over the past 5 years and matured into a relatively accessible and affordable technology. They vary in design from membrane-based filters with a few hundred cDNAs, to glass-based 'chips' with tens of thousands of genetic elements. Mammalian DNA arrays will soon allow expression analysis on a genome-wide scale, similar to that already accomplished in some lower organisms (e.g. S. cerevisiae, E. coli). These whole-genome arrays will be  powerful tools for identifying and characterizing toxicants in environmental and  pharmaceutical science. This review discusses the technology behind the production of DNA arrays, the options available to those interested in applying them to their own research, and the possible toxicological applications of this exciting new technology. 
11173827	 Combining chemotherapy with radiotherapy has improved the cure rate among patients with cancers of the cervix. Although one-half to two-thirds of the patients can be cured by radiation alone, such patients cannot be identified at present and must therefore suffer the burden of chemotherapy. Our long-range goal is to identify those cervical cancers that are radiosensitive and could be cured  by radiotherapy alone. The advent of methods that permit the simultaneous analysis of expression patterns of thousands of genes, make it feasible to attempt to identify the molecular events related to radiosensitivity and the associated regulatory pathways. We hypothesize that the sensitivity of tumor cells to ionizing radiation (IR) is determined by the level of expression of specific genes that may be identified with the aid of cDNA microarrays. As the first step in testing this hypothesis, we determined the gene expression differences between two cell lines exhibiting different degrees of radiosensitivity. These were derived from the same tumor prior to treatment from  a patient with squamous cell carcinoma of the cervix. The mRNA from these cells was subjected to cDNA analysis on a microarray of 5,776 known genes and ESTs. The expression of 52 genes of the total of 5,776 was elevated (maximum 4.1 fold) in the radioresistant cells as compared to the radiosensitive cells. Ten of the 52 sequences are known genes while 42 are ESTs. Conversely, the expression of 18 genes was elevated in the sensitive cells as compared to the resistant cells. Seven of these 18 are known genes while eleven are ESTs. Among the genes expressed differentially between the resistant and sensitive cells were several known to be associated with response to IR and many more genes and ESTs that had  not previously been reported to be related to radiosensitivity. The genes that showed the greatest overexpression in the radioresistant cell line were metal-regulatory transcription factor-1, cytochrome P450 CYP1B1, adenomatosis polyposis coli, translation elongation factor-1, cytochrome-c oxidase, whereas in the sensitive cell line, transcription factor NF-kappa-B, metalloproteinase inhibitor-1 precursor, superoxide dismutase-2, insulin-like growth factor binding protein-3, guanine nucleotide-binding protein and transforming growth factor beta-induced protein were overexpressed. 
9813115	 A major mode of gene regulation occurs via the binding of specific proteins to specific DNA sequences. The availability of complete bacterial genome sequences offers an unprecedented opportunity to describe networks of such interactions by  correlating existing experimental data with computational predictions. Of the 240 candidate Escherichia coli DNA-binding proteins, about 55 have DNA-binding sites  identified by DNA footprinting. We used these sites to construct recognition matrices, which we used to search for additional binding sites in the E. coli genomic sequence. Many of these matrices show a strong preference for non-coding  DNA. Discrepancies are identified between matrices derived from natural sites and those derived from SELEX (Systematic Evolution of Ligands by Exponential enrichment) experiments. We have constructed a database of these proteins and binding sites, called DPInteract (available at http://arep.med.harvard.edu/dpinteract). 
8652576	 T4 RegB endonuclease specifically cleaves at -GGAG- sites in several early T4 messages, rendering them nonfunctional. Not all -GGAG- sites are processed equally by RegB; those found at the Shine-Dalgarno sequences and in intercistronic regions are processed with higher efficiency than the -GGAG- sites located in coding regions. The low activity of RegB observed in vitro is enhanced by 1-2 orders of magnitude by the Escherichia coli ribosomal protein S1. We have  used SELEX (systematic evolution of ligands by exponential enrichment) on a combinatorial RNA library to obtain molecules that are specifically cleaved by T4 RegB endonuclease in the presence of S1. The sequences obtained contain the required -GGAG- tetranucleotide and were unusually enriched in adenosine and cytosine nucleotides. No consensus structure or sequence motif other than -GGAG-  was conserved among the selected molecules. The majority of the RNAs are entirely dependent on S1 for RegB-catalyzed cleavage; however, a few RNAs are found to be  S1 independent but are cleaved by RegB with much lower rates. 
8568875	 The SELEX procedure was used to study the recognition between the E. coli methionine repressor (MetJ) and its DNA binding sites. DNA ligands with high affinity for either the holo-repressor or apo-repressor were isolated from a pool of molecules randomized over 20 base-pairs. Among 90 DNA ligands selected by holo-repressor binding, roughly 90% contain variations of two tandem, perfect eight base-pair Met-boxes, which are the consensus deduced from natural met operators. Base-pairs that are important, for specific interactions with the protein are highly conserved. The data also reveal the importance of the non-contacted operator base-pairs in facilitating the conformational changes in the operator which must occur for repressor binding. There are also effects due to the sequences of the base-pairs immediately flanking the operator site. DNA ligands selected by apo-repressor share a very similar, but not identical, consensus with that selected by holo-repressor, suggesting that the corepressor does not greatly alter the specificity of repressor binding.