NsrR targets in the Escherichia coli genome: new insights
Jonathan D. Partridge ,1 Diane M. Bodenmiller ,2 † Michael S. Humphrys2 ‡ and Stephen Spiro1 * 1Department of Molecular and Cell Biology , The University of Texas at Dallas , 800 W Campbell Road , Richardson , TX 75080 , USA . 
2School of Biology , Georgia Institute of Technology , 310 Ferst Drive , Atlanta , GA 30332 , USA . 
Summary
The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription . 
The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing . 
By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter . 
We used chromatin immunoprecipitation and microarray analysis ( ChIP-chip ) to show that NsrR binds to 62 sites close to the 5 ends of genes . 
Analysis of the ChIP-chip data suggested that a single 11 bp motif ( with the consensus sequence AANATGCATTT ) can function as an NsrR-binding site in vivo . 
NsrR binds to sites in the promoter regions of the fliAZY , fliLMNOPQR and mqsR-ygiT transcription units , which encode proteins involved in motility and biofilm development . 
Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters . 
A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo . 
Assays on softagar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain ; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR . 
Accepted 6 July , 2009 . 
* For correspondence . 
E-mail stephen.spiro @ utdallas.edu ; Tel. ( +1 ) 972 883 6896 ; Fax ( +1 ) 972 883 2409 . 
Present addresses : † Lilly Research Laboratories , Eli Lilly and Company , India-napolis , IN 46285 , USA . 
‡ Centers for Disease Control and Prevention , 1600 Clifton Road , Atlanta , GA 30333 , USA . 
Introduction
Nitric oxide ( NO ) is synthesized by the inducible nitric oxide synthase in phagocytic cells and is an important component of the innate immune response to infection ( Fang , 2004 ) . 
NO is also made by some bacteria , either as a by-product of nitrite reduction to ammonia , or as an intermediate of denitrification ( Watmough et al. , 1999 ) . 
Thus , pathogenic bacteria can potentially be exposed both to endogenously generated NO and to the NO produced by host cells . 
In Escherichia coli , the transcriptional regulators NorR and NsrR mediate adaptive responses to NO by controlling the expression of genes encoding enzymes that reduce or oxidize NO to less toxic species ( Mukhopadhyay et al. , 2004 ; Bodenmiller and Spiro , 2006 ; Spiro , 2007 ) . 
The key NO detoxifying enzymes are the flavohaemoglobin ( encoded by the hmp gene ) and the flavorubredoxin ( encoded by norVW ) , which are regulated by NsrR and NorR respectively ( Hutchings et al. , 2002 ; Gardner et al. , 2003 ; Bodenmiller and Spiro , 2006 ) , and the respiratory nitrite reductase , Nrf , which reduces both nitrite and NO to ammonia ( Poock et al. , 2002 ) . 
The s54-dependent transcriptional activator NorR is stimulated by the formation of a nitrosyl species at a mono-nuclear iron site in its signalling domain ( D ′ Autréaux et al. , 2005 ) . 
In the case of NsrR , the binding site for NO is likely to be an [ Fe-S ] cluster ( Isabella et al. , 2008 ; Tucker et al. , 2008 ; Yukl et al. , 2008 ) . 
We initially identified NsrR as a repressor of the ytfE , hmp and ygbA genes ( Bodenmiller and Spiro , 2006 ) . 
The product of the ytfE gene is a di-iron protein , which has been implicated in the repair of damaged [ Fe-S ] clusters ( Justino et al. , 2007 ) . 
A YtfE homologue ( NorA ) from Ralstonia eutropha has been shown to bind NO , and has been suggested to function to lower the cytoplasmic NO concentration ( Strube et al. , 2007 ) . 
The ygbA gene is of unknown function , while hmp encodes a well-characterized NO detoxification system ( Poole and Hughes , 2000 ) . 
Subsequently described targets for NsrR regulation include the hcp-hcr and yeaR-yoaG genes , and the nrf operon that encodes Nrf ( Filenko et al. , 2007 ; Lin et al. , 2007 ) . 
Known and predicted targets for NsrR regulation have in their promoter regions an 11 bp inverted repeat with a spacing of 1 bp ( Rodionov et al. , 2005 ; Bodenmiller and Spiro , 2006 ; Lin et al. , 2007 ) . 
In this paper we confirm that this sequence is required for NsrRmediated repression of the ytfE promoter , but also present evidence to suggest that a single copy of the 11 bp motif may be sufficient for NsrR binding . 
The full extent of the NsrR regulon of E. coli has been assessed computationally ( Rodionov et al. , 2005 ) , and by analysis of the transcriptome of a strain in which NsrR was depleted by the presence of multiple copies of a cloned NsrR-binding site ( Filenko et al. , 2007 ) . 
As a complementary approach to identifying genes that might be regulated by NsrR , we describe in this paper the use of chromatin immunoprecipitation and microarray analysis ( ChIP-chip ) to locate NsrR-binding sites in the E. coli genome . 
Computational analysis of newly identified targets revealed additional insights into the requirements for a functional NsrR target site . 
Unexpectedly , we found NsrR-binding sites associated with the promoter regions of three transcription units containing genes with well-established or suspected roles in motility and/or biofilm development . 
We confirmed that two of the three promoters are subject to regulation by NsrR and NO , and showed that NsrR is a negative regulator of motility in E. coli . 
Results
Isolation of mutations in the NsrR-binding site We have previously shown that NsrR regulates the ytfE , hmp and ygbA promoters , and have predicted the sequences of the NsrR-binding sites in these promoters ( Bodenmiller and Spiro , 2006 ) . 
There is also a predicted NsrR-binding site in the promoter region of the hcp-hcr genes ( Rodionov et al. , 2005 ) , which encode the hybrid cluster ( prismane ) protein and an associated redox enzyme , and NsrR is a repressor of hcp-hcr transcription ( Filenko et al. , 2007 ) . 
We analysed the 5 ′ non-coding regions of these four transcription units for the occurrence of candidate cis-acting regulatory sequences , using the MEME algorithm ( Bailey et al. , 2006 ) . 
MEME detected the previously predicted NsrR-binding sites , and further suggested the presence of a second NsrR-binding site in the ygbA , hcp and hmp promoters . 
In each case , the primary ( previously described ) NsrR sites overlap the -10 and/or transcription start site ( Fig. 1A ) , while the secondary sites are further upstream . 
The seven predicted sites ( Fig. 1A ) give rise to the sequence logo depicted in Fig. 1B , which is very similar to the logo previously generated for NsrR-binding sites in a group of Enteric bacteria ( Rodionov et al. , 2005 ) , with the addition of two AT base pairs which are present at the 5 ′ ends of all seven E. coli sites ( Fig. 1 ) . 
A similar sequence can also be found in the yeaR promoter , which is regulated directly by NsrR ( Lin et al. , 2007 ) . 
The presence in the cell of multiple copies of the putative NsrR-binding site from the ytfE promoter causes de-repression of a ytfE -- lacZ reporter fusion , by a repressor titration effect ( Bodenmiller and Spiro , 2006 ) . 
Deletion of a single AT base pair at the centre of the NsrR-binding site eliminated repressor titration ( Bodenmiller and Spiro , 2006 ) . 
We selected the NsrR-binding site in ytfE as a a. Wild type and mutant ytfE promoter sequences cloned in pSTBlue-1 were transformed into a strain with a ytfE-lacZ reporter fusion . 
Activities reflect de-repression of the ytfE promoter by repressor titration . 
b. Wild type and mutant promoters were fused to lacZ and integrated in the chromosome . 
Cultures were grown anaerobically in a minimal medium . 
For treatment with nitrite , cultures were grown to midexponential phase , supplemented with 5 mM nitrite , then assayed 60 -- 90 min later . 
c. Wild type ytfE promoter assayed in a DnsrR background . 
d . 
The deletion mutation eliminates ytfE promoter activity , presumably because it is located in the -10 sequence . 
ND , not done . 
model for further study , and sought to isolate additional mutations in this sequence that impair NsrR binding . 
We subjected the 205 bp ytfE promoter fragment to random mutagenesis , and screened on lactose indicator media for clones with lower activities in the repressor titration assay . 
By picking Lac - colonies , we repeatedly isolated the same 1 bp deletion that we had previously made by site directed mutagenesis . 
We assume that the run of four AT base pairs in the NsrR-binding site in ytfE is prone to deletion by slipped-strand mispairing during the mutagenesis reaction . 
This mutation eliminates activity in the repressor titration assay ( Table 1 ) , and ytfE promoter activity , presumably because the deletion is in the -10 sequence ( we were unable to isolate Lac + fusion phages to assay the activity of this mutant ytfE promoter ) . 
By screening for a partial phenotype in the repressor titration assay ( pale blue colonies ) , we isolated two substitutions at positions 2 and 6 in the NsrR-binding site ( Fig. 1B ) . 
Both caused a defect in the repressor titration assay , and de-repression of the ytfE promoter in both the absence and presence of nitrite ( Table 1 ) . 
The two mutations isolated by random mutagenesis are at positions that are almost completely conserved in known and predicted NsrR-binding sites , and introduce nucleotides that never occur in known and predicted sites ( Fig. 1 ; Table 2 ) , with the single exception of a C at position 6 in the MEME-predicted second site in hmp ( Fig. 1A ) . 
Additional mutations were introduced into the NsrR-binding site in ytfE by site-directed mutagenesis . 
Mutations corresponding to those previously isolated at positions 2 and 6 were made in the right half of the inverted repeat , at positions 22 and 18 respectively ( Fig. 1B ) . 
We also made mutations at the symmetry-related positions 5 and 19 , and made three double mutants in which symmetry-related single mutations were combined ( Fig. 1B , Table 1 ) . 
Repressor titration assays showed that all single mutations significantly reduced the ability of the sites to titrate NsrR , and that single mutations at symmetry-related positions had similar effects ( mutations 2 and 22 being the most severe ) . 
In all three cases , symmetry-related double mutations had larger effects than either of the corresponding single mutations ( Table 1 ) . 
Mutant ytfE sequences made by site-directed mutagenesis were also fused to lacZ for assays of promoter activity . 
A similar pattern was found to that seen with the repressor titration assays , in that single mutations caused some de-repression of the promoter , with mutations at symmetry-related positions having similar effects ( Table 1 ) . 
Double mutations caused greater de-repression than the corresponding single mutations , showing that both halves of the site are required for optimal repression of the ytfE promoter . 
Mutations at positions 2 and 22 again caused the most severe phenotypes . 
Induction by nitrite increased the activities of promoters with mutations at positions 2 and 22 between 2.5 - and 3.6-fold , but between 8.8 - and 14.7-fold for all of the other promoters ( Table 1 ) . 
The reduced induction ratio is due to particularly high activities for the 2 , 22 and 2 + 22 mutants in cultures grown anaerobically in the absence of nitrite ( Table 1 ) . 
The reason why mutations at these positions have a disproportionate effect on promoter activity under noninducing conditions is not known . 
All of the promoters remain somewhat inducible by nitrite , demonstrating that none of the mutations completely eliminates NsrR binding . 
We tested binding of NsrR to a selection of the mutant ytfE promoters in vivo by chromatin immunoprecipitation ( ChIP ) . 
For these experiments , the NsrR protein was modified by addition of a C-terminal 3XFlag tag ; the modi-fied protein was expressed from a single-copy gene at the nsrR locus on the chromosome ( Efromovich et al. , 2008 ) . 
Cultures were transformed with pSTBlue-1 derivatives containing wild type and mutant ytfE promoters ( the same plasmids used for the repressor titration assays ) . 
After ChIP , the immunoprecipitated DNAs were used as templates for PCRs using vector-specific primers designed to amplify ytfE sequences . 
Amplification conditions were optimized to allow detection of NsrR binding to the wildtype site in ytfE ( Fig. 1D , compare lanes 1 and 2 ) . 
Under these conditions , binding to the deletion mutant was barely detectable , and all of the single and double point mutations tested showed significantly reduced binding ( Fig. 1D ) . 
Quantitative conclusions can not be drawn from this experiment , but it nevertheless shows that all of the mutations reduce binding in vivo sufficiently to severely impair detection by ChIP , under conditions that allow detection of the wild type interaction . 
Our results with the ytfE promoter are consistent with previous suggestions that the NsrR consensus-binding site in E. coli consists of two copies of the sequence 5 ′ - AAGATGCYTTT-3 ′ arranged as an inverted repeat separated by 1 bp ( Rodionov et al. , 2005 ; Bodenmiller and Spiro , 2006 ; Lin et al. , 2007 ) . 
Results presented later in this paper suggest that a single 11 bp motif can also function as an NsrR-binding site . 
Genome-wide search for NsrR-binding sites We next used ChIP-chip to identify NsrR-binding sites in the genome of E. coli K12 strain MG1655 . 
Cultures expressing the 3XFlag-tagged NsrR were grown anaerobically in the presence and absence of nitrate . 
Under anaerobic growth conditions , nitrate provides a source of endogenously generated NO and causes de-repression of NsrR targets ( Bodenmiller and Spiro , 2006 ) . 
After ChIP , the precipitated DNAs were labelled with Cy5 and Cy3 and hybridized together to a high-density microarray ( from Oxford Gene Technology ) . 
Peaks in the fluorescence ratio therefore identify regions of the chromosome that are bound by NsrR , with the degree of occupancy of sites being greater in the culture grown in the absence of nitrate . 
Full technical details of this experiment and statistical procedures used for data analysis have been published ( Efromovich et al. , 2008 ) . 
The ChIP-chip data for the three originally identified NsrR targets ( ytfE , hmp and ygbA ) are shown in Fig. 2 . 
The ChIP-chip data show only a weak signal for NsrR binding at the ygbA promoter ( Fig. 2 ) , which is known to be regulated by NsrR in vivo ( Bodenmiller and Spiro , 2006 ) . 
Thus we required rigorous methods to identify other weak signals in the data that may represent bona fide cis-acting regulatory sites . 
We adopted three approaches to this problem . 
First , we scrutinized the three datasets individually , and looked for peaks in which two or more consecutive probes showed a greater than twofold enrichment . 
If a peak met these criteria in at least two of the three samples , then it was recorded as positive . 
Twenty-nine peaks were identified in this way , of these nine were not considered further on the grounds that they were between convergently transcribed genes or deep within coding regions ( here defined as > 300 bp from the start codon ) . 
This method did not identify the ygbA peak , so may be too conservative . 
Next , we analysed the three datasets independently with ChIPOTle ( Buck et al. , 2005 ) , and scored a peak as positive if it was significant ( P < 0.0001 ) in at least two of the three datasets . 
This analysis identified an additional 41 peaks ( including ygbA ) of which 16 were discarded as internal sites or sites between convergent genes . 
Finally , we used a novel method for peak detection ( Efromovich et al. , 2008 ) which , disregarding sites deep within coding regions , between convergently transcribed genes or with very small mean enrichment ratios ( < 1.5 ) , identified an additional 17 NsrR-binding sites . 
The final output of 62 NsrR-binding sites in or close to 5 ′ non-coding regions is shown in Table 2 . 
Previously identified and novel NsrR-binding sites The presence of NsrR-binding sites in or near to 5 ′ noncoding regions identifies genes that potentially belong to the NsrR regulon . 
Of the promoters bound by NsrR in vivo ( Table 2 ) , eight not previously known to be regulated by NsrR ( hypA/hycA , acs , aceE , ydcX , putA , ndh and sodB ) show responses to nitrite in transcriptomics experiments that are consistent with positive or negative regulation by NsrR ( Constantinidou et al. , 2006 ) . 
Most known NsrR regulon members ( ytfE , yeaR-yoaG , hmp , hcp-hcr and ygbA ) show differential regulation in an asymptomatic strain of E. coli growing in the urinary tract ( Roos and Klemm , 2006 ) . 
Potential NsrR targets ydcX , dsdX , ndh and tehAB ( Table 2 ) are also upregulated in the urinary tract ( Roos and Klemm , 2006 ) and so share an expression pattern with genes known to be regulated by NsrR . 
Several of the other targets listed in Table 2 have been reported to respond to sources of NO or to S-nitrosoglutathione in other transcriptomics experiments ( Justino et al. , 2005 ; Hyduke et al. , 2007 ; Pullan et al. , 2007 ; Bourret et al. , 2008 ; Jarboe et al. , 2008 ) . 
In total , of the 62 targets implicated by the ChIP-chip data , 33 have been previously shown to be influenced by NsrR and/or sources of NO or nitrosative stress ( Table 2 ) . 
Nine transcription units were previously suggested by transcriptomics to be repressed by NsrR ( Filenko et al. , 2007 ) ; the promoter regions of seven of these ( ytfE , hmp , hcp , nrfA , yccM , ygbA and napF ) are bound by NsrR in vivo according to the ChIP-chip data ( Table 2 ) , and two ( uspF and yeaR ) are not . 
Visual inspection of the raw ChIP-chip data confirmed the absence of signals for uspF and yeaR . 
Direct regulation of yeaR by NsrR has been demonstrated ( Lin et al. , 2007 ) , hence this is a true false-negative in the ChIP-chip data . 
One possible explanation is that the 3X-Flag tag on NsrR is occluded by other proteins bound to the yeaR promoter . 
Overexpression of NsrR causes reduced expression of the small RNA RybB and of the rpoE gene encoding sE ( Thompson et al. , 2007 ) . 
Neither gene is bound by NsrR in its promoter region according to the ChIP-chip data . 
We assume that these are also false negatives , or that NsrR regulation of rybB and rpoE is indirect . 
The transcriptomics data provided good evidence of one gene positively regulated by NsrR , ydbC ( Filenko et al. , 2007 ) . 
We found no NsrR-binding site in the ydbC promoter , though there is a site in the downstream gene , ydbD ( Table 2 ) , which is upregulated by NO ( Justino et al. , 2005 ) . 
More than 50 promoters implicated as NsrR targets by ChIP-chip were not identified in a transcriptomics experiment in which an nsrR mutation was phenocopied by repressor titration ( Filenko et al. , 2007 ) . 
In some cases this may simply be because NsrR binding to DNA has no regulatory consequence . 
More likely explanations are that the repressor titration approach used was not very sensitive ( see below ) , and/or that some NsrR targets are subject to additional layers of regulation , such that they would not be identified in a straightforward analysis of the transcriptome under a limited range of growth conditions . 
The latter consideration applies , for example , to the tynA and feaB genes , which were identified as potential NsrR targets by ChIP-chip ( Table 2 ) , but which are subject to regulation by NsrR only in cultures grown on unusual carbon or nitrogen sources ( Rankin et al. , 2008 ) . 
In several cases , NsrR-binding sites are close to the 5 ′ ends of genes that are ( or are probably ) internal to single transcription units , and therefore are not associated with promoters ; examples are feoB , hcr and nrdB ( Table 2 ) . 
The regulatory significance , if any , of these sites is not known ; hcr is particularly interesting because the promoterproximal gene of the operon ( hcp ) also has an NsrR-binding site and is regulated by NsrR ( Filenko et al. , 2007 ) . 
The new potential targets for NsrR regulation ( Table 2 ) include genes and operons involved in carbon and energy metabolism ( hycA/hypA , feaB , aceE , mhpT , tynA , caiA and ndh ) , NO metabolism ( norR/norV ) , proteolysis ( clpB , ftsH and ptrA ) , transport processes ( mhpT , yhfC , dsdX and yhfC ) , stress responses ( sodB and sufA ) and motility ( mqsR , fliL and fliA ) . 
In an initial follow-up study , we have confirmed NsrR regulation of tynA and feaB ( Rankin et al. , 2008 ) . 
Computational analysis of NsrR-binding sites Non-coding regions that contain NsrR-binding sites as revealed by ChIP-chip were initially scrutinized for common potential regulatory sequences using WEEDER ( Pavesi et al. , 2004 ) . 
This search suggested that most novel potential NsrR targets do not contain a sequence resembling the long inverted repeat that is present in the ytfE , hmp and ygbA promoters ( Fig. 1 ) . 
However , a sequence resembling half of the inverted repeat could be detected in many cases . 
To extend this search , we constructed a position-specific score matrix ( PSSM ) from the six easily detectable half-sites in the ytfE , hmp and ygbA promoters and used the PSSM to search the E. coli genome with Virtual Footprint ( Münch et al. , 2005 ) . 
As new half sites in promoters known to be bound by NsrR ( Table 2 ) were detected , they were added to the PSSM and the search was repeated iteratively . 
In this way , we identified 49 potential NsrR-binding sites in 37 of the intergenic regions to which NsrR binds in vivo ( Table 2 ) . 
The sequence logo for these 49 sites is shown in Fig. 1C , which reveals that the consensus sequence for the suggested NsrR-binding site contains a 12 bp interrupted partial palindrome , 5 ′ - AANATGCATTTN-3 ′ , corresponding to one half of the previously described inverted repeat sequence ( Fig. 1A ; see above ) . 
In a number of promoters , the computational search failed to identify significant matches to the PSSM ( Table 2 ) . 
Similarly , in several other ChIP-chip studies , binding sites have been identified in chromosomal regions that do not contain a good match to the consensus sequence for the regulatory protein concerned ( reviewed in Wade et al. , 2007 ) . 
Regulation of motility genes by NsrR
We were particularly interested to observe NsrR-binding sites associated with the promoter regions of transcription units containing genes that are known ( fliAZY and fliLM-NOPQR ) or suspected ( mqsR-ygiT ) to have roles in motility and sessile growth ( Fig. 2 ) . 
The fliA gene encodes the alternative sigma factor , s28 , which is required for the transcription of Class III motility and chemotaxis genes ( Chilcott and Hughes , 2000 ) . 
The fliZ gene , which is co-transcribed with fliA , encodes a protein which acts as a positive regulator of motility , and as an inhibitor of the expression of curli fimbriae , which are required for surface-attached growth ( Pesavento et al. , 2008 ; Saini et al. , 2008 ) . 
The fliLMNOPQR operon encodes structural components of the flagellum and the flagellin export apparatus ( Chilcott and Hughes , 2000 ) . 
The mqsR gene ( which is very likely co-transcribed with ygiT , a predicted regulatory gene ) has been described as a regulator of motility and biofilm formation ( Gonzalez Barrios et al. , 2006 ) . 
The predicted NsrR-binding site in the fliA promoter overlaps the start site for transcription by RNA polymerase containing s28 , the product of the fliA gene ( Fig. 3 ) . 
The site is therefore well situated to mediate negative regulation of fliAZY expression . 
To test the functionality of this site , we substituted the highly conserved G at position 6 ( Fig. 1C ) with a C . 
We chose to mutate position 6 because the equivalent mutation in the ytfE promoter causes a severe phenotype ( Table 1 ) and this nucleotide is located such that the mutation is unlikely to affect either the s70 or the s28 promoter of fliA ( a substitution at position 2 would change the s28 transcription start site ) . 
The G to C mutation is on the bottom strand of the fliA promoter ( Fig. 3A ) ; the mutant promoter is designated fliA c. NsrR binding to the fliA promoter in vivo was examined by ChIP . 
When PCR amplification of immunoprecipitated DNA was optimized to allow detection of NsrR binding to the wild-type fliA promoter , binding to the fliA c promoter was undetectable above background levels ( Fig. 3B ) . 
Thus , these experiments confirm the presence of an NsrR-binding site in the fliA promoter as was suggested by ChIP-chip ( Fig. 2 ) and bioinformatic analysis , and provide experimental support for the revised consensus sequence for NsrR-binding sites . 
To quantify the regulation of motility genes by NsrR , we constructed lacZ reporter fusions to the fliA , fliL and mqsR promoters and measured their activities in an nsrR mutant and a strain containing multiple copies of the nsrR gene , in the presence and absence of a source of NO . 
We found no evidence for regulation of the mqsR promoter by NsrR ( data not shown ) , possibly because we have yet to identify suitable growth conditions that reveal regulation by NsrR . 
The fliA and fliL promoters were 1.9 - and 1.7-fold upregulated in an nsrR mutant respectively , and had moderately increased activities in the presence of a source of NO ( Table 3 ) . 
In the presence of multiple copies of nsrR , the activities of both promoters were 6 -- 7 fold reduced , an effect that was partially reversed by the addition of a source of NO to growth media ( Table 3 ) . 
Taken together , these data indicate that NsrR is a negative regulator of both the fliA and the fliL promoters . 
Assay of a fliA c -- lacZ fusion revealed that the single base pair mutation in the fliA c promoter mimicked the effect of NO in an otherwise wild-type strain ( Table 3 ) . 
The partial de-repression caused by the fliAc mutation could be overcome by the presence of multiple copies of nsrR ( Table 3 ) . 
These results are consistent with the fliAc mutation lowering the affinity of the NsrR-binding site in the fliA promoter , as was suggested by ChIP . 
The fliA -- lacZ reporter fusion was ~ 2-fold upregulated in a strain transformed with a high copy number plasmid containing the cloned ytfE promoter ( data not shown ) . 
This effect of the ytfE promoter was abolished by the deletion at position 12 of the NsrR-binding site ( Fig. 1B ) , suggesting that multiple copies of the NsrR-binding site in ytfE de-repress the fliA promoter by repressor titration ( hence providing additional confirmation of the presence of an NsrR-binding site in fliA ) . 
The small magnitude of the repressor titration effect on fliA likely explains why fliA was not identified as an NsrR target in the transcriptomics analysis ( Filenko et al. , 2007 ) . 
In the reciprocal experiment , multiple copies of the fliA promoter failed to cause de-repression of the ytfE -- lacZ reporter fusion ( and also failed to de-repress the fliA promoter , data not shown ) . 
One possible explanation is that the inverted repeat sequence in ytfE provides a higher affinity NsrR-binding site than the single half site in fliA . 
The same consideration may also explain why deletion of the central base pair in the inverted repeat in ytfE abolishes repressor titration despite preserving two intact half sites . 
Regulation of motility by NsrR
Flagella-based motility was assayed on soft agar plates . 
An nsrR mutant showed a small though reproducible and significant increase in motility ( 1.3-fold ; P < 0.0001 ) as compared with the wild-type strain ( measured as the diameter of the motility ring ; data not shown ) . 
Addition of an NO source caused a similar increase in motility in a wild-type strain but not in an nsrR mutant . 
These observations are consistent with the negative regulation of motility genes by NsrR that was measured in reporter fusion assays . 
In a strain containing multiple copies of nsrR , the motility ring was ~ 2-fold smaller ( P < 0.0001 ) than in a control strain with a single chromosomal copy of nsrR ( Fig. 4 ) . 
This effect of NsrR on motility was reversed in plates supplemented with a slow-releasing source of NO ( Fig. 4 ) , suggesting that inactivation of NsrR alleviates negative control of motility . 
The NsrR protein contains three conserved cysteine residues thought to be involved in the co-ordination of an [ Fe-S ] cluster , which is the likely site of NO sensing ( Isabella et al. , 2008 ; Tucker et al. , 2008 ; Yukl et al. , 2008 ) . 
We have substituted cysteine 96 with serine , and found that the NsrR-C96S variant is unable to repress fully the NsrR targets ytfE , hmp , hcp and ygbA ( J. Partridge and S. Spiro , unpubl . 
data ) . 
The C96S protein also has no negative effect on motility ( data not shown ) , confirming that the effect of NsrR on motility requires the protein to be in a form that is competent to control transcription . 
This excludes the possibility that inhibition of motility by multiple copies of nsrR is a non-specific consequence of protein over-production . 
Motility is a variable and strainspecific phenotype in E. coli . 
In similar assays to those described above , we showed that multiple nsrR copies inhibit motility to a similar extent ( ~ 2-fold ; P < 0.0001 ) in an E. coli K12 strain ( RP437 ) that is frequently used for assays of motility and chemotaxis . 
It has recently been shown that hmp mutants of E. coli are non-motile ( Stevanin et al. , 2007 ) , though on the succinate medium used the phenotype would also be consistent with a defect in aerotaxis . 
As the hmp gene is negatively regulated by NsrR ( Bodenmiller and Spiro , 2006 ) , one possible interpretation of our results is that the motility defect associated with an increased nsrR copy number results from downregulation of hmp . 
We assayed the motility of hmp mutants of MG1655 and RP437 , using both tryptone and succinate soft agar ( Stevanin et al. , 2007 ) . 
With these strains , we found no detectable effect of hmp on E. coli motility . 
Thus , the motility phenotype that we observe when nsrR is deleted or overexpressed is not a consequence of hmp up or downregulation respectively . 
NsrR regulates motility and surface attachment in a uropathogenic strain of E. coli
We were interested to determine whether the effects of NsrR ( and NO ) on motility that we observed in K12 strains are generalizable to pathogenic strains of E. coli . 
We focused on a uropathogenic strain of E. coli ( UPEC ) that is associated with urinary tract infections . 
The amino acid sequence of NsrR , and the nucleotide sequence of the fliA promoter shown in Fig. 3 are identical in the UPEC strain CFT073 and MG1655 . 
In CFT073 , flagella-based motility is important for the organism 's ability to ascend the urinary tract and disseminate further in the host ( Lane et al. , 2007 ) . 
Furthermore , some genes that are regulated by NsrR in K12 strains are upregulated during urinary tract infection , notably the hmp gene encoding the NO detoxifying haemoglobin ( Snyder et al. , 2004 ) . 
Thus , there is evidence that both motility and 
NsrR might have important roles in vivo , and transcriptomics data suggest that CFT073 is exposed to NO ( Snyder et al. , 2004 ) . 
We found that nsrR overexpression exerted a greater negative effect on motility in CFT073 ( > 3-fold ; P < 0.0001 ) than was the case in K12 strains , an effect that was reversed by addition of NO to the medium ( Fig. 4 ) . 
NO caused a small though significant ( P < 0.0001 ) stimulation of CFT073 motility ( Fig. 4 ) , as did deletion of the nsrR gene ( Fig. 4 ) . 
Thus , NsrR is a negative regulator of motility in CFT073 , and NO influences motility via NsrR . 
As for K12 strains , we found no motility phenotype associated with an hmp mutation in CFT073 ( data not shown ) . 
As motility and attached growth are typically subject to reciprocal regulation , we measured the ability of CFT073 ( and derivatives deleted for nsrR or containing multiple copies of the nsrR gene ) to adhere to the surface of glass tubes . 
Deletion of nsrR or addition of a source of NO significantly reduced attached growth ( Fig. 5 ) . 
The presence of multiple copies of nsrR stimulated attached growth , an effect that was partially reversed by the addition of NO ( Fig. 5 ) . 
These results indicate that NsrR regulates attached growth in CFT073 ( most likely indirectly ) and that NO influences attached growth via NsrR . 
Discussion
Data presented in this paper suggest that NsrR-binding sites in E. coli fall into two classes : those ( such as the site in ytfE ) comprising two copies of an 11 bp inverted repeat with 1 bp spacing , and those ( exemplified by the site in fliA ) which have a single copy of the 11 bp element . 
The available information suggests that the inverted repeat is a higher-affinity site that allows NsrR repression to operate over a larger range . 
A logical cor-ollary of this suggestion is that the two types of site are occupied by NsrR in different oligomeric states . 
As the 11 bp motif is a palindrome ( Fig. 1C ) , it may be a binding site for an NsrR dimer , in which case the 23 bp inverted repeat might be occupied by a dimer of dimers . 
In sedimentation equilibrium experiments , the Streptomyces coelicolor NsrR formed a sequence-specific complex with DNA with a molecular weight consistent with the protein being dimeric ( Tucker et al. , 2008 ) . 
However , these experiments were done with protein containing a [ 2Fe-2S ] cluster ; the physiologically relevant form of NsrR may contain a [ 4Fe-4S ] cluster ( Yukl et al. , 2008 ) . 
The [ 4Fe-4S ] NsrR from Bacillus subtilis is dimeric in solution , its oligomeric state in the presence of a DNA target has not been examined ( Yukl et al. , 2008 ) . 
Interestingly , the NsrR homologue IscR also binds to two types of site ( Type 1 and Type 2 ) , though in this case they are unrelated sequences . 
Binding to Type 2 sites does not require the [ Fe-S ] cluster of IscR , and two IscR dimers bind cooperatively to a Type 2 site ( Nesbit et al. , 2009 ) . 
We have demonstrated that the NO-sensitive repressor protein NsrR is a negative regulator of motility genes and of flagella-based motility in E. coli K12 . 
We propose that NO exerts effects on motility through NsrR-mediated regulation of the fliA promoter . 
The fliA gene product ( s28 ) is required for the expression of all Class III flagella and chemotaxis genes ( Chilcott and Hughes , 2000 ) , so by regulating fliA NsrR potentially exerts widespread indirect effects on motility and chemotaxis , both of which are involved in migration through soft agar ( Wolf and Berg , 1989 ) . 
The effects of NsrR on fliA promoter activity and on motility are quite small , and are more pronounced in strains containing multiple copies of nsrR than in an nsrR mutant . 
Similar contrasts between deletion and overexpression have been observed previously for genes regarded as negative regulators of motility . 
For example , the pefI-srgD genes of Salmonella enterica serovar Typhimurium were recently described as negative regulators of motility , despite the fact that there is no phenotype associated with deletion of the genes , which inhibit motility when expressed from the araBAD or tetA promoters ( Wozniak et al. , 2009 ) . 
We also showed that NsrR regulates motility and attached growth in a UPEC strain . 
A search of the UPEC strain CFT073 genome with the same PSSM used to search the E. coli K12 genome revealed predicted NsrR-binding sites in the promoter regions of genes involved in the production of pili ( papI and papI_2 ) and fimbriae ( sfaB , C1936 , ipbA and ipuA ) . 
Thus it is possible that the effect of NsrR on attached growth is multifactorial and indirect . 
One possible component of the effect is that changes in fliZ expression mediated by NsrR regulation of the fliAZY promoter lead to altered levels of expression of curli fimbriae . 
In E. coli K12 , FliZ acts by indirectly causing downregulation of genes involved in the expression of curli fimbriae , which are required for surface attachment ( Pesavento et al. , 2008 ) . 
Circumstantial evidence has previously implicated NO as a regulator of chemotaxis , motility and biofilm development . 
Haem-containing NO-binding domains of methyl accepting chemotaxis proteins have been characterized ( Karow et al. , 2004 ; Nioche et al. , 2004 ) , although the prediction that these proteins mediate taxis towards or away from NO has not been tested . 
In transcriptomic experiments , the expression of some motility genes has been observed to be perturbed by exposure of cultures to sources of NO or nitrosative stress ( imposed by S-nitrosothiols ) , although both positive and negative responses have been reported , and the regulators involved were not identified ( Bourret et al. , 2008 ; Constantinidou et al. , 2006 ; Jarboe et al. , 2008 ) . 
In the nonpathogenic organism Nitrosomonas europaea , NO stimulates biofilm formation ( Schmidt et al. , 2004 ) . 
In Azotobacter vinelandii , expression of the flhDC genes ( which encode the master regulator of motility ) is negatively regulated by the oxygen-sensor CydR , an orthologue of the E. coli FNR protein ( León and Espín , 2008 ) . 
CydR is sensitive to NO ( Wu et al. , 2000 ) , suggesting that exposure to NO might stimulate motility in A. vine-landii via increased expression of flhDC . 
In Pseudomo-nas aeruginosa and Staphylococcus aureus , NO inhibits biofilm formation or stimulates dispersal , and NO stimulates motility in P. aeruginosa ( Barraud et al. , 2006 ; Van Alst et al. , 2007 ; Schlag et al. , 2007 ) . 
A molecular mechanism which accounts for the effects of NO on biofilm development or motility in these organisms has not previously been described , though there has been some speculation about the regulatory proteins involved that might act as receptors for NO ( Romeo , 2006 ) . 
The mechanism we propose in this paper may not be applicable to P. aeruginosa and S. aureus , because those species do not have obvious orthologues of NsrR . 
Nevertheless , we suggest that other NO sensing transcriptional regulators ( Spiro , 2007 ; Rodionov et al. , 2005 ) might play an equivalent role in these cases . 
Experimental procedures
Strains, media and growth conditions
The strains and plasmids used in this work are listed in Table 4 . 
The rich medium was L Broth ( tryptone , 10 g l-1 yeast extract , 5 g l-1 ; NaCl , 5 g l-1 ) . 
A mineral salts medium ( Spencer and Guest , 1973 ) supplemented with glucose ( 0.5 % and 0.2 % , w/v , for anaerobic and aerobic cultures respectively ) , casamino acids ( 0.05 % , w/v ) and thiamine ( 5 mg ml-1 ) was used for growth of cultures for b-galactosidase assays . 
Ampicillin ( 100 mg ml-1 ) and kana-mycin ( 25 mg ml-1 ) were added as required . 
Cultures were grown aerobically or anaerobically as previously described ( Bodenmiller and Spiro , 2006 ) . 
For b-galactosidase assays ( Miller , 1992 ) , aerobic cultures were treated with 50 mM spermine-NONOate , and anaerobic cultures with 5 mM nitrite when in early exponential phase ( OD600 = 0.15 -- 0.3 ) , then were assayed 90 min later while still in log phase . 
Spermine-NONOate liberates two equivalents of NO with a half-life of 39 min at 37 °C ( Cayman Chemicals ) , and under our culture conditions caused little or no growth inhibition at this concentration . 
The mqsR , fliA and fliL promoter regions were amplified by PCR ( all primer sequences are available from the authors on request ) and fused to lacZ in pRS415 , transferred to lRS45 , and integrated into the chromosome as described previously ( Simons et al. , 1987 ; Bodenmiller and Spiro , 2006 ) . 
Genes were disrupted by replacing the coding region with a l kanamycin-resistance cassette using the red recombinase method with pKD4 as the template ; mutations were converted to unmarked deletions using pCP20 ( Datsenko and Wanner , 2000 ) . 
The nsrR plasmid pJP07 is a derivative of p2795 ( Husseiny and Hensel , 2005 ) and has been described previously ( Rankin et al. , 2008 ) . 
NsrR with a C96S substitution was expressed from the equivalent plasmid pJP09 . 
The fliA promoter ( on a 474 bp fragment in pSTBlue ) was mutated in the putative NsrR-binding site , and the mutant promoter was fused to lacZ in pRS415 as described above . 
Sitedirected mutants were introduced using the QuickChange Site-Directed Mutagenesis Kit ( Stratagene ) according to manufacturer 's instructions . 
For random mutagenesis , pGIT1 ( Bodenmiller and Spiro , 2006 ) was mutagenized with the GeneMorph PCR mutagenesis kit ( Stratagene ) according to the manufacturer 's instructions . 
After mutagenesis , plasmid DNA was transformed into strain JOEY19 ( lytfE-lacZ ) , and transformants were screened on L agar containing Xgal . 
Colonies with a white or pale blue phenotype were selected , plasmid DNA was purified and the sequence of the ytfE fragment determined . 
Clones with multiple mutations were not further analysed . 
Mutant DNAs of interest generated by random or site-directed mutagenesis were cloned into pRS415 , and integrated on to the chromosome at the lambda attachment site , as previously described . 
ChIP and ChIP-chip
For ChIP analysis of the ytfE and fliA promoters , chromatin was precipitated from cultures of strains expressing 3XFlagtagged NsrR ( or an untagged control ) and transformed with pSTBlue-1 derivatives containing wild type or mutant ytfE or fliA sequences . 
Precipitated DNAs were purified and equal amounts of templates ( 1 ng for ytfE , 2 ng for fliA ) were ampli-fied by 16 cycles of PCR with primers flanking the cloning site in pSTBlue . 
ChIP-chip was performed and data analysed as described previously ( Efromovich et al. , 2008 ) . 
Chromatin immunoprecipitation and microarray data have been depos-ited in the GEO database ( accession GSE11230 ) . 
Motility and attachment assays
Motility was assayed on soft agar swim plates inoculated with 4 ml of an exponential phase ( OD600 ~ 0.5 ) culture . 
The plates contained 1 % tryptone , 0.25 % NaCl , 0.3 % Difco agar , and antibiotics as required . 
The plates were incubated in a wet box for 20 h at 30 °C . 
Surface attachment to 16 mm glass tubes was assayed in standing cultures grown for 24 h at 30 °C in L broth , using the crystal violet staining method ( Pratt and Kolter , 1998 ) . 
For treatment with NO , the plates or standing cultures were supplemented with 100 mM ( for K12 strains ) or 250 mM 
( for CFT073 ) diethylenetriamine-NONOate , which liberates two equivalents of NO with a half-life of 20 -- 56 h under the conditions of these experiments ( Cayman Chemicals ) . 
Acknowledgements
We are grateful to Sandy Parkinson , Harry Mobley , Michael Hensel , Barry Wanner and Valley Stewart for generously providing strains and plasmids , to Gladys Alexandre , Sam Efromovich , Mike Manson and David Grainger for helpful discussions , and to Ray Dixon for comments on the manuscript . 
This work was supported in part by Grant MCB-0702858 from the National Science Foundation .