The constitutive phoP gene was placed under the control of an arabinose-inducible promoter to examine the kinetics of PhoP-activated gene induction .
A differential regulation of a PhoP-dependent gene during the infection period might be related to the fact that tight regulation of the phoP regulon is required for virulence .
A differential regulation of a PhoP-dependent gene during the infection period might be related to the fact that tight regulation of the phoP regulon is required for virulence .
somA require the response regulator PhoP for expression To determine whether the expression of somA is regulated by transcription factors , the GFP report-ers were transduced into Salmonella strains with mutations in one of the following genes : phoP .
VirK require the response regulator PhoP for expression To determine whether the expression of somA is regulated by transcription factors , the GFP report-ers were transduced into Salmonella strains with mutations in one of the following genes : phoP .
PhoP-regulated genes were further identified by transcriptional profiling using microarrays of strain CS022 containing a phoQ mutation that results in increased expression of PhoP-activated genes and a phoP null mutant ( W.N. , unpubl .
To distinguish between these alternatives , we first analyzed the expression of mgtA , pcgL , mgtC , and pmrC as an indirectly PhoP-regulated gene ( 21 , 43 ) in a phoP : : Tn10 strain , expressing PhoP from the IPTG-regu-lated plasmid pEG9014 .
PhoP binds to the promoter regions of the phoP , mgtA , slyB , pcgL .
The PhoP/PhoQ system is required for transcription from one of the promoters of the slyA gene ( 20 ) , suggesting that some of the phenotypes displayed phoP and phoQ mutants could be caused by their inability to express the SlyA protein and implying that SlyA participates in transcription of a subset of PhoP-regulated genes .
Summary In Salmonella enterica , the PhoP -- PhoQ two-component system governs resistance to structurally different antimicrobial peptides including the alphahelical magainin 2 , the b-sheet defensins and the cyclic lipopeptide polymyxin B. To identify the PhoP-regulated determinants mediating peptide resistance , we prepared a plasmid library from a phoP mutant , introduced it into a phoP mutant and selected for magainin-resistant clones .
The ugtL gene mediates polymyxin B resistance independently of the PmrA-PmrB system phoP Salmonella are > 20 times more polymyxin sensitive than a pmrA mutant when bacteria are grown in low ygjU 3394555 3392617 rfaB rfaI 3914096 3915562 slsA STM3760 cigR 3959077 3960805 rhaT rhaR 4260472 4262386 STM0725 STM0726 STM0724 792487 790384 Mg2 + ( Wosten et al. , 2000 ) , indicating that a PmrA-inde-pendent PhoP-regulated gene ( s ) is necessary for poly-myxin resistance .
w ild-type + y v q e jA cto + r y v q e jA cto + r pyqj PhoP-regulated magainin 2 resistance is mediated by the ugtL , pagP and yqjA genes Because the ugtL mutant was not as susceptible to magai-nin 2 as the phoP mutant ( Fig. 2A ) , we reasoned that another PhoP-regulated gene ( s ) must be involved in magainin 2 resistance .
w ild-type + y v q e jA cto + r y v q e jA cto + r pyqj PhoP-regulated magainin 2 resistance is mediated by the ugtL , pagP and yqjA genes Because the ugtL mutant was not as susceptible to magai-nin 2 as the phoP mutant ( Fig. 2A ) , we reasoned that another PhoP-regulated gene ( s ) must be involved in magainin 2 resistance .
The ugtL and pmrA genes mediate PhoP-controlled resistance to polymyxin B. A. Percentage survival of wild-type ( 14028s ) , phoP ( MS7953s ) and ugtL ( EG13682 ) strains after incubation with polymyxin B ( 1.0 mg ml-1 ) .
% surviva A B C 100 10 1 r r to to gtL c c u ve ve p e L tL + + + typ gt ug u-ild w pe P y o-t h ld p w i L e oP rA gtL h m u p p A r m p gt u yp-t ld w i A Mg2 + L L L H b-galactosidase activity YqjA-FLAG w ild-type 14028s p h o yqjA-flag P B yqjA w 10µM Mg2 + 2 + - flag ; phoP 10mM Mg ild-type 5 0 40 p Discussion The PhoP -- PhoQ regulatory system controls resistance to a variety of structurally different antimicrobial peptides ( Groisman et al. , 1992 ) , but the identity of the PhoP-regulated determinants mediating peptide resistance has remained largely unknown .
A phoP mutant is > 20 times more polymyxin B sensitive than a pmrA mutant when bacteria are grown in low Mg2 + and > 20 times more sensitive than wild-type Salmonella when bacteria are grown in the presence of Fe3 + ( Wosten et al. , 2000 ) , indicating that a PhoP-regulated PmrA-independent gene ( s ) participate ( s ) in polymyxin B resistance .
Moreover , we are able to recapitulate the susceptibility of the phoP mutant to magainin 2 and polymyxin B by constructing strains defective in different combinations of PhoP-regulated genes .
To identify PhoP-regulated genes mediating magainin 2 resistance , we prepared a genomic library from the phoP mutant strain MS7953s in the multicopy number plasmid pUC19 ( Norrander et al. , 1983 ) , introduced the library into the same phoP mutant and exposed the transformants to magainin 2 ( see Experimental procedures ) .
This strategy was based on the premise that PhoP-regulated genes might be expressed from the lac promoter in pUC19 and confer magainin 2 resistance upon the phoP mutant .
These results were unexpected because acid resistance genes were not identified as PhoP-regulated genes in two previous microarray experiments that compared expression of wild-type and phoP Escherichia coli strains ( 14 , 35 ) nor had mutations in phoP been uncovered in screenings for acid regulatory genes in Escherichia coli .
For example , one of the profiles encompassed promoters that belong to the same expression ( E1 ) , PhoP box submotif ( M2 ) , and promoter class ( P1 ) ( Fig. 1H ) and harbored not only the prototypical PhoP-regulated phoP and mgtA promoters ( 13 , 14 ) but also the yhiW promoter , which was not known to be under PhoP control .
pagC was downregulated almost 100-fold in the phoP mutant , validating this method for investigating gene regulation by PhoP inside macrophages .
To further investigate the association between tdcA with phoP , we constructed chromosomal lacZ fusion strains of PhoP-regulated ( mgtA and pagD ) and SPI2 genes ( ssaG and sseA ) ( Navarre et al. , 2005 ) .
The PhoP homodimer functions as a transcription factor by recognizing and binding to phoP boxes in promoters of PhoP-regulated genes .
The PhoP homodimer functions as a transcription factor by binding to phoP boxes in promoters of PhoP-regulated genes .
The PhoP homodimer binds to phoP boxes in promoters of PhoP-regu-lated genes to modulate virulence gene expression .
Expression of the fusions in phoP backgrounds indicated that the cloned region contained the signals necessary for PhoP-mediated regulation of sseK1 .
In contrast , intracellular induction was not observed in a phoP null mutant , giving additional support to the conclusion that PhoP is a positive regulator of the expression of sseK1 .
Subsequently , the phosphoryl of PhoQ is transferred to the conserved aspartyl of PhoP , and then the phosphorylated PhoP activates the transcription of phoP itself and PhoP-regulated genes .
It has been shown that the transcription of phoP and PhoP-regulated genes were completely inhibited by 10 mM magnesium [ 41 ] , which caused about one third of PhoP K201 was acetylated , suggesting low acetylation proportion of PhoP K201 is required for PhoP activity .
The underlying reason is that PhoP K201R kept the ability to activate phoP transcription initiated from P1 promoter and PhoP-regulated gene transcription during infection , while PhoP K201Q inhibited these genes transcription due to its low DNA-binding affinity .
Acetylation of lysine residue located in the DNA-binding motif inhibits DNA-binding ability of PhoP , and further alters the transcription of phoP and PhoP-regulated genes .
Acetylation inhibits the transcription of phoP and PhoP-regulated genes As a major transcription factor , PhoP regulates the expression of ~ 5 % of genes in S. Typhimur-ium genome , including itself , and the positive feedback boosts the level of PhoP to extend the response range of the PhoP-PhoQ circuit under conditions that strongly activate PhoQ [ 30 ] .
The quantitative real-time PCR ( qPCR ) result showed that phoP and PhoP-regulated genes were all up-regulated in the pat deletion mutant compared with those in the wild type strain ( Fig 2A ) , while the transcriptional level of phoP did not change in the cobB deletion mutant ( S2 Fig ) .
The quantitative real-time PCR ( qPCR ) result showed that phoP and PhoP-regulated genes were all up-regulated in the pat deletion mutant compared with those in the wild type strain ( Fig 2A ) , while the transcriptional level of phoP did not change in the cobB deletion mutant ( S2 Fig ) .
We next ask whether deletion of pat increases the transcription of phoP and PhoP-regulated genes in-vivo .
mRNA levels of phoP and PhoP-regulated genes in the pat deletion mutant were also elevated significantly compared with the wild type strain in macrophage cells ( Fig 2C ) .
The qPCR assay demonstrated that both K201Q and K201A mutations led to significant transcriptional reduction of phoP and PhoP-regulated genes compared with the wild type strain , while K201R mimicking non-acetylated lysine residue activated the transcription of these genes dramatically ( Fig 3C ) , suggesting acetylation of K201 is involved in regulating the binding of PhoP to its DNA site and thus its ability to regulate its regulon in S. Typhimurium in-vivo .