OmpR Binding Sites Research Articles

The Regulator OmpR in Yersinia enterocolitica Participates in Iron Homeostasis by Modulating Fur Level and Affecting the Expression of Genes Involved in Iron Uptake.

In this study, we found that the loss of OmpR, the response regulator of the two-component EnvZ/OmpR system, increases the cellular level of Fur, the master regulator of iron homeostasis in Y. enterocolitica. Furthermore, we demonstrated that transcription of the fur gene from the YePfur promoter is subject to negative OmpR-dependent regulation. Four putative OmpR-binding sites (OBSs) were indicated by in silico analysis of the fur promoter region, and their removal affected OmpR-dependent fur expression. Moreover, OmpR binds specifically to the predicted OBSs which exhibit a distinct hierarchy of binding affinity. Finally, the data demonstrate that OmpR, by direct binding to the promoters of the fecA, fepA and feoA genes, involved in the iron transport and being under Fur repressor activity, modulates their expression. It seems that the negative effect of OmpR on fecA and fepA transcription is sufficient to counteract the indirect, positive effect of OmpR resulting from decreasing the Fur repressor level. The expression of feoA was positively regulated by OmpR and this mode of action seems to be direct and indirect. Together, the expression of fecA, fepA and feoA in Y. enterocolitica has been proposed to be under a complex mode of regulation involving OmpR and Fur regulators.

Expanded roles of two-component response regulator OmpR in Escherichia coli: genomic SELEX search for novel regulation targets.

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.

Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR.

OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in β-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

ChIP‐seq and transcriptome analysis of the OmpR regulon of Salmonella enterica serovars Typhi and Typhimurium reveals accessory genes implicated in host colonization

SummaryOmpR is a multifunctional DNA binding regulator with orthologues in many enteric bacteria that exhibits classical regulator activity as well as nucleoid-associated protein-like characteristics. In the enteric pathogen Salmonella enterica, using chromatin immunoprecipitation of OmpR:FLAG and nucleotide sequencing, 43 putative OmpR binding sites were identified in S. enterica serovar Typhi, 22 of which were associated with OmpR-regulated genes. Mutation of a sequence motif (TGTWACAW) that was associated with the putative OmpR binding sites abrogated binding of OmpR:6×His to the tviA upstream region. A core set of 31 orthologous genes were found to exhibit OmpR-dependent expression in both S. Typhi and S. Typhimurium. S. Typhimurium-encoded orthologues of two divergently transcribed OmpR-regulated operons (SL1068–71 and SL1066–67) had a putative OmpR binding site in the inter-operon region in S. Typhi, and were characterized using in vitro and in vivo assays. These operons are widely distributed within S. enterica but absent from the closely related Escherichia coli. SL1066 and SL1067 were required for growth on N-acetylmuramic acid as a sole carbon source. SL1068–71 exhibited sequence similarity to sialic acid uptake systems and contributed to colonization of the ileum and caecum in the streptomycin-pretreated mouse model of colitis.

Evolving a robust signal transduction pathway from weak cross‐talk

We have evolved a robust two-component signal transduction pathway from a sensor kinase (SK) and non-partner response regulator (RR) that show weak cross-talk in vitro and no detectable cross-talk in vivo in wild-type strains. The SK, CpxA, is bifunctional, with both kinase and phosphatase activities for its partner RR. We show that by combining a small number of mutations in CpxA that individually increase phosphorylation of the non-partner RR OmpR, phosphatase activity against phospho-OmpR emerges. The resulting circuit also becomes responsive to input signal to CpxA. The effects of combining these mutations in CpxA appear to reflect complex intragenic interactions between multiple sites in the protein. However, by analyzing a simple model of two-component signaling, we show that the behavior can be explained by a monotonic change in a single parameter controlling protein–protein interaction strength. The results suggest one possible mode of evolution for two-component systems with bifunctional SKs whereby the remarkable properties and competing reactions that characterize these systems can emerge by combining mutations of the same effect.

Positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis

OmpR has been demonstrated to negatively regulate the expression of the flagellar master operon flhDC in a wide variety of bacterial species. Here we report the positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis. A sigma(70)-dependent promoter was identified by primer extension analysis and an active region with two conserved OmpR-binding sites around the flhDC promoter was confirmed. To confirm the regulation of flhDC expression by OmpR, flhDC as well as the downstream flagellar genes fliA, flgD, flgA, flgM, fliC and flaA were fused to lacZ, and decreased expression of all these genes in an ompR mutant (Delta ompR) was detected. Furthermore, Delta ompR was defective in bacterial motility and flagella synthesis. This defect was due to the low level of expression of flhDC in Delta ompR since overproduction of FlhDC in Delta ompR restored bacterial motility. The importance of two conserved OmpR-binding sites around the flhDC promoter region in the regulation of flhDC expression by OmpR was demonstrated by the fact that mutation of either one or both sites significantly decreased the promoter activity in the wild-type but not in Delta ompR. The binding of OmpR to these two sites was also demonstrated by DNA mobility shift assay. The possible mechanism underlying this positive regulation in Y. pseudotuberculosis is discussed. To our knowledge, this is the first report to demonstrate that OmpR positively regulates flhDC expression.

Amino Acids Important for DNA Recognition by the Response Regulator OmpR

Response regulators undergo regulated phosphorylation and dephosphorylation at conserved aspartic acid residues in bacterial signal transduction systems. OmpR is a winged helix-turnhelix DNA-binding protein that functions as a global regulator in bacteria and is also important in pathogenesis. A detailed mechanistic picture of how OmpR binds to DNA and activates transcription is lacking. We used NMR spectroscopy to solve the solution structure of the C-terminal domain of OmpR (OmpR(C)) and to analyze the chemical shift changes that occur upon DNA binding. There is little overlap in the interaction surface with residues of PhoB that were reportedly involved in protein/protein interactions in its head-to-tail dimer. Multiple factors account for the lack of overlap. One is that the spacing between the OmpR half-sites is shorter than observed with PhoB, requiring the arrangement of the two OmpR molecules to be different from that of the PhoB dimer on DNA. A second is the demonstration herein that OmpR can bind to its high affinity site as a monomer. As a result, OmpR(C) appears to be capable of adopting alternative orientations depending on the precise base composition of the binding site, which also contributes to the lack of overlap. In the presence of DNA, chemical shift changes occur in OmpR in the recognition alpha-helix 3, the loop between beta-strand 4 and alpha-helix 1, and the loop between beta-strands 5 and 6. DNA contact residues are Val(203) (T), Arg(207) (G), and Arg(209) (phosphate backbone). Our results suggest that OmpR binds to DNA as a monomer and then forms a symmetric or asymmetric dimer, depending on the binding site. We propose that during activation OmpR binds to DNA and undergoes a conformational change that promotes phosphorylation of the N-terminal receiver domain, the receiver domains dimerize, and then the second monomer binds to DNA. The flexible linker of OmpR enables the second monomer to bind in multiple orientations (head-to-tail and head-to-head), depending on the specific DNA contacts.

Regulatory components at thecsgDpromoter – additional roles for OmpR and integration host factor and role of the 5′ untranslated region

CsgD is a master regulator of multicellular behaviour in Salmonella enterica serovar Typhimurium. Expression of CsgD is highly regulated on the transcriptional level. A nucleo-protein complex had been defined where the global regulators OmpR and integration host factor (IHF) bind up- and downstream of the csgD core promoter. In this study, the nucleo-protein complex of PcsgD was extended through characterization of additional OmpR and IHF binding sites that influence the transcriptional activity of the csgD promoter. Furthermore, the role of the 174 bp long 5'-untranslated region on transcriptional activity was defined.

Transcription Regulation of ompF and ompC by a Single Transcription Factor, OmpR

The ompF and ompC genes of Escherichia coli are reciprocally regulated by a single transcription factor, phosphorylated OmpR (OmpR-P), depending upon medium osmolarity. This regulation involves activation of ompF and its repression with concomitant activation of ompC. This occurs through OmpR-P binding to four (F1, F2, F3, and F4) and three (C1, C2, and C3) sites located upstream of the ompF and ompC promoters, respectively, through a novel mechanism. Here we show that there is a distinct OmpR-P binding hierarchy within F1, F2, and F3 sites as well as within C1, C2, and C3 sites. Each of these sites contains two tandem 10-bp OmpR-P-binding subsites, a-site and b-site (from 5' to 3' direction). OmpR-P has higher affinity to the downstream b-site than to the upstream a-site in each case. Six OmpR-P molecules bind to F and C sites two-by-two in a discontinuous "galloping" manner. We propose that this tight hierarchical binding of a transcription factor, OmpR, allows distinct stepwise regulation of ompF and ompC transcription, which minimizes their overlapping expression upon changes in the medium osmolarity to achieve the reciprocal expression of ompF and ompC.

Imaging OmpR localization in Escherichia coli

We have used a fusion of GFP to the response regulator OmpR to image the spatial distribution of OmpR in live cells of Escherichia coli. We observed foci of increased OmpR-GFP fluorescence that appear to be due to interactions with the histidine kinase EnvZ. We also observed colocalization of OmpR-GFP with clusters of plasmids carrying OmpR binding sites, which enabled us to develop a simple method for imaging the binding of OmpR to DNA in live cells. We used the peak fluorescence intensity within cells to quantify the extent of OmpR-GFP localization either due to interactions with EnvZ or due to binding DNA. With these assays we compared the effects of osmolarity and procaine, both of which are believed to modulate EnvZ activity. Our results suggest that, at least under our growth conditions, procaine activates EnvZ-OmpR signalling whereas osmolarity has, at best, a weak effect on the EnvZ-OmpR system.

ChxR is a transcriptional activator in Chlamydia

Chlamydia spp. are obligate intracellular bacterial pathogens that alternate between two metabolically and morphologically distinct developmental forms, and differentiation depends on transcriptional regulation. Genome sequencing of Chlamydia trachomatis revealed an ORF, CT630 (chxR), whose amino acid sequence contains a winged helix-turn-helix motif similar to the DNA-binding domain of response regulators in the OmpR subfamily. ChxR differs from many response regulators in that essential residues in the receiver or phosphorylation domain are lacking. ChxR functions as a transcriptional regulator because it activated transcription of ompF and ompC when expressed in Escherichia coli. In vitro transcription combined with microarray analysis also demonstrated that ChxR activates its own expression by binding directly to sites upstream of chxR; it also activates infA, tufA, oppA, and CT084. DNase I protection studies showed that ChxR bound to sites in the ompF and ompC promoter proximal regions that overlap but were distinct from OmpR binding sites. Both proteins could bind simultaneously to their nonoverlapping binding sites. This report identifies a stage-specific transcriptional regulator and some of its target genes in Chlamydia.

The response regulator SsrB activates transcription and binds to a region overlapping OmpR binding sites at Salmonella pathogenicity island 2

OmpR activates expression of the two-component regulatory system located on Salmonella pathogenicity island 2 (SPI-2) that controls the expression of a type III secretion system, as well as many other genes required for systemic infection in mice. Measurements of SsrA and SsrB protein levels under different growth conditions indicate that expression of these two components is uncoupled, i.e. SsrB is produced in the absence of ssrA and vice versa. This result was suggested from our previous studies, in which two promoters at ssrA/B were identified. The isolated C-terminus of SsrB binds to DNA and protects regions upstream of ssrA, ssrB and srfH from DNase I digestion. Furthermore, the C-terminus of SsrB alone is capable of activating transcription in the absence of the N-terminus. Results from beta-galactosidase assays indicate that the N-terminal phosphorylation domain inhibits the C-terminal effector domain. A previous study from our laboratory reported that ssrA-lacZ and ssrB-lacZ transcriptional fusions were substantially reduced in an ssrB null strain. Results from DNase I protection assays provide direct evidence that SsrB binds at ssrA and ssrB, although the binding sites lie within the transcribed regions. Additional regulators clearly affect gene expression at this important locus, and here we provide evidence that SlyA, a transcription factor that contributes to Salmonella virulence, also affects ssrA/B gene expression.

A phosphorylation site mutant of OmpR reveals different binding conformations at ompF and ompC

In Escherichia coli, the two-component regulatory system that controls the expression of outer membrane porins in response to environmental osmolarity consists of the sensor kinase EnvZ and the response regulator OmpR. Phosphorylated OmpR activates expression of the OmpF porin at low osmolarity, and at high osmolarity represses ompF transcription and activates expression of OmpC. We have characterized a substitution in the amino-terminal phosphorylation domain of OmpR, T83I, its phenotype is OmpF− OmpC−. The mutant protein is not phosphorylated by small molecule phosphodonors such as acetyl phosphate and phosphoramidate, but it is phosphorylated by the cognate kinase EnvZ. Interestingly, the active site T83I substitution alters the DNA binding properties of the carboxyl-terminal effector domain. DNase I protection assays indicate that DNA binding by the mutant protein is similar to wild-type OmpR at the ompF promoter, but at ompC, the pattern of protection is different from OmpR. Our results indicate that all three of the OmpR binding sites at the ompC promoter must be filled in order to activate gene expression. Furthermore, it appears that OmpR-phosphate must adopt different conformations when bound at ompF and ompC. A model is presented to account for the reciprocal regulation of OmpF and OmpC porin expression.

Identification of the Bases in the ompFRegulatory Region, which Interact with the Transcription Factor OmpR

Expression of the outer membrane protein OmpF of Escherichia coliK-12 is influenced by a variety of environmental signals. Most of the signals are thought to regulate OmpF expression at the level of transcription initiation. A key element of this regulation is the interaction between the transcriptional factor OmpR and the cis-acting regulatory region of ompF. In this study, we used a combination of DNase I, dimethyl sulfate and hydroxyl radical footprinting analysis and DNA migration retardation assays to identify the bases within the ompFregulatory region that are in contact with OmpR. Our results indicate that the −107 to −39 region of ompFcontains three individual binding sites and that a single OmpR-binding site is capable of interacting with two OmpR molecules. We also establish that a single OmpR-binding site is composed of two half-sites and that both half-sites are required for the formation of stable OmpR/DNA complexes. Comparisons of the sequences protected by OmpR indicate that an OmpR-binding site spans approximately 18 bp and has two highly conserved G/C base-pairs that are separated by three nucleotides. Although the three OmpR-binding sites we identified exhibit limited sequence similarity, this may reflect the fact that two of the sites are incapable of binding OmpR independently and can bind OmpR only if adjacent to another OmpR-binding site. Finally, our DNA migration retardation assays suggest that phosphorylation stimulates the cooperative interactions between OmpR molecules bound at neighboring sites. Therefore, this study provides a detailed understanding of how OmpR interacts with its binding sites immediately upstream of ompFand serves as a foundation for studying how phosphorylation of OmpR results in the regulation of ompF expression in response to environmental signals.

Activation of the osmoregulated ompF and ompC genes by the OmpR protein in Escherichia coli: a study involving chimeric promoters.

The expression of each of the Escherichia coli ompF and ompC genes is activated by the common positive regulator, OmpR, in response to the medium osmolarity. The promoters for these genes consist of canonical -35 and -10 regions, and upstream OmpR-binding sites. In this study, we constructed a functional ompF-ompC chimeric promoter consisting of the OmpR-binding site of ompF, and the -35 and -10 regions of ompC, which was fused to the lacZ gene on a low-copy-number plasmid. It is known that the ompF and ompC genes are expressed in a different manner in cells with mutations in the ompR gene. In this respect, it was found that the ompF-ompC chimeric promoter behaves just like ompF promoter with respect to an ompR mutation (ompR472). It was revealed that, in this chimeric promoter, the OmpR-binding site must be located stereospecifically with respect to the -35 and -10 regions for OmpR-dependent transcription of the ompF-ompC chimeric promoter to occur. These results are discussed in relation to the structures and functions of the ompF and ompC promoters, the occurrence of a DNA curvature in the promoter regions being implied, which may be an important parameter for the transcription activation by the OmpR protein.

Evidence for multiple OmpR-binding sites in the upstream activation sequence of the ompC promoter in Escherichia coli: a single OmpR-binding site is capable of activating the promoter

We present experimental evidence for the existence of multiple activator-binding sites in the upstream sequence of the ompC promoter, the expression of which is activated by the positive regulator OmpR in response to the osmolarity of the medium. We also found that a single OmpR-binding site can activate the ompC promoter, providing that the binding site is close and placed stereospecifically with respect to the canonical-35 and -10 regions.