OmpR Protein Research Articles

Promiscuity of response regulators for thioredoxin steers bacterial virulence

The exquisite specificity between a sensor kinase and its cognate response regulator ensures faithful partner selectivity within two-component pairs concurrently firing in a single bacterium, minimizing crosstalk with other members of this conserved family of paralogous proteins. We show that conserved hydrophobic and charged residues on the surface of thioredoxin serve as a docking station for structurally diverse response regulators. Using the OmpR protein, we identify residues in the flexible linker and the C-terminal β-hairpin that enable associations of this archetypical response regulator with thioredoxin, but are dispensable for interactions of this transcription factor to its cognate sensor kinase EnvZ, DNA or RNA polymerase. Here we show that the promiscuous interactions of response regulators with thioredoxin foster the flow of information through otherwise highly dedicated two-component signaling systems, thereby enabling both the transcription of Salmonella pathogenicity island-2 genes as well as growth of this intracellular bacterium in macrophages and mice.

OmpR coordinates the expression of virulence factors of Enterohemorrhagic Escherichia coli in the alimentary tract of Caenorhabditis elegans.

Enterohemorrhagic Escherichia coli (EHEC), an enteropathogen that colonizes in the intestine, causes severe diarrhea and hemorrhagic colitis in humans by the expression of the type III secretion system (T3SS) and Shiga-like toxins (Stxs). However, how EHEC can sense and respond to the changes in the alimentary tract and coordinate the expression of these virulence genes remains elusive. The T3SS-related genes are known to be regulated by the locus of enterocyte effacement (LEE)-encoded regulators, such as Ler, as well as non-LEE-encoded regulators in response to different environmental cues. Herein, we report that OmpR, which participates in the adaptation of E. coli to osmolarity and pH alterations, is required for EHEC infection in Caenorhabditis elegans. OmpR protein was able to directly bind to the promoters of ler and stx1 (Shiga-like toxin 1) and regulate the expression of T3SS and Stx1, respectively, at the transcriptional level. Moreover, we demonstrated that the expression of ler in EHEC is in response to the intestinal environment and is regulated by OmpR in C. elegans. Taken together, we reveal that OmpR is an important regulator of EHEC which coordinates the expression of virulence factors during gastrointestinal infection in vivo.

Direct interaction between the transcription factors CadC and OmpR involved in the acid stress response of Salmonella enterica.

In Salmonella enterica serovar Typhimurium, the acid-sensing regulator CadC activates transcription of the cadBA operon which contributes to the acid tolerance response. The DNA-binding response regulator OmpR in two-component regulatory system with EnvZ binds to its own promoter for autoinduction. We previously reported that CadC exerts a negative influence on ompR transcription during acid adaptation. However, its underlying mechanisms remain to be elucidated. Here we show that the level of OmpR protein is gradually reduced by a gradual increase in the CadC level using an arabinose-inducible expression system, indicating there exists a negative correlation between the expression levels of two transcription factors. To explore the molecular basis for OmpR repression by CadC, we performed in vitro binding assays and determined that CadC directly interacts with OmpR. We further show that inactivation of cadC inhibits transcription of the fliC gene, which encodes the major flagellar subunit, resulting in impaired flagellar motility under acid-adaptation conditions. Together, our findings suggest that CadC may repress autoinduction of the OmpR response regulator through their direct interaction.

Engineering a chimeric malate two-component system by introducing a positive feedback loop in Escherichia coli

Previous studies constructed a chimeric MalKZ two-component system to sense environmental malate. In this study, we used a positive feedback loop to accelerate and amplify the output signal indicating malate concentration. The positive feedback loop was constructed by cloning ompR gene, which encodes ompC and induces OmpR protein; ompC promoter was used to control the process. The transcriptional expression profile showed that the expression level of ompC gene increased about two-fold after the positive feedback loop was introduced. When GFP was used as a reporter protein, a 71% increase in fluorescence level was observed. The results indicate that the signal transduction kinetics of MalKZ can be engineered by introducing the positive feedback loop.

Two-component bacterial regulation systems: Targets of a search for new antibacterial drugs

A review of the literature presents data on two-component bacterial regulatory systems that may be possible targets in the search of new antibacterial drugs because of their universality and the role in regulation of pathogenicity. We discuss (a) the structural-functional organization of two-component systems taking as example OmpR family (EnvZ/OmpR and PhoQ, PhoP) regulating a number of processes in bacteria faciliting adaptation to stress from changes in the environment and human host, providing virulence and formation of biofilms, these being a cause of many human chronic infections, and (b) the genes and functions regulated by two-component systems by EnvZ/OmpR system, OmpR protein in particular, and PhoQ/PhoP. The review speculates on possibilities of searching for inhibitors of the proteins of two-component systems and their possible role in virulence attenuation of pathogenic bacteria.

Expression of ompR gene in the acid adaptation and thermal resistance of Salmonella Enteritidis SE86.

The objective of this study was to evaluate the involvement of the ompR gene in the acid adaptation and thermal resistance of S. Enteritidis SE86, responsible agent of more than 95 % of investigated food-borne diseases, throughout the last decade in Southern Brazil. In this study, we constructed a mutant strain of S. Enteritidis SE86 (ΔompR) that was attenuated by a knockout technique. The OmpR protein expression was determined in a tagged (3XFLAG) strain of S. Enteritidis SE86. The mutant strains were cultivated separately in nutrient broth and nutrient broth supplemented with 1% glucose (NBG) to induce acid adapted cells. The organisms were exposed to different temperature such as 37 ºC, 52 ºC, and 60ºC. The survival of the SE86 wild type (WT) and attenuated strain was determined by bacterial count, and the tagged protein (ompR::3XFLAG cat::FLAG) was detected by SDS-PAGE and immunoblotting with anti-FLAG antibodies. Results showed that when exposed at 52ºC, the acid-adapted SE86 WT cells were completely inactivated after 300 minutes; however, non-adapted cells (WT and ΔompR) and acid-adapted ΔompR demonstrated higher thermal sensitivity, since they were completely inactivated in 240 minutes. At 60ºC, the acid-adapted SE86 ΔompR also demonstrated higher sensitivity that SE86 WT, being totally inactivated after 15 minutes, while the WT cells were inactivated in 20 minutes. The acid adapted cells showed increased expression of OmpR when exposed to 52 ºC and 60ºC, this confirmed the requirement of acid adaptation for S. Enteritidis SE86 to resist elevated temperatures.

Bacterial Regulon Evolution: Distinct Responses and Roles for the Identical OmpR Proteins of Salmonella Typhimurium and Escherichia coli in the Acid Stress Response

The evolution of new gene networks is a primary source of genetic innovation that allows bacteria to explore and exploit new niches, including pathogenic interactions with host organisms. For example, the archetypal DNA binding protein, OmpR, is identical between Salmonella Typhimurium serovar Typhimurium and Escherichia coli, but regulatory specialization has resulted in different environmental triggers of OmpR expression and largely divergent OmpR regulons. Specifically, ompR mRNA and OmpR protein levels are elevated by acid pH in S. Typhimurium but not in E. coli. This differential expression pattern is due to differences in the promoter regions of the ompR genes and the E. coli ompR orthologue can be made acid-inducible by introduction of the appropriate sequences from S. Typhimurium. The OmpR regulon in S. Typhimurium overlaps that of E. coli at only 15 genes and includes many horizontally acquired genes (including virulence genes) that E. coli does not have. We found that OmpR binds to its genomic targets in higher abundance when the DNA is relaxed, something that occurs in S. Typhimurium as a result of acid stress and which is a requirement for optimal expression of its virulence genes. The genomic targets of OmpR do not share a strong nucleotide sequence consensus: we propose that the ability of OmpR to recruit additional genes to its regulon arises from its modest requirements for specificity in its DNA targets with its preference for relaxed DNA allowing it to cooperate with DNA-topology-based allostery to modulate transcription in response to acid stress.

OmpR regulation of the uropathogenic Escherichia coli fimB gene in an acidic/high osmolality environment

Uropathogenic Escherichia coli (UPEC) causes more than 90 % of all human urinary tract infections through type 1 piliated UPEC cells binding to bladder epithelial cells. The FimB and FimE site-specific recombinases orient the fimS element containing the fimA structural gene promoter. Regulation of fimB and fimE depends on environmental pH and osmolality. The EnvZ/OmpR two-component system affects osmoregulation in E. coli. To ascertain if OmpR directly regulated the fimB gene promoters, gel mobility shift and DNase I footprinting experiments were performed using OmpR or phosphorylated OmpR (OmpR-P) mixed with the fimB promoter regions of UPEC strain NU149. Both OmpR-P and OmpR bound weakly to one fimB promoter. Because there was weak binding to one fimB promoter, strain NU149 was grown in different pH and osmolality environments, and total RNAs were extracted from each population and converted to cDNAs. Quantitative reverse-transcriptase PCR showed no differences in ompR transcription among the different growth conditions. Conversely, Western blots showed a significant increase in OmpR protein in UPEC cells grown in a combined low pH/high osmolality environment versus a neutral pH/high osmolality environment. In a high osmolality environment, the ompR mutant expressed more fimB transcripts and Phase-ON positioning of the fimS element as well as higher type 1 pili levels than wild-type cells. Together these results suggest that OmpR may be post-transcriptionally regulated in UPEC cells growing in a low pH/high osmolality environment, which regulates fimB in UPEC.

Molecular cloning, sequence analysis and structure modeling of OmpR, the response regulator of Aeromonas hydrophila

The ability of bacteria to survive and proliferate in changing environmental conditions, and during host cell invasion is the key to their pathogenicity. In order to achieve this, the bacteria use a signal transduction system, the two component regulatory system, which consists of a sensor kinase and a response regulator. The EnvZ/OmpR system regulates the porin genes ompF/ompC in response to changes in osmolarity. In the present study, the ompR gene of Aeromonas hydrophila (isolate Ah17) was cloned, sequenced and characterized. Further an attempt was made to analyze the structural characteristics of the OmpR protein from Aeromonas hydrophila. The three dimensional structure of the protein was predicted by homology modeling and the modeled structure was compared to other members of two component response regulators. This study would be helpful for structure based drug design approaches to generate drugs against this harmful pathogen to control its proliferation in both human and fish hosts.

Identification of outer membrane protein ompR from rickettsia-like organism and induction of immune response in Crassostrea ariakensis

Rickettsia-like organism (RLO) caused mass mortality of oysters, but little is known about the protective immune response to this microorganism. The present study was undertaken to identify a gene, ompR, encoding an outer membrane protein of rickettsia-like organism from oyster Crassostrea ariakensis. The role of this protein in promoting immune responses was characterized through analyzing the interaction between RLO and oyster. The results indicated: (i) full-length DNA of ompR is 531 bp and encodes 176 amino acid residues. Theoretical isoelectric point and molecular weight for the ompR protein are 9.76 and 19.76 kDa, respectively; (ii) the recombinant ompR was successfully expressed in Escherichia coli BL21 (DE3) cells, and the titre of anti- ompR antibody raised against rabbits was about 1:4100. A specific immunoreactive band was detected when anti- ompR antibody was opposed to the total outer membrane proteins of RLO; (iii) the expression level of TNF-α (tumor necrosis factor-alpha) and Myd88 (myeloid differentiation factor 88) in hemocytes was induced by ompR, whereas TGF-β (transforming growth factor-beta) was not; (iv) in hemocytes monolayers, a rapid and persistent increase in the level of phosphorylated P38 and a large decrease in the level of phosphorylated JNK were induced by ompR, whereas the level of phosphorylated ERK did not change with ompR incubation; (v) the DNA binding activity of NF-κB (nuclear factor κB) in hemocytes increased after ompR stimulation.

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.

Novel Mechanism of Escherichia coli Porin Regulation

A novel mechanism of Escherichia coli porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated ipeX for inhibition of porin expression, and to DNA containing a 3'-truncated ompR gene. The major effect of ipeX on ompC expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced ompC transcription. ipeX was localized within an untranslated region of 247 base pairs between the stop codon of nmpC-a remnant porin gene from the cryptic phage qsr' (DLP12) genome-and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from lc identical to that of ipeX. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the lc transcript, whose 3' end contains the corresponding ipeX sequence, inhibits OmpC expression. Overexpression of ipeX RNA inhibited both OmpC and OmpF expression but not that of OmpA. ompC-phoA chimeric gene constructs revealed a 248-bp untranslated region of ompC required for ipeX-mediated inhibition. However, no sequence complementarity was found between ipeX and this region of ompC, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of ipeX on ompC, but not on ompF, was independent of the RNA chaperone Hfq.

The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain’s Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.

Surface Plasmon Resonance Imaging of Transcription Factor Proteins: Interactions of Bacterial Response Regulators with DNA Arrays on Gold Films

Surface plasmon resonance (SPR) imaging measurements have been used to study the sequence specific adsorption of response regulator proteins to DNA arrays constructed on gold thin films. Bacteria adapt to their environment primarily through two-component signal transduction systems that consist of an environmental sensor histidine kinase and a transcription activator response regulator DNA binding protein. DNA arrays were fabricated from oligonucleotide sequences of known DNA binding regions for two response regulators: OmpR, which controls gene expression of the outer membrane porin proteins in Escherichia coli, and VanR, which is involved in the antibiotic vancomycin resistance in Enterococcus faecium. The label-free method of SPR imaging was then used to monitor the sequence specific binding of these two response regulator proteins to the DNA arrays. The promoter regions from the ompF (F1 and F2) and ompC (C1) genes for the OmpR protein, and from the vanRS (R1) and vanHAX (H1 and H2) genes for the VanR protein were studied. SPR imaging was used to (i) monitor and compare the binding of both response regulators to various promoter regions on the DNA array, (ii) compare the binding of the OmpR response regulator protein in its phosphorylated and nonphosphorylated forms, and (iii) monitor the inhibition of VanR protein binding to the DNA arrays in the presence of a small molecule DNA binding inhibitor. The proteins exhibited specificity for the known binding sequences compared to control sequences on the DNA array. For the OmpR protein, the highest amount of binding was observed at the F1 site. When OmpR was phosphorylated by a small molecule phosphodonor, acetyl phosphate, there was on average a 42% increase in protein binding. In contrast, phosphorylated VanR binding decreased by an average of 40% in the presence of a known DNA binding inhibitor, (2,3,4-trifluorophenylisothiazolone). These measurements demonstrate that SPR imaging is an effective screening method for compounds that target DNA protein interactions and can serve as a useful tool for the discovery of new therapeutic molecules that target DNA binding proteins.

OmpR regulates the two-component system SsrA-ssrB in Salmonella pathogenicity island 2.

Salmonella pathogenicity island 2 (SPI-2) encodes a putative, two-component regulatory system, SsrA-SsrB, which regulates a type III secretion system needed for replication inside macrophages and systemic infection in mice. The sensor and regulator homologs, ssrAB (spiR), and genes within the secretion system, including the structural gene ssaH, are transcribed after Salmonella enters host cells. We have studied the transcriptional regulation of ssrAB and the secretion system by using gfp fusions to the ssrA and ssaH promoters. We found that early transcription of ssrA, after entry into macrophages, is most efficient in the presence of OmpR. An ompR mutant strain does not exhibit replication within cultured macrophages. Furthermore, footprint analysis shows that purified OmpR protein binds directly to the ssrA promoter region. We also show that minimal medium, pH 4.5, induces SPI-2 gene expression in wild-type but not ompR mutant strains. We conclude that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of ssrA soon after Salmonella enters the macrophage.

A novel Bacillus subtilis gene, antE, temporally regulated and convergent to and overlapping dnaE.

A Bacillus subtilis promoter, Px, that functions in a convergent manner with the sigA operon promoter P3 has been found in the sigA operon. Promoter Px is turned on at the same time as promoter P3 during early sporulation. The transcript from promoter Px codes for a small protein with partial homology to the OmpR protein from Escherichia coli and also carries an untranslated sequence at its 3' end that is complementary to the 5' end of the P3 transcript, which codes for the ribosome binding site of dnaE. The gene controlled by Px has been called antE. The expression of antE does not require sigmaB, sigmaE, or sigmaH. Px was transcribed in vitro by the sigmaA holoenzyme and is the seventh promoter to be recognized in the sigmaA operon. A possible role for the antE gene during early sporulation is proposed.

Suppressor mutations in α-subunit of RNA polymerase for a mutant of the positive regulator, OmpR, in Escherichia coli

The OmpR protein is a positive regulator specific for the Escherichia coli ompF and ompC genes. This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase. We previously isolated OmpR mutants which were suggested to be defective in transcription activation, but not in DNA binding (the so-called positive control (PC) mutant). In this study we isolated mutants of the α-subunit of RNA polymerase which can suppress one of the putative PC mutants of OmpR. A crucial amino acid substitution was identified as [V264G] in the α-subunit, which is located in the helix H1 of the C-terminal domain, which has been claimed, based on mutational and structural analyses, to be involved in the interaction with other positive regulators including the well-characterized cAMP receptor protein.

Suppressor mutations in α-subunit of RNA polymerase for a mutant of the positive regulator, OmpR, inEscherichia coli

The OmpR protein is a positive regulator specific for the Escherichia coli ompF and ompC genes. This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase. We previously isolated OmpR mutants which were suggested to be defective in transcription activation, but not in DNA binding (the so-called positive control (PC) mutant). In this study we isolated mutants of the alpha-subunit of RNA polymerase which can suppress one of the putative PC mutants of OmpR. A crucial amino acid substitution was identified as [V264G] in the alpha-subunit, which is located in the helix H1 of the C-terminal domain, which has been claimed, based on mutational and structural analyses, to be involved in the interaction with other positive regulators including the well-characterized cAMP receptor protein.

A cyanobacterial gene that interferes with the phosphotransfer signal transduction involved in the osmoregulatory expression of ompC and ompF in Escherichia coli.

Synechococcus sp. PCC7942 is a phototrophic cyanobacterium. In this study we cloned a Synechococcus gene that has a striking effect on the production of the Escherichia coli outer membrane proteins, OmpC and OmpF, provided that this gene was introduced by a multicopy plasmid into the heterologous cells. This multicopy gene in E. coli cells was able to specifically shut off the production of both OmpC and OmpF at the level of transcription. The nucleotides were sequenced for this gene, named sis, and its gene product was purified from E. coli to near homogeneity. A computer-aided search found that the deduced amino acid sequence consisting of 138 residues is novel, with no significant similarity to any other protein in the databases. Since the transcription of ompC and ompF is regulated by the regulatory factors EnvZ and OmpR, through phosphotransfer signal transduction, we explored the inhibitory effect of Sis in various genetic backgrounds as to envZ and ompR. In particular, the inhibitory effect of Sis was observed even in an DeltaenvZ background, but was not observed in a certain background in which the ompC and ompF transcription was supported by a mutant OmpR that can function in a phosphorylation-independent manner. These results suggested that the EnvZ kinase may not be the direct target of Sis, but rather that the process(es) concerning the phosphorylation and/or dephosphorylation of the OmpR protein may be affected by Sis. However, no direct effect of Sis was seen in an in vitro OmpR-phosphorylation assay with the purified OmpR and Sis proteins. Based on these results, possible functions of Sis are discussed with special reference to the phosphotransfer signal transduction in E. coli.

Molecular analysis of OmpR binding sequences involved in the regulation of ompF in Escherichia coli

OmpR, the transcriptional regulatory protein of ompF, had not been previously shown to specifically bind to the −70 to −60-bp region of ompF. We show that the −102 to −76-bp sequence of ompF has a high affinity binding site for OmpR and produced a single OmpR/ ompF complex (complex b). Extension of this DNA fragment to include an inverted repeat sequence located between the −71 and −64-bp region resulted in the formation of a second, slower migrating complex (complex a). A −102 to −58-bp fragment containing a substitution of the −70 CG bp was able to form complex b, but not complex a. A mutant OmpR protein derived from a strain that can not repress ompF was unable to form complex a, while complex b was formed normally. Deletion of the −70 CG bp resulted in incomplete repression of OmpF. These results suggest that OmpR binds to the −71 to −64-bp region and that this sequence plays a role in the regulation of ompF in Escherichia coli.