ompR Gene Research Articles

Differential regulation of OmpC and OmpF by AtpB in Escherichia coli exposed to nalidixic acid and chlortetracycline

Little is known about mechanisms through which OmpR/EnvZ and CpxA/CpxR regulate the expression of OmpC and OmpF, and thereby regulate bacterial responses to antibiotics exposure. In this study we investigated the relationships among OmpC, OmpF and TCSs using Escherichia coli strains with the deletion of ompR, envZ, cpxA and cpxR genes and antibiotics nalidixic acid and chlortetracycline. Significant changes at expression levels of OmpC and OmpF were detected in the strains exposed to the antibiotics. Outer membrane proteins that were altered in response to the changes of OmpC and OmpF expression levels were identified using proteomics approaches and the networks involved in the regulation of protein changes in response to the antibiotic exposure were constructed. It was found that AtpB was upregulated and downregulated in parallel with the reduced and elevated expression of OmpC in response to chlortetracycline and nalidixic acid exposure, respectively and the change of AtpB was regulated by CpxR. These findings were validated by the detection of OmpC, OmpF and CpxR changes in ΔatpD and ΔphoE. Therefore, this study provides novel insight into the regulation network of TCSs and OM proteins involved in the differential responses of bacteria to different classes of antibiotics.

An Efficient Site-Directed Mutagenesis Method <i>In Vivo</i> in Escherichia Coli

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

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.

Applied an Efficient Site-directed Mutagenesis Method into Escherichia coli

A new technique for conducting site-directed mutagenesis was developed. This method allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of s-galactosidase. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. In this paper, in order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knock-out ompR gene was constructed through overlapping PCR, digested by Not and Sal I I, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompRmutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompRmutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

Comparative analysis of Salmonella enterica serovar Enteritidis mutants with a vaccine potential

If any new live Salmonella vaccine is introduced in the future, it is quite probable that detailed characterisation of its attenuation will be required. In this study we therefore compared 34 isogenic mutants of S. Enteritidis in aroA, aroD, galE, ssrA, sseA, phoP, rpoS, ompR, htrA, clpP, lon, rfaL, rfaG, rfaC, hfq, sodCI, hilA, sipA, avrA, sopB, sopA, sopE, sifA, shdA, fliC, fur, relA, spoT, rel-spoT, misL, rmbA, STM4258, STM4259 and spvBC genes for their resistance to stresses likely to be expected in the host and for their virulence and immunogenicity in Balb/C mice. We found that the cold and bile resistances essentially did not correlate with the resistances to other stress factors. Resistance to acid pH, heat, polymyxin and serum correlated with each other and also with the attenuation. When the residual virulence and immunogenicity were both considered, mutants in htrA, ompR, aroA, aroD and lon performed the best in mice. Furthermore, when a detailed comparison of polymyxin and serum sensitive mutants was performed, the serum sensitive mutants were more immunogenic.

Structural analysis of OmpR a transcription regulator in the mutualistic bacterium, Xenorhabdus nematophila

A partnership between Marquette University High School students participating in the MSOE SMART Team (Students Modeling A Research Topic) program and a researcher enabled the team to explore structure and function, and to build a physical model using 3D printing technology, of OmpR. OmpR is a transcription factor necessary for the nutrition gathering strategy of the bacterium Xenorhabdus nematophila. Bacteria absorb food through membrane pores, which change in size to optimize food intake and to protect themselves from toxins.X. nematophila has sensor proteins in the outer membrane. When food touches an outer membrane receptor (EnvZ), the receptor transfers phosphate to OmpR forming OmpR‐P, which can bind to DNA. When nutrients are abundant, OmpR‐P binds to OmpC, a gene promoting formation of a small pore, allowing food yet limiting influx of toxins. When food is scarce, OmpR‐P binds OmpF, a gene promoting formation of a large pore, allowing more food intake and growth of a flagellum enabling movement to a nutrient rich location.The OmpR gene is also responsible for production of antibiotic compounds that combat a broad range of microorganisms. X. nematophila often forms a mutualistic relationship with nematodes. The bacterium‐nematode pair seek to inhabit and eventually kill certain insects, benefiting from the nutrients provided by the insect's corpse.Supported by a grant from NIH‐NCRR‐SEPA

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.

Stimulation of biofilm formation by insertion of Tetrahymena pyriformis wells within Burkholderia cenocepacia biofilms

Biofilm formation is an important part of the bacterial life cycle. Biofilms provide bacterial resistance to external stresses and protozoan grazing. Biofilm formation by the wild type of B. cenocepacia strain 370 in the presence of the free-living ciliate Tetrahymena pyriformis was studied. T. pyriformis grazed on planktonic bacteria and reduced the planktonic bacterial subpopulation while it noticeably stimulated biofilm formation. When cultivated alone, T. pyriformis did not form visible biofilms. Confocal laser scanning microscopy was used to demonstrate the inclusion and further destruction of protozoan cells within the biofilms formed by the bacteria. The destruction of protozoan cells was accompanied by the exit of bacteria from vacuoles and intracytoplasmic multiplication; changes in the form of protozoan cells; the demolition of internal structures; and the visual exit of the cytoplasmic content from destructing cells. Microcolonies of a characteristic round shape were revealed in the biofilms formed by B. cenocepacia in the presence of T. pyriformis. These structures were absent in the biofilms formed by B. cenocepacia alone. Insertion of protozoan cells within biofilms seems to be a driving force that promotes biofilm proliferation and influences their structure. The mortality of protozoan cells in the biofilms caused a decrease in the T. pyriformis population under conditions advantageous to B. cenocepacia biofilm formation. The mutant B. cenocepacia strain Bcb-1, which is unable to form biofilms, was isolated by plasposon mutagenesis. In contrast to the parental strain, the cocultivation with Bcb-1 bacteria improved the growth of T. pyriformis. A mutation was mapped in the ompR gene.

Regulation of flhDC expression in Proteus mirabilis

Transposon insertions located 325 and 740 base pairs upstream of the transcriptional start site of the flhDC operon resulted in cells that initiated swarming 1.5 h earlier than wild-type and exhibited a 2–2.5-fold greater swarming velocity. These mutants also failed to consolidate (de-differentiate) normally and did not form the characteristic bulls-eye pattern of concentric swarming rings on solid media. The analysis of one mutant (SS-P) with an insertion at −325 revealed that the levels of flhDC mRNA were dramatically higher than wild-type during swarmer cell differentiation and failed to decrease during the consolidation period. However, the start point of flhDC transcription was identical in the SS-P mutant and wild-type cells. The presence of the flhDC upstream region on a high copy plasmid increased swarming motility and expression of a chromosomal flhDC-lacZ fusion, presumably by titrating out a repressor. To identify potential repressors and further define flhDC regulation in P. mirabilis, targeted disruptions were created in the rcsB, ompR, lrhA and hdfR genes, previously demonstrated to repress flhDC in E. coli. Of these mutations, only the loss of rcsB increased swarming and flhDC mRNA accumulation.

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.

EnvZ-OmpR Interaction and Osmoregulation in Escherichia coli

EnvZ, a histidine kinase/phosphatase in Escherichia coli, responds to the osmolarity changes in the medium by regulating the phosphorylation state of the transcription factor OmpR, which controls the expression levels of outer membrane porin proteins OmpF and OmpC. Although both ompR and envZ genes are located on the ompB locus under the control of the ompB promoter and transcribed as a single polycistronic mRNA, the expression of envZ is known to be significantly less than ompR. However, to date no accurate estimation for the amounts of EnvZ and OmpR in the cell has been carried out. Here we examined the levels of EnvZ and OmpR in the wild-type strain MC4100 by quantitative Western blot analysis using anti-OmpR and anti-EnvZc (cytoplasmic domain of EnvZ) antisera. It was observed that during exponential growth in L-broth medium there were approximately 3500 and 100 molecules per cell of OmpR and EnvZ, respectively. The levels of OmpR and EnvZ in MC4100 cells grown in a high osmolarity medium (nutrient broth with 20% sucrose) were about the same as those grown in L-broth, whereas they were 1.7-fold higher than those in a low osmolarity medium (nutrient broth). With His10-OmpR, we also determined that the K(d) value for the EnvZc-OmpR complex formation is 1.20 +/- 0.17 microm. On the basis of these results, the molecular mechanism of osmoregulation of ompF and ompC is discussed.

Cloning and characterisation of a novel ompB operon from Vibrio cholerae 569B

The ompB operon of Vibrio cholerae 569B has been cloned and fully sequenced. The operon encodes two proteins, OmpR and EnvZ, which share sequence identity with the OmpR and EnvZ proteins of a variety of other bacteria. Although the order of the ompR and envZ genes of V. cholerae is similar to that of the ompB operon of E. coli, S. typhimurium and X. nematophilus, the Vibrio operon exhibits a number of novel features. The structural organisation and features of the V. cholerae ompB operon are described.

5527529 Vaccines comprising attenuated salmonella having mutations in the OMPR genes : Dougan Gorda; Chatfield Steven N; Higgins Christopher F; Dorman Charles J Kent, United Kingdom The Wellcome Foundation Limited; The Royal Society The Lister Institute of Preventive Medicine The University Court of The University of Dund

5527529 Vaccines comprising attenuated salmonella having mutations in the OMPR genes : Dougan Gorda; Chatfield Steven N; Higgins Christopher F; Dorman Charles J Kent, United Kingdom The Wellcome Foundation Limited; The Royal Society The Lister Institute of Preventive Medicine The University Court of The University of Dund

Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi+ phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.

Microcin B17: posttranslational modifications and their biological implications.

Microcin B17: posttranslational modifications and their biological implications.

Isolation and characterization of Escherichia coli strains producing microcins of B and C types

A screening of 11956 enterobacteria isolates resulted in selection of seven active microcin-producing strains. The microcins were shown to be peptides or their derivatives with a rather broad spectrum of activity, mainly against Gram-negative bacteria. According to cross-immunity criteria, the microcins studied belonged to two of the previously suggested types, B (five strains) and C (two strains). Those of type B could be further classified into two subtypes on the account of differences in the spectrum of antibacterial activity. In five cases out of seven the microcin-producing ability has been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells was shown to depend on ompR and ompF gene products.

Isolation and characterization ofEscherichia colistrains producing microcins of B and C types

A screening of 11956 enterobacteria isolates resulted in selection of seven active microcin-producing strains. The microcins were shown to be peptides or their derivatives with a rather broad spectrum of activity, mainly against Gram-negative bacteria. According to cross-immunity criteria, the microcins studied belonged to two of the previously suggested types, B (five strains) and C (two strains). Those of type B could be further classified into two subtypes on the account of differences in the spectrum of antibacterial activity. In five cases out of seven the microcin-producing ability has been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells was shown to depend on ompR and ompF gene products.

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.

An equivalent to bacterial ompR genes is encoded on the plastid genome of red algae.

An equivalent to bacterial ompR genes is encoded on the plastid genome of red algae.

Osmoregulatory expression of the porin genes in Escherichia coli: evidence for signal titration in the signal transduction through EnvZ-OmpR phosphotransfer.

The OmpR protein of Escherichia coli is a positive regulator specific for the ompF and ompC genes. The function of OmpR is modulated through phosphotransfer signaling mediated by the kinase, EnvZ. We previously demonstrated that OmpR contains two functional domains, which are physically separable; one is responsible for the interaction with EnvZ, whereas the other participates in interactions with cognate promoter DNAs. In this study, these domains of OmpR were overproduced in wild-type cells harboring the endogenous intact ompR gene on their chromosome. It was found that when the N-terminal domain of OmpR, which contains the phosphorylation site, was overproduced, expression of the ompF and ompC genes was markedly inhibited, irrespective of the osmolarity of the growth medium. Based on our current model for the molecular mechanism underlying signal transduction through Envz-OmpR phosphotransfer (T. Mizuno and S. Mizushima, Mol. Microbiol. 4, (1990), 1077-1082), we provide evidence that this phenomenon is best interpreted by the concept of 'signal titration' in the phosphotransfer signaling pathway.