Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen that forms a symbiotic association with the nematode, Steinernema carpocapsae. Xenorhabdus is carried into the insect host by the nematode, is released into the hemolymph and participates in killing the insect. The bacteria grow to high concentrations supporting the development of the nematode in the hemolymph. OmpR is a global regulatory protein involved in the regulation of porin genes, motility, acid tolerance and virulence in several enteric bacteria. To study the role of ompR in the lifecyle of Xenorhabdus, an ompR -minus strain was constructed. The ompR strain produced markedly reduced levels of the porin protein, OpnP and was both hypermotile and exhibited a hyperhemolysis phenotype. Inactivation of flhDC, the master regulator for flagella synthesis, eliminated hemolysin production in the ompR strain, suggesting that ompR regulates hemolysin production via flhDC. The ompR mutant strain was virulent towards insect hosts. However, when nematodes were grown on a mixture of the wild-type and the ompR strain, only the wild-type strain was recovered indicating that ompR is required for competitive symbiotic interaction with the nematode. The role of ompR in the symbiosis between the bacterium and the nematode is under investigation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Regulatory Protein OmpR Influences the Serum Resistance of Yersinia enterocolitica O:9 by Modifying the Structure of the Outer Membrane

The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.     

Pleiotropic effects of a Yersinia enterocolitica ompR mutation on adherent-invasive abilities and biofilm formation

The OmpR regulator positively influences flagella synthesis and negatively regulates invasin expression in Yersinia enterocolitica. To determine the physiological consequences of this inverse regulation, we analyzed the effect of the ompR mutation on the ability of Y. enterocolitica Ye9 (serotype O9, biotype 2) to adhere to and invade human epithelial HEp-2 cells and to form biofilms. Cell culture assays with ompR, flhDC and inv mutant strains, which vary in their motility and invasin expression, confirmed the important contribution of flagella to the adherent-invasive abilities of Y. enterocolitica Ye9. However, the loss of motility in the ompR strain was apparently not responsible for its low adhesion ability. When the nonmotile phenotype of the ompR mutant was artificially eliminated, an elevated level of invasion, exceeding that of the wild-type strain, was observed. Confocal laser microscopy demonstrated a decrease in the biofilm formation ability of the ompR strain that was only partially correlated with its loss of motility. These data provide evidence that OmpR promotes biofilm formation in this particular strain of Y. enterocolitica, although additional OmpR-dependent factors are also required. In addition, our findings suggest that OmpR-dependent regulation of biofilm formation could be an additional aspect of OmpR regulatory function.     

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12

Summary The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant back-grounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC^- phenotype in an ompR ⁺background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules, and thereby support the idea that OmpR can exist as a multimeric protein.     

The <Emphasis Type="Italic">flhDC</Emphasis> gene affects motility and biofilm formation in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mutated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIIIΔflhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regulatory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium

Summary The tppB locus of Salmonella typhimurium encodes the anaerobically-induced tripeptide permease. We have demonstrated that expression of tppB requires the function of the ompR and envZ gene products, originally identified as positive regulatory proteins required for the osmotic regulation of porin expression. Significantly, tppB expression is not osmotically regulated. We have also identified three additional genes whose expression depends on OmpR. Thus OmpR and EnvZ serve a more general regulatory role than has previously been supposed. This study provides the first detailed genetic analysis of the ompB locus of S. typhimurium.     

Cloning of the regulatory locus ompB of Salmonella typhimurium LT-2

Summary We have cloned the complete functional ompB locus of Salmonella typhimurium LT-2 into Escherichia coli K-12 using a cosmid vector and in vitro packaging into . The ompB locus of Salmonella was found to complement both envZ and ompR mutations in E. coli as well as an ompR mutation of Salmonella. The ompR part of the ompB locus was further subcloned into the multicopy plasmid pKN410 as a 1.3 kb fragment. This fragment coded for a single 28.5 kd protein corresponding to about 820 bp in length. Furthermore, the OmpR proteins of S. typhimurium and E. coli were shown to be structurally and functionally highly similar.Abbreviations SDS sedium dodecyl sulfate - kb kilobase pairs - bp base pairs - kd kilodaltons     

Co-regulation of polysaccharide production,motility, and expression of type III secretion genes by EnvZ/OmpR and GrrS/GrrA systems in Erwinia amylovora

The EnvZ/OmpR and GrrS/GrrA systems, two widely distributed two-component systems in gamma-Proteobacteria, negatively control amylovoran biosynthesis in Erwinia amylovora, and the two systems regulate motility in an opposing manner. In this study, we examined the interplay of EnvZ/OmpR and GrrS/GrrA systems in controlling various virulence traits in E. amylovora. Results showed that amylovoran production was significantly higher when both systems were inactivated, indicating that the two systems act as negative regulators and their combined effect on amylovoran production appears to be enhanced. In contrast, reduced motility was observed when both systems were deleted as compared to that of grrA/grrS mutants and WT strain, indicating that the two systems antagonistically regulate motility in E. amylovora. In addition, glycogen accumulation was much higher in envZ/ompR and two triple mutants than that of grrS/grrA mutants and WT strain, suggesting that EnvZ/OmpR plays a dominant role in regulating glycogen accumulation, whereas levan production was significantly lower in the grrS/grrA and two triple mutants as compared with that of WT and envZ/ompR mutants, indicating that GrrS/GrrA system dominantly controls levan production. Furthermore, both systems negatively regulated expression of three type III secretion (T3SS) genes and their combined negative effect on hrp-T3SS gene expression increased when both systems were deleted. These results demonstrated that EnvZ/OmpR and GrrS/GrrA systems co-regulate various virulence factors in E. amylovora by still unknown mechanisms or through different target genes, sRNAs, or proteins, indicating that a complex regulatory network may be involved, which needs to be further explored.     

OmpR May Regulate the Putative YehU/YehT Two-Component System in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhi Under Hypotonic Growth Condition

Decreased expression (twofold) of a putative yehUTS operon of which yehUT encodes a putative YehU/YehT two-component system in the ompR mutant from Salmonella enterica serovar Typhi (S. Typhi) GIFU10007 under hypotonic growth condition was observed by qRT-PCR. Purified recombinant protein OmpR_His6 of GIFU10007 was shown to bind the upstream region of the yehU gene by the gel-shift assay. In addition, the yehT deletion mutant (ΔyehT) displayed differential expression (twofold or higher) of 26 genes under the condition by the DNA microarray analysis. Altogether, OmpR might regulate the YehUT system in S. Typhi under hypotonic growth condition.     

Small RNA coaR contributes to intestinal colonization in Vibrio cholerae via the two-component system EnvZ/OmpR

Vibrio cholerae is a waterborne bacterium responsible for worldwide outbreaks of acute and fatal cholera. Recently, small regulatory RNAs (sRNAs) have become increasingly recognized as important regulators of virulence gene expression in response to environmental signals. In this study, we determined that two-component system EnvZ/OmpR was required for intestinal colonization in V. cholerae O1 EI Tor strain E12382. Analysis of the characteristics of OmpR revealed a potential binding site in the intergenic region between vc1470 and vc1471, and qRT-PCR showed that expression of the intergenic region increased 5.3-fold in the small intestine compared to LB medium. Race and northern blot assays were performed and demonstrated a new sRNA, coaR (cholerae osmolarity and acidity related regulatory RNA). A ΔcoaR mutant showed a deficient colonization ability in small intestine with CI of 0.15. We identified a target of coaR, tcpI, a negative regulator of the major pilin subunit of TcpA. The ΔtcpI mutant has an increased colonization with CI of 3.16. The expression of coaR increased 2.8-fold and 3.3-fold under relative acidic and hypertonic condition. In summary, coaR was induced under the condition of high osmolarity and acid stress via EnvZ/OmpR and explained that tcpI relieves pH-mediated repression of toxin co-regulated pilus synthesis.     

Expression of Outer Membrane Proteins in Escherichia coli Growing at Acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.