OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem−1 and weaker phem−2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the −35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem−1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5′-untranslated region (5′-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts

OmpR controls Yersinia enterocolitica motility by positive regulation of flhDC expression

Flagella and invasin play important roles during the early stages of infection by the enteric pathogen Yersinia enterocolitica. Our previous study demonstrated that OmpR negatively regulates invasin gene expression at the transcriptional level. The present study focused on the role of OmpR in the regulation of flagella expression. Motility assays and microscopic observations revealed that an ompR mutant strain exhibits a non-motile phenotype due to the lack of flagella. An analysis of flhDC::lacZYA chromosomal fusions demonstrated a decrease in flhDC expression in ompR mutant cells, suggesting a role for OmpR in the positive control of flagellar master operon flhDC, which is in contrast to the negative role it plays in Escherichia coli. Moreover, high temperature or osmolarity and low pH decreased flhDC expression and OmpR was not required for the response to these factors. Evidence from an examination of the DNA binding properties of OmpR in vitro indicated that the mechanism by which OmpR regulates flhDC is direct. Electrophoretic mobility shift assays confirmed that OmpR binds specifically to the flhDC promoter region and suggested the presence of more than one OmpR-binding site. In addition, phosphorylation of OmpR by acetyl-P appeared to stimulate the binding abilities of OmpR. Together with the results of our previous studies revealing the negative role of OmpR in the regulation of invasin expression, these findings support a model in which invasion and motility might be reciprocally regulated by OmpR

The Regulatory Circuit Underlying Downregulation of a Type III Secretion System in Yersinia enterocolitica by Transcription Factor OmpR

In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of Y. enterocolitica. Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes virF and yscM1, it highly repressed the expression of yopD. OmpR was found to bind to the promoter of the lcrGVsycD-yopBD operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of flhDC, the master regulator of the flagellar-associated T3SS

Impact of OmpR on the membrane proteome of Yersinia enterocolitica in different environments: repression of major adhesin YadA and heme receptor HemR.

Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodeling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host

Urease Expression in Pathogenic Yersinia enterocolitica Strains of Bio-Serotypes 2/O:9 and 1B/O:8 Is Differentially Regulated by the OmpR Regulator

Yersinia enterocolitica exhibits a dual lifestyle, existing as both a saprophyte and a pathogen colonizing different niches within a host organism. OmpR has been recognized as a regulator that controls the expression of genes involved in many different cellular processes and the virulence of pathogenic bacteria. Here, we have examined the influence of OmpR and varying temperature (26 degrees C vs. 37 degrees C) on the cytoplasmic proteome of Y. enterocolitica Ye9N (bio-serotype 2/O:9, low pathogenicity). Differential label-free quantitative proteomic analysis indicated that OmpR affects the cellular abundance of a number of proteins including subunits of urease, an enzyme that plays a significant role in acid tolerance and the pathogenicity of Y. enterocolitica. The impact of OmpR on the expression of urease under different growth conditions was studied in more detail by comparing urease activity and the transcription of ure genes in Y. enterocolitica strains Ye9N and Ye8N (highly pathogenic bio-serotype 1B/O:8). Urease expression was higher in strain Ye9N than in Ye8N and in cells grown at 26 degrees C compared to 37 degrees C. However, low pH, high osmolarity and the presence of urea did not have a clear effect on urease expression in either strain. Further analysis showed that OmpR participates in the positive regulation of three transcriptional units encoding the multi-subunit urease (ureABC, ureEF, and ureGD) in strain Ye9N, but this was not the case in strain Ye8N. Binding of OmpR to the ureABC and ureEF promoter regions was confirmed using an electrophoretic mobility shift assay, suggesting that this factor plays a direct role in regulating the transcription of these operons. In addition, we determined that OmpR modulates the expression of a ureR-like gene encoding a putative regulator of the ure gene cluster, but in the opposite manner, i.e., positively in Ye9N and negatively in Ye8N. These findings provide some novel insights into the function of OmpR in adaptation strategies of Y. enterocolitica

The Role of OmpR in the Expression of Genes of the KdgR Regulon Involved in the Uptake and Depolymerization of Oligogalacturonides in Yersinia enterocolitica

Oligogalacturonide (OGA)-specific porins of the KdgM family have previously been identified and characterized in enterobacterial plant pathogens. We found that deletion of the gene encoding response regulator OmpR causes the porin KdgM2 to become one of the most abundant proteins in the outer membrane of the human enteropathogen Yersinia enterocolitica. Reporter gene fusion and real-time PCR analysis confirmed that the expression of kdgM2 is repressed by OmpR. We also found that kdgM2 expression is subject to negative regulation by KdgR, a specific repressor of genes involved in the uptake and metabolism of pectin derivatives in plant pathogens. The additive effect of kdgR and ompR mutations suggested that KdgR and OmpR regulate kdgM2 expression independently. We confirmed that kdgM2 occurs in an operon with the pelP gene, encoding the periplasmic pectate lyase PelP. A pectinolytic assay showed strong upregulation of PelP production/activity in a Y. enterocolitica strain lacking OmpR and KdgR, which corroborates the repression exerted by these regulators on kdgM2. In addition, our data showed that OmpR is responsible for up regulation of the kdgM1 gene encoding the second specific oligogalacturonide porin KdgM1. This indicates the involvement of OmpR in the reciprocal regulation of both KdgM1 and KdgM2. Moreover, we demonstrated the negative impact of OmpR on kdgR transcription, which might positively affect the expression of genes of the KdgR regulon. Binding of OmpR to the promoter regions of the kdgM2-pelP-sghX operon, and kdgM1 and kdgR genes was confirmed using the electrophoretic mobility shift assay, suggesting that OmpR can directly regulate their transcription. We also found that the overexpression of porin KdgM2 increases outer membrane permeability. Thus, OmpR-mediated regulation of the KdgM porins may contribute to the fitness of Y. enterocolitica in particular local environments

Characterization of the sites of Tn5-B22 transposition in <i>Y</i>. <i>enterocolitica</i> mutants.

<p>^a sequence of region flanking transposon insertion comes from the genome of <i>Y</i>. <i>enterocolitica</i> subsp. <i>palearctica</i> 105.5R(r) (NC_015224) deposited in the NCBI database.</p><p>^b<i>lacZ</i> is in the same orientation as the region with similarity to the database entry.</p><p>^c<i>lacZ</i> is in the opposite orientation to the region with similarity to the database entry.</p><p>^d + or-mean positive or negative OmpR-dependent regulation.</p><p>Characterization of the sites of Tn5-B22 transposition in <i>Y</i>. <i>enterocolitica</i> mutants.</p