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

ABSTRACT: 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.

SUBMITTER: Nieckarz M

PROVIDER: S-EPMC5559549 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Nieckarz Marta M Raczkowska Adrianna A Jaworska Karolina K Stefańska Ewa E Skorek Karolina K Stosio Dorota D Brzostek Katarzyna K

Frontiers in cellular and infection microbiology 20170815

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 <i ...[more]

PMID: 28861396

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Project description: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.

Project description:A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by Yersinia enterocolitica (G. M. Young and V. L. Miller, Mol. Microbiol. 25:319-328, 1997). An insertion in hre20, now designated rscR, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated in rscR, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in rscR was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal rscR gene and carrying rscR on a plasmid under the control of the inducible araBAD promoter was mutagenized with an mTn5Km-2 transposon containing a promoterless lacZY. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of Serratia marcescens, and a locus (designated rscBAC) with similarity to the hmwABC locus of Haemophilus influenzae. The hmwABC locus promotes adherence of H. influenzae to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320-3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993). A strain containing a deletion mutant of rscA, the hmwA homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the rscR mutant. This suggests that the phenotype of an rscR mutant is due to the loss of RscA.

Dataset Information

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

Publications

The Role of OmpR in the Expression of Genes of the KdgR Regulon Involved in the Uptake and Depolymerization of Oligogalacturonides in <i>Yersinia enterocolitica</i>.

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