Dataset Information

Phosphorylated CpxR restricts production of the RovA global regulator in Yersinia pseudotuberculosis.

ABSTRACT:

Background

RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression.

Methodology/principal findings

In this study, we characterized CpxR phosphorylation (CpxR?P) in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR?P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR?P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate.

Conclusions/significance

Cpx pathway activation restricts levels of the RovA global regulator. The regulatory influence of CpxR?P must therefore extend well beyond periplasmic quality control in the Yersinia envelope, to include genes involved in environmental survival and pathogenicity.

SUBMITTER: Liu J

PROVIDER: S-EPMC3158067 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Publications

Phosphorylated CpxR restricts production of the RovA global regulator in Yersinia pseudotuberculosis.

Liu Junfa J Obi Ikenna R IR Thanikkal Edvin J EJ Kieselbach Thomas T Francis Matthew S MS

PloS one 20110818 8

<h4>Background</h4>RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression.<h4>Methodology/principal findings</h4>In this study, we characterized CpxR pho ...[more]

PMID: 21876746

Similar Datasets

Project description:BackgroundThe CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.ResultsCpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the ?cI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.ConclusionsThis interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

Project description:BackgroundIn the enteropathogenic Yersinia species, RovA regulates the expression of invasin, which is important for enteropathogenic pathogenesis but is inactivated in Yersinia pestis. Investigation of the RovA regulon in Y. pestis at 26 °C has revealed that RovA is a global regulator that contributes to virulence in part by the direct regulation of psaEFABC. However, the regulatory roles of RovA in Y. pestis at 37 °C, which allows most virulence factors in mammalian hosts to be expressed, are still poorly understood.Methodology/principal findingsThe transcriptional profile of an in-frame rovA mutant of Y. pestis biovar Microtus strain 201 was analyzed under type III secretion system (T3SS) induction conditions using microarray techniques, and it was revealed that many cell-envelope and transport/binding proteins were differentially expressed in the ?rovA mutant. Most noticeably, many of the T3SS genes, including operons encoding the translocon, needle and Yop (Yersinia outer protein) effectors, were significantly up-regulated. Analysis of Yop proteins confirmed that YopE and YopJ were also expressed in greater amounts in the mutant. However, electrophoresis mobility shift assay results demonstrated that the His-RovA protein could not bind to the promoter sequences of the T3SS genes, suggesting that an indirect regulatory mechanism is involved. Transmission electron microscopy analysis indicated that there are small loose electron dense particle-like structures that surround the outer membrane of the mutant cells. The bacterial membrane permeability to CFSE (carboxyfluorescein diacetate succinimidyl ester) was significantly decreased in the ?rovA mutant compared to the wild-type strain. Taken together, these results revealed the improper construction and dysfunction of the membrane in the ?rovA mutant.Conclusions/significanceWe demonstrated that the RovA regulator plays critical roles in the construction and functioning of the bacterial membrane, which sheds considerable light on the regulatory functions of RovA in antibiotic resistance and environmental adaptation. The expression of T3SS was upregulated in the ?rovA mutant through an indirect regulatory mechanism, which is possibly related to the altered membrane construction in the mutant.

Dataset Information

Phosphorylated CpxR restricts production of the RovA global regulator in Yersinia pseudotuberculosis.

Background

Methodology/principal findings

Conclusions/significance

Publications

Phosphorylated CpxR restricts production of the RovA global regulator in Yersinia pseudotuberculosis.

Similar Datasets

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