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Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 8, e23314- p.Article in journal (Refereed) Published
Abstract [en]

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 similar to 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 similar to 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 similar to P must therefore extend well beyond periplasmic quality control in the Yersinia envelope, to include genes involved in environmental survival and pathogenicity.

Place, publisher, year, edition, pages
Public Library of Science , 2011. Vol. 6, no 8, e23314- p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:umu:diva-46172DOI: 10.1371/journal.pone.0023314OAI: oai:DiVA.org:umu-46172DiVA: diva2:437258
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2011-08-29 Created: 2011-08-29 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
Open this publication in new window or tab >>Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Reglering av virulens hos Yersinia pseudotuberculosis genom ackumulering av fosforylerat CpxR-protein
Abstract [en]

Like many Gram-negative bacteria, the food-borne pathogen Yersinia pseudotuberculosis harbours different regulatory mechanisms to maintain an intact bacterial envelope especially during exposure to extracytoplasmic stress (ECS). The CpxA-CpxR two component regulatory system is one such ECS-responsive regulatory mechanism. Activation of CpxA-CpxR two-component regulatory system (TCRS) accumulates phosphorylated CpxR (CpxR~P), which not only up-regulates various factors that are designed to maintain envelope integrity, but also down-regulates key determinants of bacterial virulence.

Y. pseudotuberculosis establishes close host cell contact in part through the expression of the invasin adhesin. Invasin expression is positively regulated by the transcriptional regulator RovA, which in turn is negatively regulated in response to nutrient stress by a second transcriptional regulator RovM. In Y. pseudotuberculosis, loss of CpxA phosphatase activity accumulates CpxR~P, and this represses both rovA and inv transcription directly, or indirectly via activation of rovM transcription. It is now of interest to understand the molecular mechanism behind how CpxR~P regulates gene transcription both positively and negatively.

A type III secretion system (T3SS) is a highly conserved multi-protein secretion system used by many Gram-negative bacteria to secrete protein cargo that counteracts the effects of a host cell emitted anti-bacterial activity. A typical set of proteins that make-up a functional T3SS includes structural proteins, translocators, effectors and regulatory proteins. Accumulation of CpxR~P was shown to repress the plasmid encoded Ysc-Yop T3SS of Y. pseudotuberculosis. Although yet to be confirmed experimentally, promoter-CpxR~P binding studies indicate multiple modes of regulatory control that for example, could influence levels of the plasmid-encoded Ysc-Yop system transcriptional activator, LcrF, and the chromosomal encoded negative regulators YmoA and YtxR. 

Regulatory processes of TCRS involve transient molecular interactions between different proteins and also protein with DNA. Protein-protein interaction studies using the BACTH assay showed that it can be useful in analysing the molecular interactions involving the N-terminal domain of CpxR, while the λcI homodimerization assay can be useful in analysing molecular interactions involving the C-terminal domain of CpxR. Therefore, in combination with other biochemical and physiological tests, these hybrid-based assays can be useful in dissecting molecular contacts that can be helpful in exploring the mechanism behind CpxR~P mediated transcriptional regulation.

In conclusion, this work uncovered direct involvement of CpxR~P in down-regulating virulence in Yersinia pseudotuberculosis. It also utilised genetic mutation and explored different protein-protein interaction assays to begin to investigate the mechanism behind the positive and negative regulation of gene expression mediated through active CpxR~P. 

Place, publisher, year, edition, pages
Umeå: Umeå university, 2014. 63 p.
Keyword
Y. pseudotuberculosis, CpxA, CpxR, invasin, RovA, RovM, T3SS, virulence, transcriptional regulation
National Category
Microbiology Biochemistry and Molecular Biology
Research subject
Molecular Biology; Microbiology
Identifiers
urn:nbn:se:umu:diva-97320 (URN)978-91-7601-163-8 (ISBN)
Public defence
2015-01-22, Norrlands universitetssjukhus, Auditorium E04, Unod R1, Umeå universitet, Umeå, 09:00 (English)
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Available from: 2014-12-19 Created: 2014-12-15 Last updated: 2014-12-17Bibliographically approved

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Liu, JunfaObi, Ikenna RThanikkal, Edvin JKieselbach, ThomasFrancis, Matthew S
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