Abstract
BackgroundRovA 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 FindingsIn 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/SignificanceCpx 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.
Highlights
Extracytoplasmic stress (ECS) has a deleterious effect on bacterial fitness, since it impacts on cell envelope integrity, protein folding and function
We have recently studied the function of the Cpx two-component regulatory system (TCRS) in the enteropathogen Yersinia pseudotuberculosis [19,20], a bacterium causing self-limiting gastroenteritis in infected individuals [21]
In vitro phosphorylation of CpxR by acetyl,P is a robust method for generating a high proportion of CpxR,P molecules
Summary
Extracytoplasmic stress (ECS) has a deleterious effect on bacterial fitness, since it impacts on cell envelope integrity, protein folding and function. At least in E. coli, CpxR,P is capable of up- or down-regulating the transcription of many genes that function to alleviate the effects of ECS [2,3,4] Once this has occurred, status quo is restored by the phosphatase activity of CpxA, which dephosphorylates CpxR [5]. A consequence of generating CpxA phosphatase deficient E. coli, as would result from a full length cpxA deletion mutant or so-called gain-of-function mutants (designated cpxA*), would be to accumulate CpxR,P [5,10,11,12,13,14] In these situations, acetyl phosphate (acetyl,P), a small molecular weight phosphodonor, can elevate CpxR,P levels [5,15,16,17]. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression
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