Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis

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.

Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

“…1B) (7,8), an activity that has been demonstrated in vitro (9,10). In vivo, the existence of this activity is supported by the apparent accumulation of phosphoCpxR by cpxA mutants of Yersinia pseudotuberculosis (11).…”

“…To monitor phospho-CpxR, we took advantage of a previously reported method used for the detection of phosphorylated proteins (27,28), including bacterial response regulators (11,29). This method uses a dinuclear metal complex (i.e.…”

“…Thus, if glucose-induced cpxP transcription depended strictly on AcP, then an ackA mutant should respond more robustly to glucose than its wild-type (WT) parent, whereas the pta (or pta ackA) mutant should respond more poorly. Although the latter is true (14), the former is not (8); ackA mutants do not elicit the predicted robust response, even though purified CpxR readily autophosphorylates in the presence of AcP (6,11). This puz-zling observation could mean that AcP is not the in vivo phosphoryl donor to CpxR.…”

“…On the basis of OmpR homology, phosphorylation of Asp-51 of CpxR is predicted to induce a conformational change that promotes gene regulation (17). Indeed, Asp-51 is required for AcP-dependent phosphorylation of CpxR purified from Y. pseudotuberculosis (11) and for CpxA-induced cpxP transcription (18).…”

PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43

“…To analyse the effect of urate, urate (Sigma-Aldrich) was firstly dissolved in 1 M NaOH and then added to the reaction buffer. For the interaction between CpxR and pecO, the binding buffer contained 20 mM Tris/HCl pH 7.0, 30 mM acetyl phosphate, 125 mM KCl, 10 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 0.25 mg BSA ml 21 and 0.05 mg sheared salmon sperm ml 21 DNA (Liu et al, 2011).…”