Department of Molecular Biology, Umeå University, Umeå, Sweden.
PLoS One. 2011;6(8):e23314. doi: 10.1371/journal.pone.0023314. Epub 2011 Aug 18.
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.
RovA 是病原性耶尔森氏菌中基因表达的全局转录调控因子。RovA 的水平受到复杂的转录和转录后调控机制的控制。在肠道病原体假结核耶尔森氏菌中，我们之前曾报道过细胞外应激感应 CpxA-CpxR 双组分调节系统调节 rovA 的表达。
方法/主要发现：在这项研究中，我们对 CpxR 磷酸化（CpxR∼P）进行了体外表征，并确定磷酸化对于 CpxR 有效地结合 rovA 的上游调控区 PCR 扩增产物是必要的。通过核酸酶保护实验定位了精确的 CpxR∼P 结合位点，并且定向突变证实了体内结合到 rovA 启动子上会抑制转录。在慢性 Cpx 途径激活期间，CpxR∼P 在耶尔森氏菌细胞质中的积累以及乙酰磷酸的非特异性磷酸供体作用导致 RovA 产生减少最为明显。
结论/意义：Cpx 途径的激活限制了 RovA 全局调节剂的水平。因此，CpxR∼P 的调节影响必须远远超出耶尔森氏菌包膜的周质质量控制，包括涉及环境生存和致病性的基因。
