Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA.
J Bacteriol. 2012 Jan;194(1):79-89. doi: 10.1128/JB.06209-11. Epub 2011 Oct 28.
The RNA binding protein CsrA (RsmA) represses biofilm formation in several proteobacterial species. In Escherichia coli, it represses the production of the polysaccharide adhesin poly-β-1,6-N-acetyl-D-glucosamine (PGA) by binding to the pgaABCD mRNA leader, inhibiting pgaA translation, and destabilizing this transcript. In addition, CsrA represses genes responsible for the synthesis of cyclic di-GMP, an activator of PGA production. Here we determined that CsrA also represses NhaR, a LysR-type transcriptional regulator which responds to elevated [Na(+)] and alkaline pH and activates the transcription of the pgaABCD operon. Gel shift studies revealed that CsrA binds at two sites in the 5' untranslated segment of nhaR, one of which overlaps the Shine-Dalgarno sequence. An epitope-tagged NhaR protein, expressed from the nhaR chromosomal locus, and an nhaR posttranscriptional reporter fusion (PlacUV5-nhaR'-'lacZ) both showed robust repression by CsrA. Northern blotting revealed a complex transcription pattern for the nhaAR locus. Nevertheless, CsrA did not repress nhaR mRNA levels. Toeprinting assays showed that CsrA competes effectively with the ribosome for binding to the translation initiation region of nhaR. Together, these findings indicate that CsrA blocks nhaR translation. Epistasis studies with a pgaA-lacZ transcriptional fusion confirmed a model in which CsrA indirectly represses pgaABCD transcription via NhaR. We conclude that CsrA regulates the horizontally acquired pgaABCD operon and PGA biosynthesis at multiple levels. Furthermore, nhaR repression exemplifies an expanding role for CsrA as a global regulator of stress response systems.
RNA 结合蛋白 CsrA（RsmA）在几种变形菌中抑制生物膜的形成。在大肠杆菌中，它通过与 pgaABCD mRNA 前导序列结合，抑制 pgaA 翻译并使该转录本不稳定，从而抑制多糖黏附素聚-β-1,6-N-乙酰-D-葡萄糖胺（PGA）的产生。此外，CsrA 还抑制负责合成环二鸟苷酸（c-di-GMP）的基因，c-di-GMP 是 PGA 产生的激活剂。在这里，我们确定 CsrA 还抑制了 NhaR，这是一种响应升高的 [Na(+)] 和碱性 pH 的 LysR 型转录调节因子，并激活 pgaABCD 操纵子的转录。凝胶迁移研究表明，CsrA 在 NhaR 的 5'非翻译区结合两个位点，其中一个位点与 Shine-Dalgarno 序列重叠。一个表达于染色体上的 NhaR 标签蛋白和一个 NhaR 转录后报告基因融合（PlacUV5-nhaR'-'lacZ）都显示出 CsrA 的强烈抑制作用。Northern blot 分析显示了 NhaR 基因座的复杂转录模式。然而，CsrA 并没有抑制 NhaR mRNA 水平。Toeprinting 分析表明，CsrA 能有效地与核糖体竞争结合 NhaR 的翻译起始区。总之，这些发现表明 CsrA 阻断了 NhaR 的翻译。与 pgaA-lacZ 转录融合的上位性研究证实了 CsrA 通过 NhaR 间接抑制 pgaABCD 转录的模型。我们得出结论，CsrA 以多种方式调节水平获得的 pgaABCD 操纵子和 PGA 生物合成。此外，NhaR 的抑制例证了 CsrA 作为应激反应系统的全局调节剂的作用不断扩大。
