Laboratoire Pathogenèse des Bactéries Anaérobies, Département de Microbiologie Institut Pasteur, Paris 75015, France.
Nucleic Acids Res. 2012 Nov;40(21):10701-18. doi: 10.1093/nar/gks864. Epub 2012 Sep 18.
The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.
代谢物控制蛋白 CcpA 是一种多功能调节剂，介导细菌对快速可代谢碳水化合物（如革兰氏阳性菌中的葡萄糖）的全局转录反应。通过全转录组分析，我们描述了艰难梭菌中葡萄糖依赖性和 CcpA 依赖性基因调控。大约 18%的艰难梭菌基因受葡萄糖调控，其中 50%依赖 CcpA 进行调控。CcpA 调控子包含参与糖摄取、发酵和氨基酸代谢的基因，证实了 CcpA 作为碳氮途径之间联系的作用。通过组合染色质免疫沉淀和基因组序列分析，我们检测到 55 个 CcpA 结合位点，对应约 140 个直接受 CcpA 调控的基因。我们定义了艰难梭菌 CcpA 共识结合位点（cre(CD) 基序），即“RRGAAAANGTTTTCWW”。通过电泳迁移率变动分析证实了纯化的 CcpA 蛋白与 19 个靶 cre(CD) 位点的结合。CcpA 还直接抑制孢子形成早期关键因子（Spo0A 和 SigF）。此外，艰难梭菌毒素基因（tcdA 和 tcdB）及其调节剂（tcdR 和 tcdC）是 CcpA 的直接靶标。最后，CcpA 通过调节一大组调节剂来控制一个复杂而扩展的调控网络。
