Quantitative data file.

Phase variation of C. difficile colony morphology occurs via modulation of transcription of cmrRST, which encodes a three-protein signal transduction system. Response regulators CmrR and CmrT promote rough colony development, cell elongation and chaining, surface motility, and disease in the hamster model of infection, while impairing swimming motility and biofilm formation. Using RNA-Seq, we identified the CmrR and CmrT-dependent transcriptional differences in rough and smooth colonies. Further analysis showed that CmrT, but not CmrR, is required for differential expression of most of the genes. Two CmrT-regulated genes, herein named mrpA and mrpB, were together sufficient for restoring all CmrT-dependent in vitro phenotypes in a cmrT mutant and alleviating selection of cmr phase ON cells during growth on an agar surface. MrpA and MrpB are uncharacterized proteins with no known function but are highly conserved in C. difficile. Using immunoprecipitation and mass spectrometry to identify interacting partners, we found that MrpA interacts with the septum site-determining protein MinD and several other proteins involved in cell division and cell shape determination. Ectopic expression of mrpAB resulted in atypical cell division, consistent with MrpAB interference with MinD function. Our findings reveal a potential mechanism by which phase variation of CmrRST modulates colony morphology and motility: in cmr phase ON cells, CmrT-mediated expression of mrpAB interferes with normal cell division resulting elongated cells that enable expansion of the population across a surface while limiting swimming motility.

艰难梭菌（C. difficile）菌落形态的相变异，通过调控cmrRST的转录过程发生，该基因座编码一套三蛋白信号转导系统。应答调节蛋白CmrR与CmrT可促进粗糙菌落形成、细胞伸长与成链、表面运动能力，以及仓鼠感染模型中的疾病进程；同时却会削弱游泳运动能力与生物膜形成能力。我们借助RNA测序（RNA-Seq）技术，鉴定出粗糙与光滑菌落中依赖CmrR和CmrT的转录差异。进一步分析显示，绝大多数基因的差异表达依赖CmrT，而非CmrR。我们将两个受CmrT调控的基因命名为mrpA与mrpB，二者联合即可恢复cmrT突变株中所有依赖CmrT的体外表型，并能缓解琼脂表面生长时cmr相开启细胞的选择压力。MrpA与MrpB均为功能未知的未表征蛋白，但在艰难梭菌中高度保守。我们通过免疫沉淀（immunoprecipitation）与质谱（mass spectrometry）技术鉴定互作伴侣蛋白，发现MrpA可与隔膜位点确定蛋白MinD（MinD）以及其他多种参与细胞分裂与细胞形态决定的蛋白发生互作。异位表达mrpAB会引发非典型细胞分裂，这与MrpAB对MinD功能的干扰效应相符。本研究揭示了CmrRST相变异调控菌落形态与运动能力的潜在机制：在cmr相开启的细胞中，CmrT介导的mrpAB表达会干扰正常细胞分裂，产生伸长的细胞，从而使菌群能够在表面扩散，同时限制游泳运动能力。