Phase variation of Clostridioides difficile colony morphology occurs via modulation of cell division by CmrRST, a three-component signal transduction system

The intestinal pathogen Clostridioides difficile is a leading cause of healthcare-associated infections globally, causing recurrent infections with significant morbidity, mortality, and economic burden. Like many bacterial species, C. difficile exists in phenotypically diverse populations that increase survival in dynamic environments. Phenotypic heterogeneity in C. difficile is largely attributed to phase variation in which inversion of a DNA sequence containing regulatory information (a “switch”) modulates the expression of adjacent genes in an ON/OFF manner. The cmr switch controls expression of cmrRST, an operon that encodes the signal transduction system CmrRST. CmrS is a histidine kinase predicted to respond to a signal, and CmrR and CmrT are transcriptional regulators that presumably coordinate responses to the CmrS-detected stimulus. Prior work established that CmrRST plays a broad role in C. difficile physiology and pathogenesis. CmrR decreases biofilm production and is required for colonization in the hamster model of acute infection. CmrT promotes rough colony development, increases surface motility, decreases swimming motility, and confers cell chaining and elongation. However, little is known about the regulatory mechanisms that govern CmrRST activity, how CmrRST functions, or the genes that are regulated by this signal transduction system to confer the observed cmr-associated phenotypes. In this work, we discover that multiple regulatory mechanisms control transcription of cmrRST. Beyond the cmr switch orientation, signaling by cyclic diguanylate monophosphate and autoregulation by CmrR positively regulate expression of the operon. We characterize the CmrR and CmrT proteins by showing that the regulators form both homo- and heterodimers in vitro and identify residues that are required for full CmrR/CmrT activity. Lastly, we determine the genes regulated by CmrR and CmrT and identify two CmrT-regulated genes which encode previously uncharacterized proteins, MrpA and MrpB, that promote rough colony development, increased surface motility, cell elongation, and decreased swimming motility. We find that MrpA interferes with typical cell division by interacting with septum site-determining protein MinD, resulting in elongated cells. We propose that CmrRST-mediated effects on cell division drive the cmr-associated colony morphology and motility differences in a manner resembling sliding motility.