Characterization of two co-regulated response regulators in Clostridioides difficile.

As an obligate anaerobe, Clostridioides difficile grows exclusively in the host intestinal tract, necessitating mechanisms to sense and respond to the gut environment. The atypical signal transduction system encoded by cmrRST includes two OmpR-family response regulators, CmrR and CmrT. These regulators control multiple important phenotypes in C. difficile, including cell chaining, colony morphology, swimming motility, and biofilm formation. CmrR has a typical receiver domain with key conserved residues for phosphotransfer and conformational change, while CmrT is missing conserved residues and is likely a pseudoreceiver. Here, we used multiple methods to investigate dimer formation, including bacterial two-hybrid systems in both Escherichia coli and C. difficile and pull-down assays in E. coli. We detected CmrR homodimers and found that CmrR recognizes a specific DNA sequence found in multiple places in the genome, including upstream of cmrRST. CmrT formed homodimers in multiple assays, and mutation analysis of residues in its unusual active site suggests multiple mutations are needed to maximally reduce CmrT activity. Our data also suggest that CmrR and CmrT can form heterodimers, though the biological relevance of CmrR-CmrT heterodimers remains unclear. Our results suggest that CmrR and CmrT work together in transcriptional responses to stimuli.IMPORTANCETwo-component systems are nearly ubiquitous among bacteria and are one of the primary ways that bacteria respond to their environment. Atypical two-component proteins and systems are being identified in diverse bacteria, and studying these proteins helps us to understand the underlying mechanisms of these systems. CmrRST is an unusual two-component system that regulates many important phenotypes in Clostridioides difficile. This work begins to untangle the complex regulatory mechanisms by which CmrRST controls gene expression and furthers our understanding of the fundamental biology of C. difficile.