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bioRxiv · 10.1101/2025.08.20.671228

Phase variation of Clostridioides difficile colony morphology occurs via modulation of cell division

Mehra, A.·Garrett, E. M.·Serody, C. J.·Tamayo, R.
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Abstract

Phase variation of C. difficile colony morphology occurs via modulation of transcription 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. AUTHOR SUMMARYC. difficile can reversibly switch between two colony morphology variants that differ in multiple additional phenotypes. Previous work determined that the phenotypic switch occurs through phase variation of the CmrRST signal transduction system, however the CmrRST-regulated genes that mediate these phenotypes were unknown. Here, we identified the CmrR- and CmrT-regulated genes that are differentially expressed between rough and smooth colonies and identified two genes, mrpA and mrpB, that together are sufficient to confer phenotypes associated with cmrRST expression. Protein interaction studies revealed that MrpA interacts with MinD, a protein that helps ensure symmetric cell division in bacteria. Based on these findings, we propose that MrpA disrupts MinD function leading to aberrant cell division, resulting in elongated cells that form rough colonies. This study reveals previously uncharacterized proteins that affect C. difficile cell division and broadly impact the physiology of this pathogen.

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Mehra, A., Garrett, E. M., Serody, C. J., Tamayo, R.. 2025-08-20. Phase variation of Clostridioides difficile colony morphology occurs via modulation of cell division. https://doi.org/10.1101/2025.08.20.671228

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