A single DNA methylation site regulates cell fate during Clostridioides difficile sporulation.

DNA methylation is a widespread phenomenon in bacteria that can regulate gene expression, although the mechanisms underlying this epigenetic regulation are often poorly understood. In Clostridioides difficile, the orphan DNA methyltransferase CamA promotes sporulation, a process critical for the persistence and transmission of this nosocomial pathogen. However, the specific CamA target genes that drive this increased sporulation phenotype were unknown. Here, we show that methylation of a single CamA motif in the promoter region of spoIIE, which encodes a factor critical for activating the early-acting sporulation sigma factor {sigma}F, is sufficient to promote spoIIE transcription, {sigma}F activation, and spore formation. Surprisingly, the CamA-dependent increase in spoIIE expression also increases the frequency of premature {sigma}F activation prior to asymmetric division, resulting in miscompartmentalization of {sigma}F activity. While this premature activation event triggers cell lysis in the well-studied spore-former Bacillus subtilis, we show that C. difficile cells retain developmental plasticity: predivisional cells that have prematurely activated {sigma}F can abort sporulation and resume vegetative growth, whereas cells that activate {sigma}F in the forespore after asymmetric division remain committed to sporulation. Thus, DNA methylation controls a critical cell fate decision in C. difficile without compromising its capacity to adapt to fluctuating environmental conditions. Finally, we show that CamA confers a significant fitness advantage during murine infection through mechanisms largely independent of its ability to promote sporulation. Since CamA is specific to C. difficile and epigenetically regulates multiple pathways critical for pathogen persistence, these analyses imply that CamA could be a promising antimicrobial target. SignificanceWhile DNA methylation of the bacterial genome is ubiquitous and can regulate diverse processes, identifying the methylation sites that drive a specific phenotype remains challenging. The Clostridioides difficile-specific orphan DNA methyltransferase CamA promotes sporulation and persistence in mice, but the mechanisms underlying these phenotypes were unknown. We identified a single methylation site in the promoter of a key sporulation gene that enhances sporulation, a process critical for this clinically relevant pathogen to transmit disease. Our analyses uncovered an unexpected developmental plasticity to C. difficiles sporulation program while also revealing that CamA regulates processes beyond sporulation that are important for maintaining long-term infection. Thus, targeting CamA may offer therapeutic benefit.