SUMMARY ABSTRACT Clostridioides difficile is among the most common causes of nosocomial infections with disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Secreted toxins are largely responsible for disease development, yet many aspects of C. difficile physiology and virulence remain poorly understood. Recent work has revealed that C. difficile produces two colony morphotypes—a rough colony variant and a smooth colony variant—and can reversibly switch them. This phenomenon is conserved among diverse C. difficile strains. The two morphological variants differ in several ways. Bacteria from rough colonies are longer and often found in chains, exhibit greater surface motility and diminished swimming motility, produce less biofilm biomass, and show greater pathogenicity in animal models compared to the smooth colony counterpart. Our prior work linked colony morphology and the correlated phenotypes to the expression of genes encoding a signal transduction system, CmrRST, but the molecular mechanisms by which C. difficile develops rough and smooth colony morphologies have yet to be defined. The objective of this study is to identify genes required for formation of each colony morphotype and to determine the roles of these genes in cell morphology, motility, biofilm formation, and virulence. In Aim 1, we propose three complementary yet independent mutagenesis and screening strategies to identify genes required for rough and smooth colony development. This work takes advantage of mutants designed to yield only one colony morphotype. The in vitro phenotypes of the mutants obtained through the genetic screens will be evaluated to determine the broader impact of the identified genes on C. difficile physiology and virulence traits. In Aim 2, we will examine how one identified mutation results in strictly rough colonies and assess its ability to colonize and cause disease in a mouse model of C. difficile infection. Similar strategies will be used to characterize additional mutants obtained in Aim 1. Pilot studies with the proposed approaches have identified multiple candidate genes predicted to affect cell division. As such, these gene products may serve as new targets for therapeutic development, yet almost none of these genes have been previously studied. The proposed research will identify factors contributing to disease relevant phenotypes including cell division proteins, facilitating efforts to combat C. difficile infection.