Project Summary Clostridioides difficile is a gram-positive, spore-forming, obligate anaerobic bacterium and opportunistic pathogen. C. difficile infection (CDI) is associated with hospital antibiotic use and can cause severe diarrhea, pseudomembranous colitis, and death. The primary causative agents of CDI symptoms are the secreted toxins TcdA and TcdB, which are regulated by the flgB operon through sigma factor sigma-D. The bacterial transcription termination factor Rho regulates the flgB operon by repressing transcription only when an invertible genetic “switch” mapping to the flgB transcript 5’ UTR is in the “off” orientation. Genetic deletion of Rho functionality restores flgB expression when the switch is oriented off. Loss of Rho functionality in C. difficile results in growth, sporulation, and virulence defects independent of loss of regulation at the flgB operon, indicating additional Rho-dependent regulation of C. difficile virulence factors. Efforts to identify a Rho recognition sequence in C. difficile, including at the flagellar switch, based on a multi-organism consensus have been unsuccessful, possibly due to the unusually GC-poor C. difficile genome (~29%). Rho-dependent regulation is also likely to be highly influenced by interactions with termination/antitermination factors NusA and NusG. Nus factors are essential for C. difficile growth but their influence on Rho-dependent termination and effect on the virulence factors expression is unknown. I hypothesize that Rho is a key factor in C. difficile pathogenesis through direct regulation of critical virulence genes in addition to the flagellar operon, and that this regulation is dependent on, or attenuated by, Nus factors on a gene-by-gene basis. The objective of this proposal is to identify and annotate Rho, NusA, and NusG dependent terminators in C. difficile to identify key Rho and Nus factor regulated growth, sporulation, and virulence genes for further study, and determine a predictive rut site sequence in C. difficile. I will compare RNA-seq and Term-seq data between the Rho-null and control C. difficile strains to identify Rho dependent terminators and verify these sites in vitro. I will use CRISPRi to generate NusA and NusG dependent transcript-depleted strains for Term-seq analysis. Completion of this study will improve our understanding of C. difficile virulence regulation, provide further annotation of the C. difficile transcriptome, and may reveal new potential targets for developing therapeutics to combat CDI. Dr. Tamayo is a leader in the field of C. difficile biology and her expertise in genetic approaches complements my graduate training in biochemical techniques. The University of North Carolina at Chapel Hill and the Department of Microbiology and Immunology both contribute to a productive, collegial research environment with numerous opportunities for career development.