ABSTRACT: Purpose: The goal of this study was to identify genes whose expression is induced during sporulation in a Spo0A-, M-OM-^CF-, M-OM-^CE-, M-OM-^CG-, and M-OM-^CK-dependent manner. Methods: Whole genome RNA sequencing was performed on wildtype, spo0A-, sigF-, sigE-, sigG-, and sigK- C. difficile strains (strain 630 background; JIR8094 = parent strain), and the transcriptional profiles of the different mutants during growth on 70:30 agar plates were determined using an Illumina HiSeq1000. Results: This analysis identified 185 genes whose expression is collectively activated by sporulation sigma factors: 150 were M-OM-^CF-dependent, 150 were M-OM-^CE-dependent, 30 were M-OM-^CG-dependent, and 31 were M-OM-^CK-dependent. A total of 237 genes were identified as requiring Spo0A for their expression when sporulation was induced on the 70:30 plates. Conclusions: These results provide the first genome-wide transcriptional analysis of genes whose expression is induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes. For example, in contrast with the B. subtilis sporulation pathway, C. difficile M-OM-^CE was not required to fully activate M-OM-^CG, and M-OM-^CG was not required to activate M-OM-^CK. Wildtype, spo0A-, sigF-, sigE-, sigG-, and sigK- were streaked onto 70:30 plates in triplicate, and sporulation was induced. RNA from the three biological replicate samples was harvested, DNAse-I treated, and ribosomally depleted using Ribo-Zero kits. This RNA was transformed into cDNA, and the resulting libraries were subjected to paired end sequencing using an Illumina HiSeq 1000.
