ABSTRACT: Clostridium acetobutylicum, the endospore-forming anaerobe best known for its ABE (acetone-butanol-ethanol) fermentation, has received renewed attention recently for the biological production of butanol, both for bulk chemical production and as a potential biofuel. With butanol production in mind, most of the recent research on C. acetobutylicum has focused on increasing butanol production, tolerance to butanol, and optimizing it for various substrates. However, an equally important trait, though less understood, is its sporulation program, which it coupled to solvent formation. The model organism for endospore formation is Bacillus subtilis, but significant physiological, metabolic, and genomic differences exist between the two organisms. Despite these differences, the major sporulation-related transcription/sigma factors are conserved between the two species. These transcription/sigma factors are activated in a cascade manner such that Spo0A becomes active first, followed by M-OM-^CF, then M-OM-^CE, then M-OM-^CG, and finally M-OM-^CK. The goal of this study is to determine the regulons of 4 of these transcription/sigma factors (Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG) and compare them to those in B. subtilis. To accomplish this goal, individual mutant strains were created for Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG, in which the transcription/sigma factor is silenced. These mutants were then compared transcriptionally using microarrays to determine the regulon of each transcription/sigma factor. To help avoid false positives, comparisons were made between strains in which the downstream transcription/sigma factor is silenced (e.g., for the Spo0A regulon, the Spo0A mutant was compared against the M-OM-^CF mutant and the M-OM-^CE mutant, since they are both upregulated by Spo0A) rather than just the WT. For each regulon, 4 timepoints were taken, since it is very difficult to synchronize sporulation in C. acetobutylicum cultures, and dye-swaps were prepared for each timepoint. Four transcription/sigma factor regulons were investigated: Spo0A, M-OM-^CF, M-OM-^CE, and M-OM-^CG. For Spo0A, two comparison were made: Spo0A mutant vs M-OM-^CF mutant and Spo0A mutant vs M-OM-^CE mutant. For M-OM-^CF, two comparison were also made: M-OM-^CF mutant vs M-OM-^CE mutant and M-OM-^CF mutant vs M-OM-^CG mutant. Finally for M-OM-^CE and M-OM-^CG, only one comparison for each was made: M-OM-^CE mutant vs M-OM-^CG mutant and M-OM-^CG mutant vs wild-type, respectively. For each comparison, 4 timepoints were analyzed, and dye-swaps were prepared for each timepoint comparison. Timepoints were chosen based on when each transcription/sigma factor was expected to be active, based on a previous study [Jones, SW, et al. Genome Biol. 2008;9(7):R114.].
