| The ancient soil bacterium Clostridium acetobutylicum is important both as a potential industrial platform for chemical and biofuel production and as a model organism for the study of sporulation in solventogenic clostridia. Sporulation is a common trait to all clostridia and is a simple differentiation process in which a cell undergoes asymmetric division to form a resilient and stable endospore. Of particular importance is how solventogenesis and sporulation, both stationary phase events, are interconnected, since sporulation is not a desirable trait from an industrial point of view. In order to investigate the transcriptional regulation of sporulation and how that regulation impacts solventogenesis, several inactivation mutants for key sporulation-related transcriptional regulators were created, and their gene expression patterns were compared to the wild-type strain using genomic microarrays. In this way, all the genes controlled and influenced by these transcriptional regulators was determined.;It was found that the order in which each of these sporulation-related transcriptional regulators became active was similar to that in the model organism for endospore formation Bacillus subtilis, in that Spo0A becomes active first, followed by sigmaF, then sigma E and G, and finally sigmaK. However, beyond this central pathway, the regulation of sporulation in C. acetobutylicum and B. subtilis varied dramatically. Inactivation of the first sigma factor sigmaF in C. acetobutylicum abolished sporulation prior to asymmetric division, while in B. subtilis inactivation of sigmaF stalls sporulation after asymmetric division. In addition, the transcription of both of the downstream sigma factors, sigE and sigG, appeared to be a sigmaF-directed process, though there may be some positive feedback between sigmaF and sigmaE to boost their expression. Overall, there was almost no similarity between C. acetobutylicum and B. subtilis in the genes controlled by the first three transcriptional regulators (Spo0A, sigmaF, and sigmaE), and a new sporulation-related sigma factor was identified, the sigmaF/E-family sigma factor CAP0167. Though the sigmaF inactivation mutant produced solvents, the level of production was dependent upon the physiological state of the inoculum used. Finally, a technique was developed to integrate multiple functional genes into the clostridial chromosome to alter cellular metabolism and differentiation. |
