Complex Organization and Dynamic Regulation of the pks Gene Cluster in Bacillus subtilis

The pks genes are the largest antibiotic- encoding gene cluster in Bacillus subtilis and encode the Pks enzymatic complex that produces bacillaene. Bacillaene plays important roles in the fitness of B. subtilis during competition with other bacterial species.

In this dissertation, I investigate the regulatory mechanisms used by B. subtilis to control the expression of the pks genes and the production of bacillaene. First, I focus on understanding the transcriptional regulatory network that coordinates the activation of the pks genes. My results indicate that multiple transcriptional regulators, in particular the stationary phase regulators Spo0A and CodY, coordinate the control of the pks gene activation. Also, cells dedicated to the formation of biofilms and spores but not motility induce the expression of the pks genes. I discuss these findings in light of their roles during bacterial competition.

I also identified multiple regulatory elements along the pks genes. Promoters upstream of pksB, pksC and pksS are active during vegetative growth while a promoter upstream of pksG is active only during spore formation. The activity of the pksG promoter is exclusive to the nascent spores and not the mother cells. In addition to promoters, a cis-regulatory element at the intergenic region of pksC and pksD promotes readthrough of transcription terminators along the pks genes.

Finally, I focus on the function of PksA, previously presumed to regulate the pks genes. I have found that PksA is not involved in the control of the pks gene expression. Instead, PksA negatively regulates the expression of ymcC. My data suggests that YmcC is not involved in bacillaene production but, consistent with structural prediction, I have found that YmcC is a membrane protein produced during sporulation. I hypothesize the function of YmcC during spore maturation or germination and propose experiments to elucidate this role.

In general, this dissertation contributes to the understanding of pks gene regulation and its implications in the competitive fitness of B. subtilis. This work also provides a model for the activation of Type I trans-AT PKSs encoded in gene clusters with similar organization to the pks genes.