(566d) Signaling Pathways Affecting Sporulation and Stationary Phase Phenomena in Clostridium Acetobutylicum

Bacteria thrive in a variety of environments and have developed complex signaling pathways to coordinate adaptive responses to changing conditions. The most common manner for signal recognition and response is through a two-component system, which involves the phosphate transfer from a histidine kinase to a response regulator that then alters gene expression. Recent transcriptional examination of C. acetobutylicum has identified several histidine kinases that are regulated in a sporulation-specific manner. C. acetobutylicum is an anaerobic endospore former capable of producing the industrially relevant solvents acetone and butanol. However, little is known regarding the entry into stationary phase, characterized by solvent formation and sporulation. Using asRNA technology, we have investigated signaling pathways that affect these important cellular programs. Our experimental results suggest that the two-component system of CAC0654 and CAC0653 (a kinase and its cognate response regulator) is a putative regulator of sporulation. pH-controlled fermentations of strain 824(pHK654) carrying an asRNA construct targeting CAC0654 show reduced glucose uptake and an 80% reduction in butanol production compared to a plasmid control strain. Transcriptional profiling using microarrays shows a profound upregulation of the whole sporulation cascade (after Spo0A) down to sigK, suggesting CAC0654 and CAC0653 act as repressors of sporulation. Using this information, we reversed engineered a new strain to overexpress this two-component system to reduce sporulation, while increasing the solvent production capabilities. Possible mechanisms of interactions and gene regulation of CAC0654 and CAC0653 are proposed based on the phenotypic and transcriptional data of these strains. Through this study and future experiments we aim to better understand the regulatory networks of C. acetobutylicum, which are used to engineer improved strains for solvent production. Additionally, this information can be applied to related pathogenic clostridia, which regulate toxin production in manners analogous to solvent production in C. acetobutylicum.