Genetic factors affecting the regulation of solventogenesis in Clostridium acetobutylicum ATCC824
Scotcher, Miles Christopher
Bennett, George N.
Doctor of Philosophy
The obligate anaerobe Clostridium acetobutylicum produces the acids acetate and butyrate during exponential growth. At the transition to stationary growth, these acids are converted to the solvents acetone and butanol. The genes involved in these metabolic pathways have been identified and characterized, but little is known about the control of their expression. This thesis focuses on the identification and further investigation of genes involved in the control of solventogenesis. solR was previously identified through genetic studies as a controller of solventogenesis. We have shown that the original construct used to increase solR expression can complement the high solvent phenotype of the solR-deleted mutants B and H when the solR open reading frame is either intact or partially deleted, thus demonstrating that the solR open reading frame is not required to restore solvent levels. Using a chloramphenicol acetyl-transferase reporter system harboring fragments of the alcohol dehydrogenase ( adhE) promoter, we have shown that a region beyond the 0A box is needed for full induction of the promoter, and that the presence of sequences around a possible processing site designated P2 may have a negative role in adhE expression. We identified two transcription factors, sinR and abrB310, which may be involved in the control of solventogenesis. We showed that abrB310 is expressed strongly and transiently at the onset of solventogenesis, and antisense RNA targeted against abrB310 caused a delay and decrease in solvent production, and also in sporulation. We propose that abrB310 is a regulator of the transition between acidogenic and solventogenic growth. We identified a homologue to the Bacillus subtilis early sporulation phosphatase and membrane protein, SpoIIE. Using reporter vectors, we showed that spoIIE is expressed in wild type C. acetobutylicum cells during late solventogenesis, but not in strain SKO1, where the sporulation initiator spo0A is deleted. Antisense RNA targeted against spoIIE caused a prolongation of solventogenesis characterized by increased solvent production and defective sporulation.
Molecular biology; Genetics; Microbiology