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Knockout of Transcriptional Regulators for Decoupling Solvent Formation from Sporulation In Clostridium Acetobutylicum

Bryan P. Tracy, Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208 and Eleftherios Terry Papoutsakis, Dept. of Chemical Engineering & Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE 19711.

Understanding and manipulating bacteria differentiation are intriguing goals motivated by pure scientific curiosity, medical microbiology, concerns of biological warfare, and industrial biotechnology. Of particular interest is endospore formation, which is well understood in the facultative anaerobe Bacillus subtilis (B. subtilis), but far less understood in related endospore formers such as clostridia. Clostridium is a genus of Gram-positive, obligate anaerobic, endospore-forming bacteria that includes many revered pathogenic species (C. botulinum, difficile, tetani and perfringens) and numerous commercially desired cellulolytic and/or solvent forming species (C. acetobutylicum, thermocellum, cellulolyticum, beijerinckii, etc.). Interestingly solvent formation and some toxin production are suggested to be genetically coupled with endospore formation. Thus we are interested in characterizing the genetic connections and controlling these coupled events.

To accomplish these tasks, we applied a novel gene disruption approach to individually knock out the function of sigE and sigG, which are suspected to be involved in either or both endospore and solvent formation in C. acetobutylicum. In both cases we successfully and absolutely decouple solvent formation from the completion of endospore differentiation. Both strains exhibited near wild-type (ATCC824) solvent production characteristics. Results also reinforced the suspected similarities between the transcriptional regulation events responsible for B. subtilis endospore formation by arresting endospore formation at stages II and III of asymmetric cell division for sigE and sigG knockouts respectively. This is the first report to purposefully decouple solvent formation from endospore differentiation. Results are significant for all clostridia and both strains serve as ideal commercial platforms for future optimization in converting cellulosic feedstocks to transportation fuels.

Web Page: www.papoutsakis.org/page.php?id=63