|Stevenson, David - UW-MADISON|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 5, 2005
Publication Date: August 5, 2005
Citation: Stevenson, D., Weimer, P.J. 2005. Expression of seventeen genes in clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture. Applied and Environmental Microbiology. 71(8):4672-4678. Interpretive Summary: Clostridium thermocellum is a type of fiber-degrading bacteria that has promise as an agent for converting fiber-containing crops and agricultural wastes to ethanol. The regulation of genes important in fiber degradation and fermentation product formation by this organism has not been systematically examined. We measured the degree to which this bacterium turned on 19 different genes important in energy metabolism and cell growth when the bacterium was grown at different rates on either cellulose (a major component of fiber) or cellobiose (a soluble sugar). We identified four genes whose expression was strongly dependent on growth conditions. We also demonstrated major changes in the amount of ethanol and acetic acid that resulted from changes in growth rate. The data provide information important for developing strategies to improve fiber degradation and ethanol production by this organism.
Technical Abstract: Clostridium thermocellum is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in C. thermocellum ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation at dilution rates ranging from 0.016 to 0.16 h-1. Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of seventeen genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes (recA and 16S rDNA) to determine relative expression. Thirteen genes displayed modest (five-fold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffolidin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one type IV alcohol dehydrogenase), two putative cAMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed substantially (ten-fold or more) reduced levels of expression when grown on cellobiose at dilution rates > 0.05 h-1: cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase) and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated, but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.