Expression of 17 genes in Clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture

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 over a approximately 10-fold range of dilution rates (0.013 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 17 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 the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >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.

FIG. 1.

Effect of dilution rate on fraction of cellobiose and cellulose consumed by continuous cultures of C. thermocellum ATCC 27405. Consumption was corrected for soluble sugars remaining in the culture medium.

FIG. 2.

Effect of dilution rate on fermentation end products in continuous cultures of C. thermocellum ATCC 27405 fed cellobiose (A) or cellulose (B).

FIG. 3.

Gene expression as a function of growth rate of C. thermocellum ATCC 27405 cultures grown in cellobiose- or cellulose-limited continuous culture. Expression is shown relative to two calibrator genes, recA and the 16S rRNA gene. In order to permit visualization of the relative recA and 16S rRNA expression on the same graph, values for expression relative to 16S rRNA were multiplied by a constant (5,589.9), calculated from the ratio of the average recA expression divided by the average 16S rRNA expression. celS, exoglucanase; cipA, scaffoldin protein of the cellulosome; manA, mannanase; adhY, putative type IV alcohol dehydrogenase.