Regulation of cellulase synthesis by C. thermocellum is an important feature of the physiology of this microorganism, particularly in light of the substantial investment of ATP that cellulase synthesis represents (34,35). Johnson and coworkers (36) reported that true cellulase activity (i. e., degradation of crystalline cellulose) synthesis was markedly repressed by cellobiose. mRNAs corresponding to endoglucanases CelA, CelF, and CelD were found to be regulated at the level of transcription by a mechanism analogous to catabolite repression (37). The number of CelS (the most dominant cellulolytic enzyme of the cellulosome) and CipA (the cellulosomal scaffolding protein) transcripts per cell were shown to decrease with increasing growth rate (38, 39). CelS transcripts were found to be higher for growth under cellobiose — limitation as compared to growth under nitrogen limitation (38) and control of scaffoldin and CelS transcription was shown to involve a housekeeping Sigma-A factor (39).

Stevenson and Weimer (40) investigated expression profiles of 17 genes involved in cel­lulose hydrolysis, intracellular phosphorylation, catabolite repression, and fermentation end product formation as determined by real-time PCR in continuous cultures grown on cellobiose and cellulose. Thirteen genes displayed modest (fivefold or less) differences in expression in response to varied growth rate or substrate. By contrast, cipA, celS, and manA genes displayed 10-fold reduced levels when grown on cellobiose at dilution rates of >0.05/h, suggesting that at least some cellulosomal components are transcriptionally regulated.

Zhang and Lynd (32) investigated the regulation of cell-specific cellulase synthesis (de­fined as mg cellulase/g cell dry weight) by C. thermocellum using an ELISA protocol based on an antibody raised against a peptide sequence from the scaffoldin protein (31). We found that cellulase synthesis in Avicel-grown batch cultures was nine times greater than in cellobiose — grown batch cultures. In substrate-limited continuous cultures, however, cellulase synthesis with Avicel-grown cultures was greater than that in cellobiose-grown cultures by 1.3- to 2.4- fold, depending on the dilution rate. Continuous cellobiose-grown cultures maintained at either high dilution rates or high feed substrate concentration resulted in decreased cellulase synthesis, with a large (sevenfold) decrease between 0 and 0.2 g/L cellobiose and a much more gradual further decrease for cellobiose concentrations >0.2 g/L. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by carbon catabolite repression (CCR). These factors include: 1) substantially higher cellulase yields observed during batch growth on Avicel as compared to cellobiose, 2) a strong negative correlation between cellobiose con­centration and cellulase yield in continuous cultures with varied dilution rate at constant feed substrate concentration and also with varied feed substrate concentration at constant dilution rate, and 3) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome. CCR-mediated control of cellulosome synthesis in C. thermocellum is supported by the observation that the three key components of a CCR system — an LacI/GalR family regulatory protein, an HPr protein and an HPr kinase, and a 14-bp cis-acting catabolite responsive element (CRE) binding sequence — are presentinthe C. thermocellum genomic sequence (http://genome. ornl. gov/microbial/cthe/). Several putative LacI/GalR family genes are found in C. thermocellum. We were able to lo­cate many (>100) putative CRE sequences, including two putative CREs inside the cipA
structural gene (+953, +5231), using the more degenerate CRE consensus sequence (WG — WNANC/GNTNNCW). Substantial degeneracy of CRE sequences is supported by results from B. subtilis. For example, Chaveaux (41) found only 29 CRE sequences based on a consensus sequence with 7 of the 14 bases degenerate, whereas Moreno and coworkers (42) found, using DNA arrays, that ~330 genes were regulated by CCR. Moreover, whole genome analysis of B. subtilis indicates that the CREs sequence is not strictly conserved, and that CRE variation provides a means to alter the affinities of regulatory proteins to CRE sequences thereby modulating regulation (42, 43).