Regulation of Major Cellulosomal Endoglucanases of Clostridium thermocellum Differs from That of a Prominent Cellulosomal Xylanase

Abstract

The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CelS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 h ؊1 , the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 h ؊1 ) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate-the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.

Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis

“…The detection of CelN and CelQ on Avicel and not cellobiose may be taken as another indication of increased GH9 endoglucanase production on Avicel. The differential expression of GH9 versus GH5 endoglucanases poses an apparent discrepancy with the recent transcript analysis of Dror et al (9), who observed increased transcript levels per cell of each of the endoglucanase genes celB and celG from GH5 and celD from GH9 when cells were grown at a low versus a high growth rate and also on cellulose versus cellobiose. Thus, while our results with respect to GH9 endoglucanases agree with these previous findings at the transcript level, the increase of GH5 endoglucanases and of CelA on cellobiose was a somewhat unanticipated result.…”

“…Finally, with respect to hemicellulases, all subunits with xylanase or xyloglucanase activity decreased on Avicel, as per RelEx and emPAI analysis. XynC production has previously been shown to increase on cellobiose (4,9), and xynC transcript levels have been found to increase on cellobiose in a growth rate-independent fashion (9). In this study, XynZ, XynA, XynC, and XghA were among the five most abundant docking components in cellobiose-grown cellulosomes, along with CelA.…”

“…The immediate availability of energy results in an increased growth rate and leads to the repression of genes required to mine energy from crystalline cellulose. Lower growth rates and cellulose as a substrate seem to promote cellulase production, as has been demonstrated for the processive glycoside hydrolase family 48 (GH48) exoglucanase CelS, both at the protein (4) and the mRNA level (7,38), as well as for the transcription of the GH5 endoglucanases celB and celG and the GH9 endoglucanase celD (9). Transcription of the scaffoldin gene cipA and cell surface anchoring genes olpB and orf2p is likewise controlled by growth rate and/or carbon source, which is not the case for another cell surface gene, sdbA (8,38).…”

Induction of the celC operon of Clostridium thermocellum by laminaribiose

“…The expression of celS is growth rate-dependent as revealed by chemostat experiments (25,27). Similarly, the transcript levels of cipA, olpB, orf2p, celB, celG, and celD are dependent on growth rate (28,29). In contrast, the expression of sdbA and xynC are independent from growth rate.…”

Global Gene Expression Patterns in Clostridium thermocellum as Determined by Microarray Analysis of Chemostat Cultures on Cellulose or Cellobiose

“…Thus, scaffoldin and cellulase genes have similar transcriptional responses to growth rate and substrate (10,11,37), and transcriptional regulators have been associated with some cellulase genes (22). Previously, Stevenson and Weimer conducted a real-time PCR survey of the expression of 17 C. thermocellum genes involved in cellulose hydrolysis, intracellular phosphorylation, and catabolite repression and correlated their expression profiles with the formation of fermentation products (31).…”