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Induction of the celC operon of Clostridium thermocellum by laminaribiose

Newcomb, Michael, Chen, Chun-Yu, Wu, J.H. David
Proceedings of the National Academy of Sciences of the United States of America 2007 v.104 no.10 pp. 3747-3752
Clostridium thermocellum, bacteria, binding sites, biomass, biosynthesis, cellulosome, deoxyribonuclease I, dose response, endo-1,4-beta-glucanase, gel electrophoresis, glucans, glucose, multiprotein complexes, operon, promoter regions, transcription factors
Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic, and ethanogenic bacterium. It produces an extracellular multiprotein complex termed the cellulosome, which consists of >70 subunits, most of them glycosyl hydrolases. It also produces many free glycosyl hydrolases. How the organism commands such a large number of genes and proteins for biomass degradation is an intriguing yet unresolved question. We identified glyR3, which is cotranscribed with the cellulase/hemicellulase genes celC and licA, as a potential cellulase transcription regulator. The gel-shift assay (EMSA) revealed that the recombinant GlyR3 bound specifically to the celC promoter region. GlyR3 was also identified from the lysate of the lichenan-grown cells, which bound to the same sequence. DNase I footprinting and competitive EMSA showed the binding site to be an 18-bp palindromic sequence with one mismatch. The DNA-binding activity was specifically inhibited by laminaribiose, a β-1-3 linked glucose dimer, in a dose-dependent manner. In in vitro transcription analysis, celC expression was repressed by rGlyR3 in a dose-dependent manner. The repression was relieved by laminaribiose, also in a dose-dependent manner. These results indicate that GlyR3 is a negative regulator of the celC operon consisting of celC, glyR3, and licA, and inducible by laminaribiose. Thus, the bacterium may modulate the biosynthesis of its enzyme components to optimize its activity on an available biomass substrate, in this case, β-1-3 glucan, because both CelC and LicA are active on the substrate. The results further indicate that, despite the insolubility of the biomass substrate, regulation of the degradative enzymes can be accomplished through soluble sugars generated by the action of the enzymes.