Induction of the celC operon of Clostridium thermocellum by laminaribiose

Abstract

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 ␤-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, ␤-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.cellulase ͉ cellulosome ͉ transcription regulation

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

“…Deletion of the previously described C. thermocellum LacI transcriptional regulator gene, glyR3 (Cthe_2808/Clo1313_0396), resulted in 31, 65, and 75 genes considered significantly differentially expressed at mid-exponential, late exponential, and early stationary phases, respectively ( Table 1). Upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with the activity of this regulator as previously reported (11,19), and each showed similar trends across all three growth stages ( Fig. S3 and Table S3 in the supplemental material).…”

“…However, only a small proportion of C. thermocellum transcriptional regulators have been studied. Previous work has demonstrated the inducible expression of the celC-glyR3-licA operon (11,19), the involvement of alternative sigma factors with their cognate anti-sigma factors in the regulation of cellulolytic genes, including celS (17,20,21), and histidine kinase genes involved in the regulation of sporulation (22).…”

“…The GlyR3 regulator on this three-gene operon was demonstrated to have repressor activity, which was relieved in the presence of laminaribiose, a ␤-1,3-linked glucose dimer. GlyR3 was shown to bind to an 18-bp near-palindromic sequence upstream of the start codon of Cthe_2807 (19).…”

Thermobifida fusca exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

“…In nature, cellulosome-producing bacteria incorporate enzymes into multi-component complexes in a highly regulated process (Dror et al 2003a(Dror et al , b, 2005Bayer et al 2004;Newcomb et al 2007). Since the specificities of the scaffoldin-borne cohesins and enzyme-borne dockerins are virtually identical (Yaron et al 1995;Pagès et al 1997), selective incorporation of a given enzyme in situ is not determined at the biochemical level of the cohesin-dockerin interaction itself, but by a different undetermined cellular mechanism.…”

Mangiferin activates the Nrf2-ARE pathway and reduces etoposide-induced DNA damage in human umbilical cord mononuclear blood cells

“…EMSA gels were electroblotted onto a nylon membrane. Signal development was carried out by following the LightShift Chemiluminescent EMSA kit protocol (Pierce, Rockford, IL) with BioMax films (Kodak, Rochester, NY) for luminescence detection (Kumar et al, 2008;Newcomb et al, 2007).…”