Co-transcription of the celC gene cluster in Clostridium thermocellum

Abstract

Clostridium thermocellum, an anaerobic, thermophilic, and ethanogenic bacterium produces a large cellulase complex termed the cellulosome and many free glycosyl hydrolases. Most cellulase genes scatter around the genome. We mapped the transcripts of the six-gene cluster celC-glyR3-licA-orf4-manB-celT and determined their transcription initiation sites by primer extension. Northern blot showed that celC-glyR3-licA were co-transcribed into a polycistronic messenger with the transcription initiation site at -20 bp. Furthermore, RT-PCR mapping showed that manB and celT, two cellulosomal genes immediately downstream, were co-transcribed into a bicistronic messenger with the initiation site at -233 bp. In contrast, rf4 was transcribed alone with the two initiation sites at -130 and -138 bp, respectively. Finally, quantitative RT-PCR analysis showed that celC, glyR3, and licA were coordinately induced by growing on laminarin, a β-1,3 glucan. Gene expression peaked at the late exponential phase. Taking together with our previous report that GlyR3 binds to the celC promoter in the absence of laminaribiose, a β-1,3 glucose dimer, these results indicate that celC, glyR3, and licA form an operon repressible by GlyR3 and inducible by laminaribiose, signaling the availability of β-1,3 glucan. The celC operon is the first glycosyl hydrolase operon reported in this bacterium.

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).…”

“…The regulon of GlyR3 appears to be limited to the celC-glyR3-licA operon, as previously described (11,19). GlyR1 affected the expression of licB (Clo1313_2022), containing a glycoside hydrolase 16 domain.…”

“…Expression studies across growth stages have identified a peak in the gene expression from this operon at late exponential phase as the cell transitions into early stationary phase (11,35,36). The activity of 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.…”

“…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 expression of the cellulosomal proteins is influenced by substrate availability and extracellular polysaccharides (8). Most cellulase and hemicellulase genes of C. thermocellum are scattered throughout the genome in small clusters, in contrast to the larger operons in mesophilic cellulolytic clostridia (9)(10)(11)(12)(13)(14)(15)(16). Methods of sensing and coordinating gene expression must therefore exist to allow the organism to utilize cellodextrins in plant biomass (17).…”