The alternative σ^I5 factor regulate cellulosomal arabinoxylan utilization genes in Clostridium thermocellum

Clostridium (Ruminiclostridium) thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that is capable of efficiently hydrolyzing crystalline cellulose via an extracellular multienzyme complex, termed the cellulosome. The bacterium can also produce ethanol, which makes him an attractive candidate for a consolidated process of converting biomass into bioethanol in a single fermentation step. C. thermocellum regulates its cellulosomal genes in response to extracellular biomass composition via a unique σ^I/RsgI biomass sensing system. The bacterium contains a set of six operons, encoding for an alternative σ^I-factors and a multi-modular proteins, RsgIs (regulator of SigI). In the present study, we identified the promoter region of σ^I5, and the cellulosomal genes regulated by σ^I5. Unlike the previously characterized sigI-rsgI operons that contain two genes, the sigI5-rsgI5 operon is composed of three genes including a gene upstream to sigI5, for a putative arabinofuranosidase from glycoside hydrolase family 43 (GH43). Transcription analysis using RT-PCR confirmed that gh43, sigI5 and rsgI5 are co-transcribed. The apparent transcription start site of the gh43-rsgI5 operon was identified and assigned to a G nucleotide, 67 bases upstream from the gh43 ATG initiation codon. The σ^I5 promoter region contains conserved sequence of the “extended -35” (AAA), CAACCCAAA, which is separated by 14-bp from the -10 region (CGTA). The predicted σ^I5 promoter sequence was identified in five arabinoxylan utilization genes, suggesting that their expression is controlled by σ^I5. Thus, the alternative sigma factor σ^I5 appears to regulate arabinoxylan utilization genes consistent with the binding properties of the RsgI5 arabinoxylan sensing domain (CBM42).