Clostridium thermocellum solubilizes cellulose and other polysaccharides with the aid of extracellular multienzyme cellulolytic complex called the cellulosome. Systematic analysis of C. thermocellum genomes revealed over 80 genes encoding cellulosomal proteins. Currently there is only limited information on the regulation of a few cellulosomal genes, and there are only hypotheses about possible transcriptional regulators that control their expression. We have recently discovered multiple two-component regulatory systems in C. thermocellum, which resemble the Bacillus subtilis alternative sigma factor σI and its cognate negative membrane-associated regulator (anti-sigma) RsgI. Six predicted C. thermocellum RsgI-like proteins possess an extracellular carboxy-terminal carbohydrate-binding module (CBM), and we have suggested that these modules can function as extracellular sensors recognizing corresponding polysaccharides. Our previous characterization of the two C. thermocellum σI-like factors, σI1 and σI6, led us to hypothesize that they might be involved in specific regulation of genes encoding key cellulosomal enzymes. In order to characterize other representatives of the C. thermocellum σI/RsgI systems, we performed a complete deletion of the rsgI3 gene encoding a putative anti-σI3 factor RsgI3 that carries a C-terminal tandem of two PA14 modules binding to pectin. We also mapped the transcriptional start site of the sigI3-rsgI3 operon, and its putative auto-regulated σI3-dependent promoter has been predicted. Several cellulosomal pectin-associated enzyme genes were predicted to possess σI3-dependent promoters. Using real-time RT-PCR, we demonstrated a significant up-regulation of selected genes in theC. thermocellum ∆rsgI3 mutant compared to the wild type. These results support our hypothesis that σI3/RsgI3 system may regulate genes encoding cellulosomal genes.
