Clostridium thermocellum secretes a multi-enzyme system called the cellulosome that rapidly solubilizes cellulose and other polysaccharides. The cellulosomal composition is modulated depending on the type of polysaccharide present in the biomass. Currently, the regulation of the cellulosomal genes is poorly understood. However, recently, we discovered a set of eight C. thermocellum alternative sigma factors sharing a strong relationship to the Bacillus subtilis σI. We hypothesized that these multiple σI–like factors are involved in the regulation of cellulosomal genes. Each sigI-like gene is arranged in operon with another gene encoding a putative membrane-associated protein that is similar to the B. subtilis anti-σI factor RsgI. Additionally, extracellular C-terminal domains of six RsgI-like proteins have a carbohydrate binding module (CBM), suggesting that RsgI-like proteins might be sensors of the environmental polysaccharides. To demonstrate that C. thermocellum σI–like factors regulate cellulosomal genes, and the cognate RsgI CBMs are the polysaccharide sensors, we performed a study using the σI6/RsgI6 system as a model. Because of the lack of genetic tools in C. thermocellum, we used B. subtilis as a heterologous host since this bacterium is a well-known model system for GC-low Gram-positive microorganisms. We constructed a host strain with an entire sigI-rsgI operon deletion and then expressed the C. thermocellum σI6 which efficiently regulated a reporter yellow fluorescence protein (YFP) gene that was fused to several predicted σI6–dependent promoters. Moreover, co-expression of both σI6 and RsgI6 allowed the sequestration of σI6 activity, down-regulating the reporter YFP gene.
