Clostridium thermocellum secretes a multi-enzyme system called the cellulosome to solubilize polysaccharides. The cellulosomal composition is modulated according to the polysaccharide. In general, regulation of the cellulosomal genes is poorly understood.
Recently, we discovered a set of multiple C. thermocellum alternative sigma factors sharing a strong sequence similarity 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 individual C. thermocellum σI–like factors may regulate specific target cellulosomal genes, we performed a study by using C. thermocellum σI2, σI3 and σI6 as models because their promoters were experimentally identified in our group. We used a B. subtilis ΔsigI-rsgI strain as a heterologous host because of the lack of genetic tools in C. thermocellum. A bioinformatics analysis of the upstream regions of sigI2, sigI3, sigI6, and some key cellulosomal genes reveals a putative promoter consensus motif for each σI. To corroborate the functionality of the predicted promoters, we fused them to the promoterless lacZ reporter gene. Expression of each C. thermocellum σI factor with their cognate putative promoters in B. subtilis, confirms their functionality, supporting our hypothesis that these multiple σI–like factors regulate cellulosomal genes.
