Genome analysis of the Gram-positive cellulolytic bacterium Clostridium thermocellum revealed the presence of multiple negative regulators of alternative sigma factors. Nine of the deduced proteins share a strong similarity in their N-terminal sequences to the Bacillus subtilis membrane-associated anti-sigma(I) factor RsgI and have an unusual domain organization. In six RsgI-like proteins, the C-terminal sequences contain predicted carbohydrate-binding modules. Three of these modules were overexpressed and shown to bind specifically to cellulose and/or pectin. Bioinformatic analysis of >1200 bacterial genomes revealed that the C. thermocellum RsgI-like proteins are unique to this species and are not present in other cellulolytic clostridial species (e.g. Clostridium cellulolyticum and Clostridium papyrosolvens). Eight of the nine genes encoding putative C. thermocellum RsgI-like anti-sigma factors form predicted bicistronic operons, in which the first gene encodes a putative alternative sigma factor, similar to B. subtilis sigma(I), but lacking in one of its domains. These observations suggest a novel carbohydrate-sensing mechanism in C. thermocellum, whereby the presence of polysaccharide biomass components is detected extracellularly and the signal is transmitted intracellularly, resulting in the disruption of the interaction between RsgI-like proteins and sigma(I)-like factors, the latter of which serve to activate appropriate genes encoding proteins involved in cellulose utilization.