UNRAVELING THE CLOSTRIDIUM THERMOCELLUM ALTERNATIVE Σ^I6 FACTOR BIOMASS SENSING REGULON

The Gram-positive anaerobic thermophilic cellulolytic bacterium Clostridium (Ruminiclostridium) thermocellum secretes a multi-enzyme system called the cellulosome to solubilize plant cell wall polysaccharides. During the saccharolytic process, the enzymatic composition of the cellulosome is modulated according to the type of polysaccharide(s) present in the environment. C. thermocellum has a set of eight alternative sigma factors that are activated in response to polysaccharides and share sequence similarity to the Bacillus subtilis alternative σ^I factor. The aim of the present work was to demonstrate that individual C. thermocellum σ^I–like factors likely regulate specific target cellulosomal genes, focusing on the C. thermocellum σ^I6 factor because the sigI6 promoter was previously identified. To search for putative σ^I6-dependent promoters, a bioinformatics analysis of the upstream regions of cellulosomal genes has been performed. Because of the lack of genetic tools in C. thermocellum, the functionality of the predicted σ^I6-dependent promoters was studied in B. subtilis as a heterologous host. This system allowed us to observe the activation of the nine predicted σ^I6-dependent promoters associated with the following genes: cipA (Clo1313_0627), sigI6 (Clo1313_2778), xyn11B (Clo1313_0522), xyn10D (Clo1313_0177), xyn10Z (Clo1313_2635), xyn10Y (Clo1313_1305), cseP (Clo1313_2188), sigI1 (Clo1313_2174), and rsgI5 (Clo1313_0985). These results suggest a possible regulon of σ^I6 in C. thermocellum as well as a σ^I6 promoter consensus sequence that provides important clues to understand better the mechanism and regulation of C. thermocellum` adaptive response to extracellular carbon source utilization.