Decoding Biomass-Sensing Regulons of Clostridium thermocellum Alternative Sigma-I Factors in a Heterologous Bacillus subtilis Host System

The Gram-positive, anaerobic, cellulolytic, thermophile 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 RNA polymerase sigma (σ) factors that are activated in response to extracellular polysaccharides and share sequence similarity to the Bacillus subtilis σ I factor. The aim of the present work was to demonstrate whether individual C . thermocellum σ I -like factors regulate specific cellulosomal genes, focusing on C . thermocellum σ I6 and σ I3 factors. To search for putative σ I6 - and σ I3 -dependent promoters, bioinformatic analysis of the upstream regions of the cellulosomal genes was performed. Because of the limited genetic tools available for C . thermocellum , the functionality of the predicted σ I6 - and σ I3 -dependent promoters was studied in B . subtilis as a heterologous host. This system enabled observation of the activation of 10 predicted σ I6 -dependent promoters associated with the C . thermocellum genes: sigI6 (itself, Clo1313_2778), xyn11B (Clo1313_0522), xyn10D (Clo1313_0177), xyn10Z (Clo1313_2635), xyn10Y (Clo1313_1305), cel9V (Clo1313_0349), cseP (Clo1313_2188), sigI1 (Clo1313_2174), cipA (Clo1313_0627), and rsgI5 (Clo1313_0985). Additionally, we observed the activation of 4 predicted σ I3 -dependent promoters associated with the C . thermocellum genes: sigI3 (itself, Clo1313_1911), pl11 (Clo1313_1983), ce12 (Clo1313_0693) and cipA . Our results suggest possible regulons of σ I6 and σ I3 in C . thermocellum , as well as the σ I6 and σ I3 promoter consensus sequences. The proposed -35 and -10 promoter consensus elements of σ I6 are CNNAAA and CGAA, respectively. Additionally, a less conserved CGA sequence next to the C in the -35 element and a highly conserved AT sequence three bases downstream of the -10 element were also identified as important nucleotides for promoter recognition. Regarding σ I3 , the proposed -35 and -10 promoter consensus elements are CCCYYAAA and CGWA, respectively. The present study provides new clues for understanding these recently discovered alternative σ I factors.