Decoding biomass-sensing regulons of clostridium thermocellum alternative sigma-I factors in a heterologous bacillus subtilis host system

Abstract

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 (δ) factors that are activated in response to extracellular polysaccharides and share sequence similarity to the Bacillus subtilis δI factor. The aim of the present work was to demonstrate whether individual C.Thermocellum δI-like factors regulate specific cellulosomal genes, focusing on C.Thermocellum δI6 and δI3 factors. To search for putative δI6-And δI3-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 δI6-And δI3-dependent promoters was studied in B. subtilis as a heterologous host. This system enabled observation of the activation of 10 predicted δI6-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 δI3-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 δI6 and δI3 in C.Thermocellum, as well as the δI6 and δI3 promoter consensus sequences. The proposed-35 and-10 promoter consensus elements of δI6 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 δI3, 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 δI factors.