ALTERNATIVE σ FACOTRS ARE INVOLVED IN THE REGULATION OF CELLULOSOMAL GENES IN CLOSTRIDIUM THERMOCELLUM

Clostridium thermocellum is a gram positive, anaerobic, thermophilic soil bacterium that secretes a high molecular weight protein complex, the cellulosome, which is capable of hydrolyzing crystalline cellulose. Recently, we identified a set of σ and anti- σ factors that plays a role in the regulation of cellulosomal genes in C. thermocellum . These alternative σ-factors are located upstream to a gene, encoding to a trans-membrane protein (RsgI) with an intracellular anti - σ domain at its N-terminus, and at its C-terminus an extracellular polysaccharide-related function module such as carbohydrate binding modules, family 10 glycoside hydrolase domain and a PA14 module that is known to bind pectin. This arrangement provides a novel regulatory mechanism, in which the expression of the cellulosomal genes can be controlled by the composition of high molecular weight, extracellular polysaccharides. In this study, the regulatory mechanism was directly challenged by disrupting two sigI – rsgI operons and the transcription level of the suspected genes was measured by real – time RT PCR. Deletion of the rsgI gene resulted in an increased transcript level of the target cellulosomal genes and the sigI gene itself. The two rsgI deleted strains were also used for the identification of transcriptional start sites by primer extension analysis. The expression analysis together with the identification of transcriptional start sites enabled us to identify the promoter sequences of these sigma factors and their regulated genes. Taken together, these results indicate the vital role of alternative σ factors in mediating between the extracellular polysaccharides and the regulation of the expression level of cellulosomal genes.