Revisiting the Regulation of the Primary Scaffoldin Gene in Clostridium thermocellum.

Cellulosomes are considered to be one of the most efficient systems for the degradation of plant cell wall polysaccharides. The central cellulosome component comprises a large, noncatalytic protein subunit called scaffoldin. Multiple saccharolytic enzymes are incorporated into the scaffoldins via specific high-affinity cohesin-dockerin interactions. Recently, the regulation of genes encoding certain cellulosomal components by multiple RNA polymerase alternative σ factors has been demonstrated in () In the present report, we provide experimental evidence demonstrating that the gene, which encodes the primary cellulosomal scaffoldin, is regulated by several alternative σ factors and by the vegetative σ factor. Furthermore, we show that previously suggested transcriptional start sites (TSSs) of are actually posttranscriptional processed sites. By using comparative bioinformatic analysis, we have also identified highly conserved σ- and σ-dependent promoters upstream of the primary scaffoldin-encoding genes of other clostridia, namely, , , , and sp. strain Bc-iso-3. Interestingly, a previously identified TSS of the primary scaffoldin CbpA gene of matches the predicted σ-dependent promoter identified in the present work rather than the previously proposed σ promoter. With the exception of , both σ and σ promoters of primary scaffoldin genes are located more than 600 nucleotides upstream of the start codon, yielding long 5'-untranslated regions (5'-UTRs). Furthermore, these 5'-UTRs have highly conserved stem-loop structures located near the start codon. We propose that these large 5'-UTRs may be involved in the regulation of both the primary scaffoldin and other cellulosomal components. Cellulosome-producing bacteria are among the most effective cellulolytic microorganisms known. This group of bacteria has biotechnological potential for the production of second-generation biofuels and other biocommodities from cellulosic wastes. The efficiency of cellulose hydrolysis is due to their cellulosomes, which arrange enzymes in close proximity on the cellulosic substrate, thereby increasing synergism among the catalytic domains. The backbone of these multienzyme nanomachines is the scaffoldin subunit, which has been the subject of study for many years. However, its genetic regulation is poorly understood. Hence, from basic and applied points of view, it is imperative to unravel the regulatory mechanisms of the scaffoldin genes. The understanding of these regulatory mechanisms can help to improve the performance of the industrially relevant strains of and related cellulosome-producing bacteria to the consolidated bioprocessing of biomass.