Some Gram-positive anaerobic bacteria produce extracellular multi-enzyme systems, highlighted by a cellulosome complex, for degradation of plant cell wall polysaccharides. Such bacteria can produce a variety of carbohydrate-active enzymes, which can be incorporated into a cellulosomal integrating subunit. The cellulosome assemblies are tremendously heterogeneous and their content is regulated during the degradation process. The regulation of enzyme incorporation into the cellulosome remains a mystery. We have recently discovered an unusual carbohydrate-sensing regulatory mechanism in the Clostridium thermocellum, purported to sense the distribution of the extracellular carbohydrates in the environment and regulate the activation of genes coding for polysaccharide-degrading enzymes. This mechanism includes a unique set of membrane-associated anti-sigma factors, which are proposed to be negative regulators of their cognate sigma factors. Multiple sigma factors are regulated in response to different environmental conditions. We embarked on a broad preliminary bioinformatic study which has revealed a similarity among the multiple anti-sigma-like proteins in some cellulosome-producing bacteria to the Bacillus subtilis RsgI, a negative regulator of an alternative factor σI. I plan to take advantage of B. subtilis, in which the native rsgI gene or the full sigI- rsgI operon were knocked out, as a model system for expressing recombinant σI and anti-σI-like factors from several cellulolytic bacteria. My research is concentrated on the regulatory mechanisms of expression of the extracellular sensing components and comparative aspects among the cellulosome-producing bacteria. Deep understanding of the specific regulatory factors will enable us to design high-efficiency cellulosomes for conversion of plant-derived biomass and improved production of biofuels.
