Clostridium thermocellum produces a highly efficient cellulolytic extracellular complex, termed the cellulosome, for hydrolyzing plant cell wall biomass. The composition of the cellulosome is affected by the presence of extracellular polysaccharides; however, the regulatory mechanism is unknown. Recently, we have identified in C. thermocellum a set of putative σ and anti-σ factors that include extracellular polysaccharide-sensing components [KahelRaifer et al. (2010) FEMS Microbiol Lett 308:84-93]. These factorencoding genes are homologous to the Bacillus subtilis bicistronic operon sigI-rsgI, which encodes for an alternative σ I factor and its cognate anti-σ I regulator RsgI that is functionally regulated by an extracytoplasmic signal. In this study, the binding of C. thermocellum putative anti-σ I factors to their corresponding σ factors was measured, demonstrating binding specificity and dissociation constants in the range of 0.02 to 1 μM. Quantitative real-time RT-PCR measurements revealed three-to 30-fold up-expression of the alternative σ factor genes in the presence of cellulose and xylan, thus connecting their expression to direct detection of their extracellular polysaccharide substrates. Cellulosomal genes that are putatively regulated by two of these σ factors, σ I1 or σ I6, were identified based on the sequence similarity of their promoters. The ability of σ I1 to direct transcription from the sigI1 promoter and from the promoter of celS (encodes the family 48 cellulase) was demonstrated in vitro by runoff transcription assays. Taken together, the results reveal a regulatory mechanism in which alternative σ factors are involved in regulating the cellulosomal genes via an external carbohydratesensing mechanism.biomass | carbohydrate binding modules | Clostridium regulation | glycoside hydrolases | anti-sigma factors

Genome analysis of the Gram-positive cellulolytic bacterium Clostridium thermocellum revealed the presence of multiple negative regulators of alternative sigma factors. Nine of the deduced proteins share a strong similarity in their N-terminal sequences to the Bacillus subtilis membrane-associated anti-sigma(I) factor RsgI and have an unusual domain organization. In six RsgI-like proteins, the C-terminal sequences contain predicted carbohydrate-binding modules. Three of these modules were overexpressed and shown to bind specifically to cellulose and/or pectin. Bioinformatic analysis of >1200 bacterial genomes revealed that the C. thermocellum RsgI-like proteins are unique to this species and are not present in other cellulolytic clostridial species (e.g. Clostridium cellulolyticum and Clostridium papyrosolvens). Eight of the nine genes encoding putative C. thermocellum RsgI-like anti-sigma factors form predicted bicistronic operons, in which the first gene encodes a putative alternative sigma factor, similar to B. subtilissigma(I), but lacking in one of its domains. These observations suggest a novel carbohydrate-sensing mechanism in C. thermocellum, whereby the presence of polysaccharide biomass components is detected extracellularly and the signal is transmitted intracellularly, resulting in the disruption of the interaction between RsgI-like proteins and sigma(I)-like factors, the latter of which serve to activate appropriate genes encoding proteins involved in cellulose utilization.

The composition of the cellulase system in the cellulosome-producing bacterium, Clostridium thermocellum, has been reported to change in response to growth on different carbon sources. Recently, an extensive carbohydrate-sensing mechanism, purported to regulate the activation of genes coding for polysaccharide-degrading enzymes, was suggested. In this system, CBM modules, comprising extracellular components of RsgI-like anti-σ factors, were proposed to function as carbohydrate sensors, through which a set of cellulose utilization genes are activated by the associated σ(I)-like factors. An extracellular module of one of these RsgI-like proteins (Cthe_2119) was annotated as a family 10 glycoside hydrolase, RsgI6-GH10, and a second putative anti-σ factor (Cthe_1471), related in sequence to Rsi24, was found to contain a module that resembles a family 5 glycoside hydrolase (termed herein Rsi24C-GH5). The present study examines the relevance of these two glycoside hydrolases as sensors in this signal-transmission system. The RsgI6-GH10 was found to bind xylan matrices but exhibited low enzymatic activity on this substrate. In addition, this glycoside hydrolase module was shown to interact with crystalline cellulose although no hydrolytic activity was detected on cellulosic substrates. Bioinformatic analysis of the Rsi24C-GH5 showed a glutamate-to-glutamine substitution that would presumably preclude catalytic activity. Indeed, the recombinant module was shown to bind to cellulose, but showed no hydrolytic activity. These observations suggest that these two glycoside hydrolases underwent an evolutionary adaptation to function as polysaccharide binding agents rather than enzymatic components and thus serve in the capacity of extracellular carbohydrate sensors.

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