3c) on both crystalline and amorphous cellulose as well as complex cellulosic substrates, for example, alfalfa cell walls, wheat straw and banana fruit stem. Both recombinant CBM3s underwent partial cleavage by E. coli native proteases during the purification procedure. Nevertheless, the full-length recombinant CBM3 of Cthe_0059 bound strongly to all of the cellulosic target substrates; the recombinant CBM3 of Cthe_0404 showed much weaker binding characteristics to the various substrates, particularly to amorphous cellulose and alfalfa cell

walls, perhaps indicating the different recognition properties of the two CBM3 variants. The cellulose-degradation process commences with the binding of the cellulolytic enzymes and/or the entire organism to the cellulosic substrate, mediated by a separate cellulosome-borne component, the CBM3 (Poole et al., 1992; Bayer et al., 1996; Tormo et al., 1996). CBMs can serve as targeting agents for the catalytic modules of free cellulases SB203580 nmr or can act as a separate

Daporinad supplier targeting module, for example, as part of the noncatalytic scaffoldin subunit of the cellulosome (Bayer et al., 1998). The C. thermocellum genome contains 20 genes encoding proteins, which carry 23 CBM3s. The CBM3s are known to either bind strongly to crystalline cellulose, thus playing a substrate-targeting role, or serve to modulate the apparent mode of action of the parent cellulase. Thirteen of these proteins are GHs and other enzymes involved in polysaccharide degradation; one is the main cellulosomal structural protein (scaffoldin CipA), and six others are hypothetical proteins of unknown function. All in all, the functional Methane monooxygenase connection between carbohydrate-active proteins and the huge majority

of genes encoding for CBM3-containing proteins could be accounted for, with the notable exception of Cthe_0059, Cthe_267 and Cthe_404. This anomaly deserved further attention (Lamed, 2010), and upon meticulous bioinformatic examination, we discovered that the N-terminal portions of the latter hypothetical proteins bore tenuous homology to the B. subtilisσI-modulating protein RsgI (Fig. S1). Systematic analysis of the C. thermocellum genome revealed another six hypothetical proteins whose N-terminal regions also exhibited homology to those of the abovementioned CBM3-containing proteins and, hence, also shared a relationship with the B. subtilis RsgI. Intriguingly, the C-terminal regions of additional proteins contained other types of carbohydrate-active modules, i.e., CBM42, PA14 and GH10 (Fig. 1). Moreover, in each case, a gene located immediately upstream of the rsgI-like ORF encoded a putative alternative σ factor resembling B. subtilisσI (Fig. 2). Such an operon-like organization of the B. subtilis and C. thermocellum sigI and rsgI genes matches perfectly one of the main criteria for the ECF σ factors (Helmann, 2002). Preliminary analysis of putative σI-related promoter sequences of the C.