The binding specificity of the duplicated segments borne by Clostridium thermocellum endoglucanase CelD and by the cellulosome-integrating protein CipA was investigated. The fusion protein CelC-DSCelD, in which the duplicated segment of CelD was fused to the COOH terminus of endoglucanase CelC, bound with an affinity of 4.7 x 10(7) M-1 to the fusion protein MalE-RDCipA, in which the seventh receptor domain of CipA was grafted onto the COOH terminus of the Escherichia coli maltose-binding protein MalE. The affinity of CelC-DSCelD for the homologous chimeric protein MalE-RDORF3p, carrying the receptor of the surface protein ORF3p, was 6.9 x 10(6) M-1. The fusion protein CelC-DSCipA, in which the duplicated segment of CipA was grafted onto the COOH terminus of CelC, did not bind detectably to MalE-RDCipA or MalE-RDORF3p. However, Western blotting (immunoblotting) experiments indicated that the duplicated segment of CipA was able to bind to a set of C. thermocellum proteins which are different from those recognized by the duplicated segment of CelD. These results argue against the hypothesis that ORF3p interacts with the duplicated segment of CipA. More probably, ORF3p binds to individual cellulases and hemicellulases harboring duplicated segments.

Two independent collections of clones containing Clostridium thermocellum genes involved in cellulose have been previously obtained at IAPGR, Cambridge, and at the Pasteur Institute, Paris. The two collections were compared for cross‐hybridization, restriction maps and enzyme phenotypes. Truly distinct genes were one β‐glucosidase gene, two xylanase genes, and fifteen endogluconase genes. Two of the cloned fragments contained extraneous DNA which was absent from their respective counterparts isolated in the other collection. The dicrepancies resulted from in vivo rearrangements which had occurred in either of the C. thermocellum NCIB 10682 stocks used to generate the two gene banks.

Primary structure of cellulasesBacterial cellulase genes are easier to clone than fungal genes. It is therefore not surprising that most of the sequence information about cellulase genes and their products comes from bacteria. Over 50 cellulase genes have now been sequenced, and a first set of conclusions can be drawn by comparing the sequences.Volume 20

An Escherichia coli clone encoding a single cohesin domain of the cellulosome-integrating protein CipA from Clostridium thermocellum was constructed, and the corresponding polypeptide was purified, treated with papain, and crystallized from a PEG 8000 solution. Crystals exhibit orthorhombic symmetry, space group P212121, with cell dimensions a = 37.7 A , b = 80.7 A , c = 93.3 A, and four or eight molecules in the unit cell. The crystals diffract X-rays to beyond 2 A resolution and are suitable for further crystallographic studies.

Ten distinct Eco RI fragments of Clostridum thermocellum DNA have been cloned in Escherichia coli and shown to express enzymatic activities related to cellulose degradation. Two of the cloned fragments appeared to carry the previously characterized celA and celB genes, which code for the endoglucanases (EG) A and B. Five other cloned fragments code for hitherto unidentified EGs, which can be detected by the Congo red test for hydrolysis of carboxymethylcellulose (CMC). In addition, three separate clones hydrolyzed methylumbelliferyl‐β‐cellobioside (MUC) but not CMC, hinting that they may express three different cellobiohydrolase genes.