No abstract

The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied. The adherence phenomenon was determined to be selective for cellulose. The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars. A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS. Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells. By using these CBF-specific

The microbial degradation of cellulose is one of the most important processes on Earth, and it affects the human condition in many direct and indirect ways. If it did not occur, there would be an inexhaustible accumulation of plant cell refuse, and herbivorous life forms would largely vanish.The multienzyme cellulosome complex is a major mechanism by which some cellulolytic bacteria efficiently degrade cellulose and related plant cell wall polysaccharides (2,4,5,8,9,18,20,32,58,59). To date, cellulosomes have been found in several strains of anaerobic bacteria and fungi obtained from very different types of ecosystems. The first cellulosome was discovered in studies on the anaerobic thermophile Clostridium thermocellum (3,36,37). The cellulosome system of this organism consists of a variety of different enzymes bound to a noncatalytic scaffoldin subunit, which can, in turn, bind to one of several cell surface anchoring proteins. In this organism, both the attachment of the enzymes to the scaffoldin and the attachment of the scaffoldin to the anchoring proteins are accomplished by a special kind of protein-protein interaction, the cohesin-dockerin interaction. In this context, the enzyme subunits include a dockerin domain, and the scaffoldin contains multiple copies of cohesin modules for collective incorporation into the complex. The scaffoldin subunit itself harbors a single dockerin variant that interacts selectively with corresponding cohesin variants on the anchoring proteins. In C. thermocellum, the primary scaffoldin is trifunctional in that it also contains (in addition to the cohesins and dockerin) a substrate-targeting cellulose-binding domain (CBD). The anchoring proteins are bifunctional; in addition to the cohesins, they have another type of domain, the S-layer homology domain (SLH), which is known to bind strongly to the cell surface (12,40). Thus, the sequential cohesin-dockerin-mediated set of interactions among the enzymes, scaffoldin, and anchoring

A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an "X" domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature.

Sequencing of a cellulosome-integrating gene cluster in Acetivibrio cellulolyticus was completed. The cluster contains four tandem scaffoldin genes (scaA, scaB, scaC, and scaD) bounded upstream and downstream, respectively, by a presumed cellobiose phosphorylase and a nucleotide methylase. The sequences and properties of scaA, scaB, and scaC were reported previously, and those of scaD are reported here. The scaD gene encodes an 852-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. The calculated molecular weight of the mature ScaD is 88,960; a 67-residue linker segment separates cohesins 1 and 2, and two ϳ30-residue linkers separate cohesin 2 from 3 and cohesin 3 from the SLH module. The presence of an SLH module in ScaD indicates its role as an anchoring protein. The first two ScaD cohesins can be classified as type II, similar to the four cohesins of ScaB. Surprisingly, the third ScaD cohesin belongs to the type I cohesins, like the seven ScaA cohesins. ScaD is the first scaffoldin to be described that contains divergent types of cohesins as integral parts of the polypeptide chain. The recognition properties among selected recombinant cohesins and dockerins from the different scaffoldins of the gene cluster were investigated by affinity blotting. The results indicated that the divergent types of ScaD cohesins also differ in their preference of dockerins. ScaD thus plays a dual role, both as a primary scaffoldin, capable of direct incorporation of a single dockerin-borne enzyme, and as a secondary scaffoldin that anchors the major primary scaffoldin, ScaA and its complement of enzymes to the cell surface.Cellulosomes are multienzyme complexes that are designed to efficiently degrade cellulose and related plant cell wall polysaccharides (5-10, 18-20, 22, 28, 51, 52). Bacterial cellulosomes are organized by means of a special type of subunit, the scaffoldin, which is comprised of an array of cohesin modules. The cohesin interacts selectively and tenaciously with a complementary type of domain, the dockerin, which is borne by each of the cellulosomal enzyme subunits. The integrity of the complex is thus maintained by the cohesin-dockerin interaction (4).In Clostridium thermocellum, the first cellulosome to have been described, the scaffoldin gene product (CipA) is located in a gene cluster (24) which also includes a series of genes that encode cohesin-containing anchoring proteins downstream of CipA. These anchoring proteins contain one or more cohesins and a C-terminal S-layer homology (SLH) module that mediates attachment to the cell surface (21). In contrast to the arrangement of the gene cluster in C. thermocellum, for other cellulosome-producing species, such as Clostridium cellulolyticum and Clostridium cellulovorans, gene clusters that comprise a series of genes coding for cellulosomal (dockerin-containing) enzymes instead of anchoring proteins have been described (2,11,19,53). The difference between the scaffoldins of the latter bacteria and...