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A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin. The gene immediately downstream of cipV was sequenced and designated scaB. The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin. Scab cohesins 1 and 2 are very closely linked. Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin. The scaC gene downstream of scab codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. A long, ca. 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times. The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162. The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein. The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins. Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative Scab cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the Scab dockerin. The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA. In addition, Scab is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the Scab adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture.

Research reveals two linked genes in Ruminococcus flavefaciens 17, which encode key structural components of a cellulosome complex. Data indicate that the gene products ScaA and ScaB represent scaffoldin-related proteins with several copies of cellulosome sequences, which exhibit dockerin-cohesin interactions.

Results show that the protein CipBc is a 2,292-amino-acid residue polypeptide exhibiting 11 type II cohesin domains all of which possessing similar sequence. Data further reveal that one of the domains interacts with the cellulosomal enzyme which contains components of a cellulosomal scaffoldin.

The cellulosomal scaffoldin has a molecular weight of 199,496 with cellulose-binding domains and a catalytic module belonging to family 9 glycosyl hydrolases. Comparative sequence and phylogenetic analyses of different functional modules of other scaffoldins indicate that the scaffolding gene seem to have evolved from different microbial sources.