A Scaffoldin of the Bacteroides cellulosolvens Cellulosome That Contains 11 Type II Cohesins

Abstract

A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobe Bacteroides cellulosolvens. The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437. CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins. In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II. This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component. The results attest to the emerging diversity of cellulosomes and their component sequences in nature.

Cellulase, Clostridia, and Ethanol

“…At least eight scaffoldin genes from cellulolytic bacteria have been sequenced (74). These include cipA from C. thermocellum, cbpA from C. cellulovorans (306), cipC from C. cellulolyticum (258), cipA from C. josui (149), cipA from C. acetobutylicum (254,280), scaB from R. flavefaciens (71), cipBc from B. cellulosolvens (70), and cipV from A. cellulolyticus (69). The last two named organisms are closely related to the clostridia (191).…”

Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors

“…Primary scaffoldins are large noncatalytic cohesin-containing proteins, which characteristically include a carbohydrate-binding module that directs the cellulosome and therefore the cellulosomal enzymes to its cellulosic substrate. A sequence-divergent type II dockerin, located at the C terminus of some primary scaffoldins, tethers the cellulosome to the peptidoglycan layer of the bacterial cell envelope through an interaction with type II cohesin modules located in cell-surface anchoring scaffoldins (1,3 somes are therefore orchestrated by the specificity of the different cohesin and dockerin modules (4). Cohesin-dockerin (CohDoc) 3 pairs exhibit one of the strongest protein-protein affinities found in nature, and their interaction is crucial for both cellulosome assembly and cell-surface attachment (5,6).…”

ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome

“…Moreover, CipA possesses a family 3a CBM with high affinity toward cellulose fibrils and is thought to mediate the specific interactions of the bacterium with its substrate. A similar overall arrangement, with distinctive variations on the theme, exists in the cellulosome systems of A. cellulolyticus and B. cellulosolvens (12,13,33,34). In contrast, in R. flavefaciens the catalytic subunits are organized in a different way.…”

“…For example, the cellulosome system of Bacteroides cellulosolvens has a two-component scaffoldin arrangement similar to that of C. thermocellum, except that the types of cohesins carried by the primary and anchoring scaffoldins are reversed (13,33). In Acetivibrio cellulolyticus, the number of enzymes incorporated into the cellulosome complex is amplified by the involvement of a multi-cohesin-bearing adaptor scaffoldin which mediates between the primary and anchoring scaffoldins (12,34).…”