A Novel Acetivibrio cellulolyticus Anchoring Scaffoldin That Bears Divergent Cohesins

Abstract

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...

Structure–function analyses generate novel specificities to assemble the components of multienzyme bacterial cellulosome complexes

“…Previous studies have shown that the type I Doc of A. cellulolyticus ScaB binds specifically to the type I Cohs of ScaC, but not to those of ScaA (10)(11)(12). Similarly, enzyme-borne type I Docs specifically bind to the seven type I Cohs of ScaA (and to one in ScaD), but not to those of ScaC (21).…”

“…Within the genome .A. cellulolyticus is a cluster of four tandem scaffoldin genes (scaA, scaB, scaC, and scaD) (10,11). The primary scaffoldin ScaA (where the enzymes of the cellulosome are recruited) shares the main traits found in the primary ScaA scaffoldin of the canonical cellulosome of Clostridium thermocellum.…”

Exploration of New Geometries in Cellulosome-Like Chimeras

“…These properties indeed match native cellulosome organization selected by mesophilic clostridia but do not apply to the elaborate cellulosomes produced by A. cellulolyticus or ruminal bacteria that form a vast network of interacting scaffoldins, catalytic subunits, and cell-surface-anchoring proteins (29,36,37). Nevertheless, the GH48 and GH9 enzymes, selected as the model components for our studies on designer cellulosomes, are common components of the relatively simple, natural cellulosomes produced by the mesophilic clostridia.…”

Conservation and Divergence in Cellulosome Architecture between Two Strains ofRuminococcus flavefaciens

“…1C). The FD-1 ScaB is but the second reported occurrence of a scaffoldin that bears divergent cohesins, the previous report being that of the ScaD scaffoldin of Acetivibrio cellulolyticus (22). Owing to the additional cohesins in the FD-1 ScaB scaffoldin, its overall size is proportionately larger than that of strain 17 (Fig.…”