A Novel Cell Surface-Anchored Cellulose-Binding Protein Encoded by the sca Gene Cluster of Ruminococcus flavefaciens

Abstract

Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sca cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein.Proteomic analysis showed that the native CttA protein (ϳ130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species.Ruminococcus flavefaciens is an important anaerobic, cellulolytic bacterium found in the rumen and hindgut of domestic and wild mammals (13, 17, 19, 22-24, 34, 35). Our understanding of the enzyme systems responsible for degradation of plant cell wall polymers by R. flavefaciens has advanced recently with the identification of cellulosome-like high-molecular-weight enzyme complexes (12,25,26,27). In R. flavefaciens 17, the structural protein ScaA is known to interact via cohesindockerin pairings with a range of catalytic polypeptides possessing xylanase, endoglucanase, and esterase activities (2,18,26,28). An additional structural protein, ScaC, acts as an adaptor scaffoldin, binding via its own dockerin to ScaA but also, via an unusual cohesin, to a range of other, so far unidentified proteins (27). ScaA, in turn, connects to a large structural protein, ScaB, which was recently shown to mediate attachment of the cellulosome to the bacterial cell surface through its interaction with the cell wall-anchored protein ScaE (25).It has long been recognized that noncatalytic carbohydratebinding modules (CBMs) play an important role in the initial stages of degradation of crystalline cellulose (8,29). CBMs enhance the hydrolysis of cellulose by targeting and increasing the effective concentration of either individual glycoside hydrolases or multienzyme cellulosome complexes on the surfaces of carbohydrate substrate...

Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components

“…This modular arrangement represents a common theme shared by XDoc dyads from the CttA and ScaB proteins of both R. flavefaciens strains 17 and FD-1. The dockerin sequences of the latter proteins and their inserts are highly conserved (24). The conserved insertions are unique to the type IIIe dockerins of CttA and ScaB and absent from other known dockerin modules.…”

“…Gene clustering analysis of several R. flavefaciens strains revealed that the cellulosome system is generally composed of four major scaffoldin subunits, ScaA, ScaB, ScaC, and ScaE, and of a cellulose-binding protein, CttA (19,20). The cohesin, dockerin, and X-modules of these cellulosomal components were found to be divergent in sequence from previously known type I and type II cellulosomal modules, and the Coh-Doc interactions were therefore collectively designated type III on the respective phylogenetic trees (21)(22)(23)(24).…”

“…The XDoc modular dyads of ScaB and CttA share high sequence homology (47% identity over a 222-residue stretch) and comprise an X-module of unknown function and an unconventional dockerin sequence that includes three similarly conserved insertions unique to this type of protein; one of these insertions disrupts the putative second calcium (Ca 2ϩ )-binding site (24).…”

In-feed antibiotic effects on the swine intestinal microbiome

“…In addition, Ruminococcus spp. are common in ruminants and are frequently found in the hindgut of pigs (42). Adept at degrading cellulose, an increase in Ruminococcus spp.…”

Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass

“…Spent Avicel with substrate-bound (SB) proteins was isolated by decanting the supernatant (SN) and whole cells (WC) and washing the SB fraction twice with icecold DSMZ medium 640, following which the medium was decanted and combined with the SN-plus-WC fraction. Further washing of the SB fraction was done, as described previously (72), with TBS-Ca-T buffer (25 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM CaCl 2 , and 0.05% [vol/vol] Tween 20). Cell-free SN fraction was obtained by centrifugation at 4°C and 5,000 rpm for 15 min, followed by bottle-top filtration through a 0.22-m-pore-size filter (Millipore).…”