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Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

ABSTRACT: Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

SUBMITTER: Ding SY

PROVIDER: S-EPMC95089 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

Ding S Y SY Rincon M T MT Lamed R R Martin J C JC McCrae S I SI Aurilia V V Shoham Y Y Bayer E A EA Flint H J HJ

Journal of bacteriology 20010301 6

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. Sc ...[more]

PMID: 11222592

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Project description:BackgroundThe cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown.Methodology/principal findingsWe have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca(2+)-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e.g., cohesin-containing proteins, belonging to the scaffoldins.Conclusions/significanceThis report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the parent protein, in response to their role in the rumen environment.

Project description:A novel cellulosomal scaffoldin gene, termed cipV, was identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of Clostridium thermocellum. The cipV gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted "recognition codes" differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of C. thermocellum and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain. Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from C. thermocellum as opposed to the known scaffoldins from the mesophilic clostridia. The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.

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Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

Publications

Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

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