Abundance and Diversity of Dockerin-Containing Proteins in the Fiber-Degrading Rumen Bacterium, Ruminococcus flavefaciens FD-1

Rincón, Marco T.; Dassa, Bareket; Flint, Harry J.; Travis, Anthony J.; Jindou, Sadanari; Borovok, Ilya; Lamed, Raphael; Bayer, Edward A.; Henrissat, Bernard; Coutinho, Pedro M.; Antonopoulos, Dion; Miller, Margret E. Berg; White, Bryan A.

doi:10.1371/journal.pone.0012476

Cited by 66 publications

(83 citation statements)

References 35 publications

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“…Commensurate with this proposed cellulosomal complexity, the genome of R. flavefaciens FD-1 encodes ∼230 dockerin-containing proteins, which are likely to integrate into the multienzyme complex (6). A large number of the protein modules identified in the R. flavefaciens cellulosome are of unknown function and may reflect an extended capacity to recognize carbohydrates through an extended CBM profile.…”

Section: Significancementioning

confidence: 99%

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Venditto

Luís

Rydahl

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

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Section: Significancementioning

confidence: 99%

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Venditto

Luís

Rydahl

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…It also confirms the presence of a multi-enzyme cellulosome complex in which enzymes are linked to a non-catalytic scaffold structure via dockerin domains. The cellulosome organisation in FD-1 is extremely complex and more than 200 dockerin-containing proteins have been identified from the draft genome sequence (Rincon et al, 2010). Analysis of gene expression in this organism indicates that the type of substrate used by R. flavefaciens FD-1 can influence the enzymatic composition of the cellulosome.…”

Section: Rumen Microbial Genome Projectsmentioning

confidence: 99%

Rumen microbial (meta)genomics and its application to ruminant production

Morgavi

Kelly

Janssen

et al. 2013

Animal

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Meat and milk produced by ruminants are important agricultural products and are major sources of protein for humans. Ruminant production is of considerable economic value and underpins food security in many regions of the world. However, the sector faces major challenges because of diminishing natural resources and ensuing increases in production costs, and also because of the increased awareness of the environmental impact of farming ruminants. The digestion of feed and the production of enteric methane are key functions that could be manipulated by having a thorough understanding of the rumen microbiome. Advances in DNA sequencing technologies and bioinformatics are transforming our understanding of complex microbial ecosystems, including the gastrointestinal tract of mammals. The application of these techniques to the rumen ecosystem has allowed the study of the microbial diversity under different dietary and production conditions. Furthermore, the sequencing of genomes from several cultured rumen bacterial and archaeal species is providing detailed information about their physiology. More recently, metagenomics, mainly aimed at understanding the enzymatic machinery involved in the degradation of plant structural polysaccharides, is starting to produce new insights by allowing access to the total community and sidestepping the limitations imposed by cultivation. These advances highlight the promise of these approaches for characterising the rumen microbial community structure and linking this with the functions of the rumen microbiota. Initial results using high-throughput culture-independent technologies have also shown that the rumen microbiome is far more complex and diverse than the human caecum. Therefore, cataloguing its genes will require a considerable sequencing and bioinformatic effort. Nevertheless, the construction of a rumen microbial gene catalogue through metagenomics and genomic sequencing of key populations is an attainable goal. A rumen microbial gene catalogue is necessary to understand the function of the microbiome and its interaction with the host animal and feeds, and it will provide a basis for integrative microbiome–host models and inform strategies promoting less-polluting, more robust and efficient ruminants.

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“…6). Similarly to what was observed for group 3 Docs, the majority of group 6 Doc-containing enzymes were previously annotated as hemicellulases (7). To understand why groups 3 and 6 Docs have similar Coh specificities, six representative members of R. flavefaciens FD-1 group 6 Docs (out of a total of 45) were produced recombinantly, and their affinities for CohScaC were probed through ITC.…”

Section: Table 4 Thermodynamics Of Interaction Between Wild Type Cohsmentioning

confidence: 89%

“…1) (6). Based on primary structure identity, R. flavefaciens Docs have been organized into six major groups (7). Recently, classification of Ruminococcus Docs into groups was shown to be functionally relevant as members of the same Doc group present similar Coh specificities (8).…”

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confidence: 99%

Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome

Bule¹,

Alves²,

Leitão³

et al. 2016

Journal of Biological Chemistry

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Edited by Gerald HartThe assembly of one of Nature's most elaborate multienzyme complexes, the cellulosome, results from the binding of enzymeborne dockerins to reiterated cohesin domains located in a noncatalytic primary scaffoldin. Generally, dockerins present two similar cohesin-binding interfaces that support a dual binding mode. The dynamic integration of enzymes in cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins to assemble the most intricate cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. Here, we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Groups 3 and 6 dockerins are predominantly appended to hemicellulose-degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated cellulosomes acting in the gastrointestinal tract of mammals.Plant cell wall polysaccharides, primarily cellulose and hemicellulose, are the most abundant organic molecules produced in Nature, thus constituting a major reservoir of carbon and energy (1). The intricate organization of structural carbohydrates in plant cell walls and their inherent heterogeneity pose significant constraints to polysaccharide degradation, which usually requires a wide array of catalytic activities acting cooperatively (2, 3). In highly competitive anaerobic environments, such as the rumen of mammals, enzymatic systems that recycle the carbon stored in plant cell walls are organized in high molecular mass multienzyme complexes termed cellulosomes (4, 5). Molecular integration of microbial biocatalysts into these extremely elaborate nanomachines results from the binding of enzyme-borne dockerin modules (Doc) 3 to reiterated cohesin domains (Coh) located in large non-catalytic scaffoldins, a mechanism that promotes enzyme synergy and stability. In addition, recruitment of cellulosomes to the bacterial cell surface via divergent Coh-Doc interactions allows the immediate uptake of released sugars, which are used by microbes as an energy source.Ruminococcus flavefaciens is a Gram-positive, anaerobic bacterium of the Firmicutes phylum and the only species in the...

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Abundance and Diversity of Dockerin-Containing Proteins in the Fiber-Degrading Rumen Bacterium, Ruminococcus flavefaciens FD-1

Cited by 66 publications

References 35 publications

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Rumen microbial (meta)genomics and its application to ruminant production

Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome

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