Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

Ravachol, Julie; Borne, Romain; Tardif, Chantal; Philip, Pascale de; Fierobe, Henri‐Pierre

doi:10.1074/jbc.m113.545046

Cited by 73 publications

(90 citation statements)

References 44 publications

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“…This supports reports that species of Clostridium may utilise histidine as part of a strategy to overcome nutrient limitation during the stationary growth phase (Fonknechten et al, 2010). Finally, the class of glycosylases to which the cellulosome enzymes from Clostridium species belong (EC 3.2; Ravachol et al, 2014), was more functionally divergent in Clostridium than in Prevotella. This suggests Clostridium possess a greater range of diverse functional isoforms involved in catabolism of these polysaccharides than Prevotella and may provide an advantage in competitive conditions.…”

Section: Metabolic Adaptations In Clostridiumsupporting

confidence: 85%

Erratum: Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

Rubino¹,

Carberry²,

Waters³

et al. 2017

The ISME Journal

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Many microbes in complex competitive environments share genes for acquiring and utilising nutrients, questioning whether niche specialisation exists and if so, how it is maintained. We investigated the genomic signatures of niche specialisation in the rumen microbiome, a highly competitive, anaerobic environment, with limited nutrient availability determined by the biomass consumed by the host. We generated individual metagenomic libraries from 14 cows fed an ad libitum diet of grass silage and calculated functional isoform diversity for each microbial gene identified. The animal replicates were used to calculate confidence intervals to test for differences in diversity of functional isoforms between microbes that may drive niche specialisation. We identified 153 genes with significant differences in functional isoform diversity between the two most abundant bacterial genera in the rumen (Prevotella and Clostridium). We found Prevotella possesses a more diverse range of isoforms capable of degrading hemicellulose, whereas Clostridium for cellulose. Furthermore, significant differences were observed in key metabolic processes indicating that isoform diversity plays an important role in maintaining their niche specialisation. The methods presented represent a novel approach for untangling complex interactions between microorganisms in natural environments and have resulted in an expanded catalogue of gene targets central to rumen cellulosic biomass degradation.

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Section: Metabolic Adaptations In Clostridiumsupporting

confidence: 85%

Erratum: Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

Rubino¹,

Carberry²,

Waters³

et al. 2017

The ISME Journal

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show abstract

“…1). The transcripts for GH9, a family of proteins with soluble or insoluble (amorphous) cellulose as their favored substrate (38), existed in the greatest abundance among the cel-GH transcripts (accounting for 34% and 42% of the total cel-GH reads in CS9007 and CS9036, respectively) (see Fig. S3b in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

Dai

Tian

et al. 2015

Appl Environ Microbiol

189

178

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gThe bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ϳ1% and ϳ0.1% of the total non-rRNAs, respectively. The majority (ϳ98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (ϳ82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen. In nature, the cow rumen represents a highly specialized bioreactor wherein plant cell wall polysaccharides (PCWPs) are efficiently deconstructed. The extraordinary efficiency results from the concerted action of various enzymes produced by rumen-resident bacteria, archaea, fungi, and protozoa. Three rumen bacteria, i.e., Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes, which can be isolated and cultivated in the laboratory, have been thought to serve a predominant role in the degradation of cellulosic PCWPs in this niche (1, 2). However, metagenomic quantitations based on 16S rRNA gene analysis indicate that these three species of bacteria account for only less than 5% of the total rumen microorganisms (3). In addition, Koike et al. estimated that ϳ77% of the rumen microorganisms attached to solid fibers are uncultured, as their 16S rRNA gene sequences share less than 97% similarity with those of known isolates (4).To bypass the cultivation step, metagenomic approaches involving the direct analysis of total DNA sequences have been extensively used to investigate the PCWP-degrading gastrointestinal microbes in a variety of herbivores, such as termite hindguts (5); cow (6-8), yak (9), and Svalbard reindeer (10) ...

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“…Endo-xyloglucanase activity, which cleaves b(1?4)-Dglucosidic linkages in the XyG backbone, has been demonstrated previously in GH5, GH7, GH9, GH12, GH16, GH44 and GH74 [24][25][26]. C. japonicus does not encode GH7, GH12, nor GH44 members, but encodes multiple GH5 (n = 15), GH9 (n = 3) and GH16 (n = 9) members (http://www.cazy.org/genomes.…”

Section: Bioinformatic Analysismentioning

confidence: 99%

Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

et al. 2016

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The heteropolysaccharide xyloglucan (XyG) comprises up to one‐quarter of the total carbohydrate content of terrestrial plant cell walls and, as such, represents a significant reservoir in the global carbon cycle. The complex composition of XyG requires a consortium of backbone‐cleaving endo‐xyloglucanases and side‐chain cleaving exo‐glycosidases for complete saccharification. The biochemical basis for XyG utilization by the model Gram‐negative soil saprophytic bacterium Cellvibrio japonicus is incompletely understood, despite the recent characterization of associated side‐chain cleaving exo‐glycosidases. We present a detailed functional and structural characterization of a multimodular enzyme encoded by gene locus CJA_2477. The CJA_2477 gene product comprises an N‐terminal glycoside hydrolase family 74 (GH74) endo‐xyloglucanase module in train with two carbohydrate‐binding modules (CBMs) from families 10 and 2 (CBM10 and CBM2). The GH74 catalytic domain generates Glc4‐based xylogluco‐oligosaccharide (XyGO) substrates for downstream enzymes through an endo‐dissociative mode of action. X‐ray crystallography of the GH74 module, alone and in complex with XyGO products spanning the entire active site, revealed a broad substrate‐binding cleft specifically adapted to XyG recognition, which is composed of two seven‐bladed propeller domains characteristic of the GH74 family. The appended CBM10 and CBM2 members notably did not bind XyG, nor other soluble polysaccharides, and instead were specific cellulose‐binding modules. Taken together, these data shed light on the first step of xyloglucan utilization by C. japonicus and expand the repertoire of GHs and CBMs for selective biomass analysis and utilization. Database Structural data have been deposited in the RCSB protein database under the Protein Data Bank codes: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKR, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKS, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKT and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5FKQ.

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Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

Cited by 73 publications

References 44 publications

Erratum: Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

Erratum: Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

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