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Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci.

Pope PB, Mackenzie AK, Gregor I, Smith W, Sundset MA, McHardy AC, Morrison M, Eijsink VG - PLoS ONE (2012)

Bottom Line: Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature.Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation.Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway. phil.pope@umb.no

ABSTRACT
Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

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Comparative gene organization of presumptive cellulolytic PULs identified in the Svalbard reindeer rumen and other gut environments.Gene clusters were recovered from the cow rumen metagenome (partial genome of an as-yet uncultured Bacteroidales phylotype AC2a) and sequenced cellulolytic fosmids constructed from environmental DNA originating from the gut microbiomes of the wallaby foregut and the reindeer and buffalo rumen (upper four clusters). All fosmid sequences originate from Bacteroidetes, according to PhyloPythiaS binning, and for all cellulolytic activity has been detected in functional screens. Green genes represent SusE/SusF-like genes predicted to encode outer-membrane proteins whose function is currently unknown. Black genes encode putative response-regulators. BACON: indicates a carbohydrate binding domain identified by [40]. TonB: indicates members of the TonB-dependent receptor family, a group of outer membrane spanning β-barrel proteins that transport solutes and macromolecules. TW-64 and TW-33 correspond to sample IDs for the GH9 and GH5 genes (respectively) encoded within the AC2a PUL, from which expressed proteins were tested positive for hydrolytic activity on various cellulosic substrates by Hess et al., [7]. GenBank accession numbers and/or IMG Gene Object ID numbers are provided. Gene IDs for the AC2a PUL are provided in Table S5.
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pone-0038571-g002: Comparative gene organization of presumptive cellulolytic PULs identified in the Svalbard reindeer rumen and other gut environments.Gene clusters were recovered from the cow rumen metagenome (partial genome of an as-yet uncultured Bacteroidales phylotype AC2a) and sequenced cellulolytic fosmids constructed from environmental DNA originating from the gut microbiomes of the wallaby foregut and the reindeer and buffalo rumen (upper four clusters). All fosmid sequences originate from Bacteroidetes, according to PhyloPythiaS binning, and for all cellulolytic activity has been detected in functional screens. Green genes represent SusE/SusF-like genes predicted to encode outer-membrane proteins whose function is currently unknown. Black genes encode putative response-regulators. BACON: indicates a carbohydrate binding domain identified by [40]. TonB: indicates members of the TonB-dependent receptor family, a group of outer membrane spanning β-barrel proteins that transport solutes and macromolecules. TW-64 and TW-33 correspond to sample IDs for the GH9 and GH5 genes (respectively) encoded within the AC2a PUL, from which expressed proteins were tested positive for hydrolytic activity on various cellulosic substrates by Hess et al., [7]. GenBank accession numbers and/or IMG Gene Object ID numbers are provided. Gene IDs for the AC2a PUL are provided in Table S5.

Mentions: The frequency of fosmid clones testing positive for CMCase activity was the highest reported so far (48 positive from ∼5,000 screened, corresponding to ∼1% of the clones and a hit rate of 1 per 3.5 Mb screened; [20]). The majority of the sequenced fosmid clones were predicted to be derived from Bacteroidetes-affiliated lineages (Table 2). More than 70% of the scaffolds constructed carry one or more genes encoding a presumptive GH5 endoglucanase. However, two scaffolds (Sc00001 & Sc Sc00021) did not possess any GH families representing known endoglucanases, suggesting members of the Bacteroidales might also possess novel cellulose-degrading mechanisms. Further studies, e.g. employing random transposon mutagenesis, are needed to identify exactly which genes are responsible for the enzymatic activity encoded by these fosmids. The presumptive GH5 endoglucanase genes were most often located in close proximity to multi-gene PULs which were all found on fosmids assigned to Bacteroidetes, including one fosmid assigned to SRM-1 (see Figure 2, Table 2). Their gene-organisation included Sus-like outer membrane proteins homologous to SusC and SusD, which are essential for the import and degradation of starch by the Sus initially described in the human gut bacterium Bacteroides thetaiotaomicron[21]. SusC- and SusD- like genes which typify a Sus-like PUL, function together with other hypothetical outer-membrane proteins (SusE/F-like) and linked carbohydrate-active enzymes [11]. Whilst originally characterised on starch, Sus-like PULs encoded in human gut Bacteroidetes have since been described with capabilities to degrade other plant polysaccharides that include pectins and hemicellulosic substrates [22]. Predictions of polysaccharide degradation by Sus-like PULs in other environments have previously been made [11], [23], but so far there are only few studies addressing their importance in the herbivore gut. Their involvement in xylan degradation by the rumen bacterium Prevotella bryantii has been illustrated by gene transcriptome studies [23], whereas their potential role in cellulose degradation has been suggested on the basis of the metagenome described for the anaerobic microbiome of the Tammar wallaby foregut [5].


Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci.

Pope PB, Mackenzie AK, Gregor I, Smith W, Sundset MA, McHardy AC, Morrison M, Eijsink VG - PLoS ONE (2012)

Comparative gene organization of presumptive cellulolytic PULs identified in the Svalbard reindeer rumen and other gut environments.Gene clusters were recovered from the cow rumen metagenome (partial genome of an as-yet uncultured Bacteroidales phylotype AC2a) and sequenced cellulolytic fosmids constructed from environmental DNA originating from the gut microbiomes of the wallaby foregut and the reindeer and buffalo rumen (upper four clusters). All fosmid sequences originate from Bacteroidetes, according to PhyloPythiaS binning, and for all cellulolytic activity has been detected in functional screens. Green genes represent SusE/SusF-like genes predicted to encode outer-membrane proteins whose function is currently unknown. Black genes encode putative response-regulators. BACON: indicates a carbohydrate binding domain identified by [40]. TonB: indicates members of the TonB-dependent receptor family, a group of outer membrane spanning β-barrel proteins that transport solutes and macromolecules. TW-64 and TW-33 correspond to sample IDs for the GH9 and GH5 genes (respectively) encoded within the AC2a PUL, from which expressed proteins were tested positive for hydrolytic activity on various cellulosic substrates by Hess et al., [7]. GenBank accession numbers and/or IMG Gene Object ID numbers are provided. Gene IDs for the AC2a PUL are provided in Table S5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368933&req=5

pone-0038571-g002: Comparative gene organization of presumptive cellulolytic PULs identified in the Svalbard reindeer rumen and other gut environments.Gene clusters were recovered from the cow rumen metagenome (partial genome of an as-yet uncultured Bacteroidales phylotype AC2a) and sequenced cellulolytic fosmids constructed from environmental DNA originating from the gut microbiomes of the wallaby foregut and the reindeer and buffalo rumen (upper four clusters). All fosmid sequences originate from Bacteroidetes, according to PhyloPythiaS binning, and for all cellulolytic activity has been detected in functional screens. Green genes represent SusE/SusF-like genes predicted to encode outer-membrane proteins whose function is currently unknown. Black genes encode putative response-regulators. BACON: indicates a carbohydrate binding domain identified by [40]. TonB: indicates members of the TonB-dependent receptor family, a group of outer membrane spanning β-barrel proteins that transport solutes and macromolecules. TW-64 and TW-33 correspond to sample IDs for the GH9 and GH5 genes (respectively) encoded within the AC2a PUL, from which expressed proteins were tested positive for hydrolytic activity on various cellulosic substrates by Hess et al., [7]. GenBank accession numbers and/or IMG Gene Object ID numbers are provided. Gene IDs for the AC2a PUL are provided in Table S5.
Mentions: The frequency of fosmid clones testing positive for CMCase activity was the highest reported so far (48 positive from ∼5,000 screened, corresponding to ∼1% of the clones and a hit rate of 1 per 3.5 Mb screened; [20]). The majority of the sequenced fosmid clones were predicted to be derived from Bacteroidetes-affiliated lineages (Table 2). More than 70% of the scaffolds constructed carry one or more genes encoding a presumptive GH5 endoglucanase. However, two scaffolds (Sc00001 & Sc Sc00021) did not possess any GH families representing known endoglucanases, suggesting members of the Bacteroidales might also possess novel cellulose-degrading mechanisms. Further studies, e.g. employing random transposon mutagenesis, are needed to identify exactly which genes are responsible for the enzymatic activity encoded by these fosmids. The presumptive GH5 endoglucanase genes were most often located in close proximity to multi-gene PULs which were all found on fosmids assigned to Bacteroidetes, including one fosmid assigned to SRM-1 (see Figure 2, Table 2). Their gene-organisation included Sus-like outer membrane proteins homologous to SusC and SusD, which are essential for the import and degradation of starch by the Sus initially described in the human gut bacterium Bacteroides thetaiotaomicron[21]. SusC- and SusD- like genes which typify a Sus-like PUL, function together with other hypothetical outer-membrane proteins (SusE/F-like) and linked carbohydrate-active enzymes [11]. Whilst originally characterised on starch, Sus-like PULs encoded in human gut Bacteroidetes have since been described with capabilities to degrade other plant polysaccharides that include pectins and hemicellulosic substrates [22]. Predictions of polysaccharide degradation by Sus-like PULs in other environments have previously been made [11], [23], but so far there are only few studies addressing their importance in the herbivore gut. Their involvement in xylan degradation by the rumen bacterium Prevotella bryantii has been illustrated by gene transcriptome studies [23], whereas their potential role in cellulose degradation has been suggested on the basis of the metagenome described for the anaerobic microbiome of the Tammar wallaby foregut [5].

Bottom Line: Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature.Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation.Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway. phil.pope@umb.no

ABSTRACT
Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation.

Show MeSH
Related in: MedlinePlus