Metagenomic Insights into the Carbohydrate-Active Enzymes Carried by the Microorganisms Adhering to Solid Digesta in the Rumen of Cows

Wang, Lingling; Hatem, Ayat; Çatalyürek, Ümit V.; Morrison, Mark; Yu, Zhongtang

doi:10.1371/journal.pone.0078507

Cited by 93 publications

(88 citation statements)

References 37 publications

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“…More recently, metagenomic studies have revealed enzymes from uncultured and deeply branched lineages, which constitute the majority of microorganisms within rumen microbiomes (15). While PULs originating from uncultured rumen bacteria have previously been detected (12,14,33,34), here we have taken additional measures to partially describe the enzymatic capabilities of one such PUL encoded within a numerically abundant uncultured phylotype. The GH5_g -GH5_h…”

Section: Resultsmentioning

confidence: 96%

A Polysaccharide Utilization Locus from an Uncultured Bacteroidetes Phylotype Suggests Ecological Adaptation and Substrate Versatility

Mackenzie

Naas

Kračun

et al. 2015

Appl Environ Microbiol

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bRecent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against ␤-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward ␤-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome. Microbes belonging to the Bacteroidetes phylum are numerically dominant in the gut of mammals, where they contribute to plant cell wall degradation. Our current understanding regarding their enzymatic mechanisms, regulated by what are otherwise known as polysaccharide utilization loci (PULs), has largely been developed from in-depth analysis of cultivated Bacteroidetes strains within the human distal gut (1-3) and a single Prevotella rumen isolate (4). PULs have been defined as gene clusters that encode cell envelope-associated enzyme systems that enable the bacterium to respond to, bind, and degrade specific glycans and import the released oligosaccharides (2). The original PUL was described as a starch utilization system (Sus) and was built around two core proteins that are anchored on the outer membrane (SusC/D). The SusC proteins are a group of outer membrane-spanning proteins that can import solutes and macromolecules into the periplasm (5, 6), whereas the SusD protein coordinates polysaccharide binding at the cell surface (7). The remaining genes often encoded by PULs include (and ...

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Section: Resultsmentioning

confidence: 96%

A Polysaccharide Utilization Locus from an Uncultured Bacteroidetes Phylotype Suggests Ecological Adaptation and Substrate Versatility

Mackenzie

Naas

Kračun

et al. 2015

Appl Environ Microbiol

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“…1) through (meta)genomic, transcriptomic, and proteomic approaches (93)(94)(95)(96). PULs have been identified in species outside the Bacteroides genus in diverse environments, including the ruminal Prevotella bryantii (97), the freshwater dweller Flavobacterium johnsoniae (98), and the gut microbiota of the termite Pseudacanthotermes militaris (99).…”

Section: Pulomicsmentioning

confidence: 99%

Polysaccharide Utilization Loci: Fueling Microbial Communities

et al. 2017

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The complex carbohydrates of terrestrial and marine biomass represent a rich nutrient source for free-living and mutualistic microbes alike. The enzymatic saccharification of these diverse substrates is of critical importance for fueling a variety of complex microbial communities, including marine, soil, ruminant, and monogastric microbiota. Consequently, highly specific carbohydrate-active enzymes, recognition proteins, and transporters are enriched in the genomes of certain species and are of critical importance in competitive environments. In Bacteroidetes bacteria, these systems are organized as polysaccharide utilization loci (PULs), which are strictly regulated, colocalized gene clusters that encode enzyme and protein ensembles required for the saccharification of complex carbohydrates. This review provides historical perspectives and summarizes key findings in the study of these systems, highlighting a critical shift from sequence-based PUL discovery to systems-based analyses combining reverse genetics, biochemistry, enzymology, and structural biology to precisely illuminate the molecular mechanisms underpinning PUL function. The ecological implications of dynamic PUL deployment by key species in the human gastrointestinal tract are explored, as well as the wider distribution of these systems in other gut, terrestrial, and marine environments.

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“…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)(7)(8), yak (9), and Svalbard reindeer (10) rumens; and the foreguts of Australian macropods (11). It has been demonstrated that PCWP-degrading enzymes that exist in the rumen are far more diverse than was previously believed.…”

mentioning

confidence: 99%

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

Dai

Tian

et al. 2015

Appl Environ Microbiol

194

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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Metagenomic Insights into the Carbohydrate-Active Enzymes Carried by the Microorganisms Adhering to Solid Digesta in the Rumen of Cows

Cited by 93 publications

References 37 publications

A Polysaccharide Utilization Locus from an Uncultured Bacteroidetes Phylotype Suggests Ecological Adaptation and Substrate Versatility

A Polysaccharide Utilization Locus from an Uncultured Bacteroidetes Phylotype Suggests Ecological Adaptation and Substrate Versatility

Polysaccharide Utilization Loci: Fueling Microbial Communities

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

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