Dataset Information

Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics.

ABSTRACT: The mechanisms by which specific anaerobic microorganisms remain firmly attached to lignocellulosic material, allowing them to efficiently decompose organic matter, have yet to be elucidated. To circumvent this issue, microbiomes collected from anaerobic digesters treating pig manure and meadow grass were fractionated to separate the planktonic microbes from those adhered to lignocellulosic substrate. Assembly of shotgun reads, followed by a binning process, recovered 151 population genomes, 80 out of which were completely new and were not previously deposited in any database. Genome coverage allowed the identification of microbial spatial distribution in the engineered ecosystem. Moreover, a composite bioinformatic analysis using multiple databases for functional annotation revealed that uncultured members of the Bacteroidetes and Firmicutes follow diverse metabolic strategies for polysaccharide degradation. The structure of cellulosome in Firmicutes species can differ depending on the number and functional roles of carbohydrate-binding modules. In contrast, members of the Bacteroidetes are able to adhere to and degrade lignocellulose due to the presence of multiple carbohydrate-binding family 6 modules in beta-xylosidase and endoglucanase proteins or S-layer homology modules in unknown proteins. This study combines the concept of variability in spatial distribution with genome-centric metagenomics, allowing a functional and taxonomical exploration of the biogas microbiome.IMPORTANCE This work contributes new knowledge about lignocellulose degradation in engineered ecosystems. Specifically, the combination of the spatial distribution of uncultured microbes with genome-centric metagenomics provides novel insights into the metabolic properties of planktonic and firmly attached to plant biomass bacteria. Moreover, the knowledge obtained in this study enabled us to understand the diverse metabolic strategies for polysaccharide degradation in different species of Bacteroidetes and Clostridiales Even though structural elements of cellulosome were restricted to Clostridiales species, our study identified a putative mechanism in Bacteroidetes species for biomass decomposition, which is based on a gene cluster responsible for cellulose degradation, disaccharide cleavage to glucose, and transport to cytoplasm.

SUBMITTER: Kougias PG

PROVIDER: S-EPMC6121989 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics.

Kougias Panagiotis G PG Campanaro Stefano S Treu Laura L Tsapekos Panagiotis P Armani Andrea A Angelidaki Irini I

Applied and environmental microbiology 20180831 18

The mechanisms by which specific anaerobic microorganisms remain firmly attached to lignocellulosic material, allowing them to efficiently decompose organic matter, have yet to be elucidated. To circumvent this issue, microbiomes collected from anaerobic digesters treating pig manure and meadow grass were fractionated to separate the planktonic microbes from those adhered to lignocellulosic substrate. Assembly of shotgun reads, followed by a binning process, recovered 151 population genomes, 80 ...[more]

PMID: 30006398

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Project description:Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: ?-glucosidase, ?-L-arabinofuranosidase, ?-xylosidase, and endo-1,4-?-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative ?-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl ?-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading ?-xylosidase and ?-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-?-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 ?M, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.

Project description:Two bacterial strains, denoted so4 and w15, isolated from wheat straw (WS)-degrading microbial consortia, were found to grow synergistically in media containing WS as the single carbon and energy source. They were identified as Citrobacter freundii so4 and Sphingobacterium multivorum w15 based on 16S rRNA gene sequencing and comparison to the respective C. freundii and S. multivorum type strains. In order to identify the mechanisms driving the synergistic interactions, we analyzed the draft genomes of the two strains and further characterized their metabolic potential. The latter analyses revealed that the strains had largely complementary substrate utilization patterns, with only 22 out of 190 compounds shared. The analyses further indicated C. freundii so4 to primarily consume amino acids and simple sugars, with laminarin as a key exception. In contrast, S. multivorum w15 showed ample capacity to transform complex polysaccharides, including intermediates of starch degradation. Sequence analyses revealed C. freundii so4 to have a genome of 4,883,214 bp, with a G + C content of 52.5%, 4,554 protein-encoding genes and 86 RNA genes. S. multivorum w15 has a genome of 6,678,278 bp, with a G + C content of 39.7%, 5,999 protein-encoding genes and 76 RNA genes. Genes for motility apparatuses (flagella, chemotaxis) were present in the genome of C. freundii so4, but absent from that of S. multivorum w15. In the genome of S. multivorum w15, 348 genes had regions matching CAZy family enzymes and/or carbohydrate-binding modules (CBMs), with 193 glycosyl hydrolase (GH) and 50 CBM domains. Remarkably, 22 domains matched enzymes of glycoside hydrolase family GH43, suggesting a strong investment in the degradation of arabinoxylan. In contrast, 130 CAZy family genes were found in C. freundii so4, with 61 GH and 12 CBM domains identified. Collectively, our results, based on both metabolic potential and genome analyses, revealed the two strains to harbor complementary catabolic armories, with S. multivorum w15 primarily attacking the WS hemicellulose and C. freundii so4 the cellobiose derived from cellulose, next to emerging oligo- or monosaccharides. Finally, C. freundii so4 may secrete secondary metabolites that S. multivorum w15 can consume, and detoxify the system by reducing the levels of (toxic) by-products.

Dataset Information

Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics.

Publications

Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics.

Similar Datasets

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