Dataset Information

Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics.

ABSTRACT: Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after growing on [13C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [13C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (<95%) with any cultured representative species, indicating that it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for acetoclastic methanogenesis, indicating that it was enriched in 13C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to identify in situ interspecies metabolic cooperation within syntrophic consortia important to anaerobic waste treatment as well as global carbon cycling.IMPORTANCE Predicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in situ metabolic activities of uncultured species may enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in situ function of mixed microbiomes by combining stable-isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve "who is doing what" by obtaining confirmatory assimilation of the labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.

SUBMITTER: Ziels RM

PROVIDER: S-EPMC6687939 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics.

Ziels Ryan M RM Nobu Masaru K MK Sousa Diana Z DZ

mSystems 20190806 4

Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to reco ...[more]

PMID: 31387934

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Project description:The functioning, health, and productivity of soil are intimately tied to a complex network of interactions, particularly in plant root-associated rhizosphere soil. We conducted a stable-isotope-informed, genome-resolved metagenomic study to trace carbon from Avena fatua grown in a 13CO2 atmosphere into soil. We collected paired rhizosphere and nonrhizosphere soil at 6 and 9 weeks of plant growth and extracted DNA that was then separated by density using ultracentrifugation. Thirty-two fractions from each of five samples were grouped by density, sequenced, assembled, and binned to generate 55 unique bacterial genomes that were ≥70% complete. We also identified complete 18S rRNA sequences of several 13C-enriched microeukaryotic bacterivores and fungi. We generated 10 circularized bacteriophage (phage) genomes, some of which were the most labeled entities in the rhizosphere, suggesting that phage may be important agents of turnover of plant-derived C in soil. CRISPR locus targeting connected one of these phage to a Burkholderiales host predicted to be a plant pathogen. Another highly labeled phage is predicted to replicate in a Catenulispora sp., a possible plant growth-promoting bacterium. We searched the genome bins for traits known to be used in interactions involving bacteria, microeukaryotes, and plant roots and found DNA from heavily 13C-labeled bacterial genes thought to be involved in modulating plant signaling hormones, plant pathogenicity, and defense against microeukaryote grazing. Stable-isotope-informed, genome-resolved metagenomics indicated that phage can be important agents of turnover of plant-derived carbon in soil. IMPORTANCE Plants grow in intimate association with soil microbial communities; these microbes can facilitate the availability of essential resources to plants. Thus, plant productivity commonly depends on interactions with rhizosphere bacteria, viruses, and eukaryotes. Our work is significant because we identified the organisms that took up plant-derived organic C in rhizosphere soil and determined that many of the active bacteria are plant pathogens or can impact plant growth via hormone modulation. Further, by showing that bacteriophage accumulate CO2-derived carbon, we demonstrated their vital roles in redistribution of plant-derived C into the soil environment through bacterial cell lysis. The use of stable-isotope probing (SIP) to identify consumption (or lack thereof) of root-derived C by key microbial community members within highly complex microbial communities opens the way for assessing manipulations of bacteria and phage with potentially beneficial and detrimental traits, ultimately providing a path to improved plant health and soil carbon storage.

Project description:BackgroundThe transformation of plant photosynthate into soil organic carbon and its recycling to CO2 by soil microorganisms is one of the central components of the terrestrial carbon cycle. There are currently large knowledge gaps related to which soil-associated microorganisms take up plant carbon in the rhizosphere and the fate of that carbon.ResultsWe conducted an experiment in which common wild oats (Avena fatua) were grown in a 13CO2 atmosphere and the rhizosphere and non-rhizosphere soil was sampled for genomic analyses. Density gradient centrifugation of DNA extracted from soil samples enabled distinction of microbes that did and did not incorporate the 13C into their DNA. A 1.45-Mbp genome of a Saccharibacteria (TM7) was identified and, despite the microbial complexity of rhizosphere soil, curated to completion. The genome lacks many biosynthetic pathways, including genes required to synthesize DNA de novo. Rather, it requires externally derived nucleotides for DNA and RNA synthesis. Given this, we conclude that rhizosphere-associated Saccharibacteria recycle DNA from bacteria that live off plant exudates and/or phage that acquired 13C because they preyed upon these bacteria and/or directly from the labeled plant DNA. Isotopic labeling indicates that the population was replicating during the 6-week period of plant growth. Interestingly, the genome is ~ 30% larger than other complete Saccharibacteria genomes from non-soil environments, largely due to more genes for complex carbon utilization and amino acid metabolism. Given the ability to degrade cellulose, hemicellulose, pectin, starch, and 1,3-β-glucan, we predict that this Saccharibacteria generates energy by fermentation of soil necromass and plant root exudates to acetate and lactate. The genome also encodes a linear electron transport chain featuring a terminal oxidase, suggesting that this Saccharibacteria may respire aerobically. The genome encodes a hydrolase that could breakdown salicylic acid, a plant defense signaling molecule, and genes to interconvert a variety of isoprenoids, including the plant hormone zeatin.ConclusionsRhizosphere Saccharibacteria likely depend on other bacteria for basic cellular building blocks. We propose that isotopically labeled CO2 is incorporated into plant-derived carbon and then into the DNA of rhizosphere organisms capable of nucleotide synthesis, and the nucleotides are recycled into Saccharibacterial genomes.

Project description:Project description In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane, and is accomplished through obligate mutualistic cross-feeding between SAO bacteria and methanogenic archaea. This pathway is particularly important in anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of the SAO functional guild to the stability and efficiency of the AD process, little is known about their in situ ecophysiologies due to their difficulty of isolation as well as low biomass abundances. Here, we performed a long-term (300 day) continuous enrichment of a thermophilic (55C) SAO community from a municipal AD system. Over 80% of the enriched bioreactor metagenome collectively belonged to a three-member consortium, including an acetate-oxidizing DTU068 bacterium encoding for CO2, H2, and formate production, along with two methanogenic Methanothermobacter_A archaeal species. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. Sample processing After 300 days of chemostat operation, batch microcosms were established in 40 ml glass serum bottles flushed with 80:20 N2:CO2 by anoxically transferring 18 ml of digestate from a single bioreactor (R2) and sealing with butyl rubber septa. Four different incubation conditions were established in triplicate: (1) blank control (e.g. no amendment); (2) 50 mM [12C]-acetate; (3) 50 mM [2 13C]-acetate (e.g. methyl-labeled); (4) 50 mM [1,2 13C]-acetate (universally labeled). Acetate was added to the microcosms (2 ml) as anoxic sterilized basal medium containing [12C], [2 13C], or [1,2 13C] sodium acetate (isotope purity >98%, Cambridge Isotopes). Twelve replicate bottles were established for all universally-labeled acetate-amended microcosms, allowing for three triplicate sets to be sacrificed for protein extraction at 24h, 144 hr, and 408 hr, and a single triplicate set for liquid sampling throughout for VFA analysis. Biomass was pelleted from 10 ml liquid samples via centrifugation (10,000xg) and stored at minus 20C until protein extraction. Protein from cell pellet samples were extracted in an 8M urea solution, then reduced and alkylated. Proteins were then digested with trypsin and subsequently desalted using C18 solid phase extraction. MS analysis was performed using 0.1 ug/ul of peptide solution injected into a Q Exactive HF X mass spectrometer (Thermo Scientific). Data processing Mass spectrometry (MS) data for each biological replicate at all time points (n=18) were analyzed using an implementation of OpenMS implemented in KNIME. Briefly, MS/MS spectra were searched using the MS-GF+ tool against a protein database consisting of all ORFs from the de-replicated set of MAGs, concatenated with reversed (decoy) sequences of all protein entries. Peptide spectra matches (PSMs) were filtered at a 5% false discovery rate (FDR) with Percolator.

Dataset Information

Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics.

Publications

Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics.

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