Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially processed plant derived carbon

Background: The 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. Results: We 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 labelled plant DNA. Isotopic labeling indicates that the population was replicating during the six-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 or lactate. The genome 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 make a variety of isoprenoids, including the plant hormone zeatin. Conclusions: Rhizosphere 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

Plant roots alter microbial functional genes supporting litter decomposition

Decomposition of soil organic carbon is central to the global carbon cycle and profoundly affected by plant roots. While root “priming” of decomposition has been extensively investigated, it is not known how plants alter the molecular ecology of soil microbial decomposers. We disentangled the effects of Avena fatua, a common annual grass, on 13C-labeled root litter decomposition and quantified multiple genetic characteristics of soil bacterial and fungal communities. In our study, plants consistently suppressed rates of litter decomposition. Plant uptake N may cause soil microbes under N limitation and reduce their activities. Microbes from planted soils had relatively more genes coding for low molecular weight compound degradation enzymes, while those from unplanted had more macromolecule degradation genes. Higher abundances of “water stress” genes in planted soils suggested plant-induced water stress for microbes. We used a quantitative model to integrate our extensive data set; it indicated that the multiple soil environmental and microbial mechanisms involved in litter decomposition all acted through changing the functional gene profiles of microbial decomposers living near plant roots

Additional file 5: of Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon

Figure S3. The same tree as in Fig. 2 with accessions. (PDF 93 kb

Additional file 1: of Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon

Table S1. Stable isotope probing and sequencing statistics. (XLSX 9 kb

Additional file 3: of Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon

Figure S1. Plot of GC skew (black) and cumulative GC skew (green, window 1000 bp, slide of 10 bp) of the T. rhizospherense genome. The plot shows the predicted locations of the origin (red line, 1201 bp) and terminus (blue line, 757,370 bp) of replication. The form of the plot is as expected for a correctly assembled, circularized genome that undergoes bi-directional replication from a single origin. (PDF 69 kb

Additional file 2: of Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon

Table S2. Genome summary, including HMM-based annotations. (XLSX 1076 kb

Additional file 6: of Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon

Figure S4. Stable isotope fraction determination from Fig. 1 with added pre-planted bulk soil. (PDF 283 kb

Additional file 4: of Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon

Figure S2. Protein modeling of T. rhizospherense. Proteins with Swiss-Model in Clustal colors with DSSP secondary structure overlaid. a The top row is the T. rhizospherense NADH dehydrogenase II and the bottom row is the reference protein from Caldalkalibacillus thermarum. The symbols above the alignment indicate: F FAD binding site, N NADH binding site, and U Ubiquinone or menaquinone binding site. b The top row represents the T. rhizospherense cytochrome bo3 ubiquinol oxidase subunit I and the bottom row is the reference sequence from E. coli. The symbols above the alignment represent key residues: D D-channel, U Ubiquinol binding site, *. Ion binding site, K K-channel, bulky hydrophobic residues which differentiate between cytochrome c oxidase and cytochrome bo3 ubiquinol oxidase. (PNG 3927 kb

Fungal-bacterial cooccurrence patterns differ between arbuscular mycorrhizal fungi and nonmycorrhizal fungi across soil niches

Soil bacteria and fungi are known to form niche-specific communities that differ between actively growing and decaying roots. Yet almost nothing is known about the cross-kingdom interactions that frame these communities and the environmental filtering that defines these potentially friendly or competing neighbors. We explored the temporal and spatial patterns of soil fungal (mycorrhizal and nonmycorrhizal) and bacterial cooccurrence near roots of wild oat grass, Avena fatua, growing in its naturalized soil in a greenhouse experiment. Amplicon sequences of the fungal internal transcribed spacer (ITS) and bacterial 16S rRNA genes from rhizosphere and bulk soils collected at multiple plant growth stages were used to construct covariation-based networks as a step toward identifying fungal-bacterial associations. Corresponding stable-isotope-enabled metagenome-assembled genomes (MAGs) of bacteria identified in cooccurrence networks were used to inform potential mechanisms underlying the observed links. Bacterial-fungal networks were significantly different in rhizosphere versus bulk soils and between arbuscular mycorrhizal fungi (AMF) and nonmycorrhizal fungi. Over 12 weeks of plant growth, nonmycorrhizal fungi formed increasingly complex networks with bacteria in rhizosphere soils, while AMF more frequently formed networks with bacteria in bulk soils. Analysis of network-associated bacterial MAGs suggests that some of the fungal-bacterial links that we identified are potential indicators of bacterial breakdown and consumption of fungal biomass, while others intimate shared ecological niches

The interconnected community: microbial networks in complex and simple natural habitats

Microbes do not live alone in nature, instead they live in the interconnected community. The interactions among microbes are an important characteristic of the community, however, very little is known about microbial networks owing to the lack of appropriate experimental data and analytical tools. Recent advances in metagenomics technologies (such as high throughput sequencing) has provided revolutionary tools for analyzing microbial community networks. We used random matrix theory (RMT)-based network analysis to identify potential bacterial interactions (co-occurrence patterns) in two different natural systems: diverse rhizosphere communities associated with wild oat (Avena fatua) and relatively simple legume (Medicago sativa L.) nodule endophytic communities. Rhizosphere networks were substantially more complex than those in surrounding bulk soils. Network complexity increased as plants grew, even as diversity indices decreased, highlighting that interactions are a crucial dimension of community organization overlooked by univariate diversity. Consistent with the hypothesis that extensive mutualistic interactions occur among rhizosphere bacteria, covariations were predominantly positive (>80%); we identified quorum-based signaling as one potential rhizosphere interaction strategy. Putative keystone species often had low relative abundances, suggesting low abundance taxa may significantly contribute to rhizosphere structure and function. Currently, we are analysing the microbial network in a legume nodule endophytic communities. Although nodule communities contain much fewer microbial taxa compared to a rhizosphere community, defined microbial networks are evident. These studies show that network complexity appears to be a defining characteristic of the interconnected microbiome, and is a previously uncharacterised property of these habitats