Proteogenomic analyses indicate bacterial methylotrophy and archaeal heterotrophy are prevalent below the grass root zone
- Published
- Accepted
- Subject Areas
- Bioinformatics, Environmental Sciences, Genomics, Microbiology, Soil Science
- Keywords
- Genome-resolved metagenomics, methanol dehydrogenase, soil bacteria, soil archaea, proteomics, metabolomics
- Copyright
- © 2016 Butterfield et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2016. Proteogenomic analyses indicate bacterial methylotrophy and archaeal heterotrophy are prevalent below the grass root zone. PeerJ Preprints 4:e2473v1 https://doi.org/10.7287/peerj.preprints.2473v1
Abstract
Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO2. However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10 – 20 cm and 30 – 40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. We also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation. Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. The new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Measurements of soil characteristics
(A) total organic carbon (TOC), (B) soil moisture, (C) soil pH, and (D) particle size. Measurements of TOC, moisture and pH were performed at each depth before and after rain. One inch of rain fell on Day 30 and 3 inches of rain fell on Day 39. TOC decreased by depth and appeared to slightly increase at all depths after the second rainfall. Soil moisture in the upper soil zones was very low before the rainfall and increased with each rain event. Soil pH was fairly uniform between the depths and rose from about 4.6 to 4.9 after the first rain then held steady. The soil was a mixture of clay- and silt-sized particles (~ 45% each) with a minor component of sand-sized particles (8-10%).
Phylogenetic Trees
a) Bacterial and archaeal16S rRNA phylogenetic tree. b) Bacterial and archaeal rpS3 phylogenetic tree. All rpS3 proteins or 16S rRNA genes from every sample (colored branches) and the references (black branches, chosen based on closest hits to the sample sequences and representatives of in the NCBI database) were aligned and a phylogenetic tree was constructed using RAxML and visualized in Geneious (version 7.0 by Biomatters).
Depiction of the Gemmatimonadetes contig that encodes the methanol utilization proteins
The genetic neighborhood of the methanol dehydrogenase in Gemmatimonadetes encodes for most of the proteins involved in the metabolism of methanol, including the tetrahydromethanopterin (THMPT) biosynthesis (orange), formaldehyde capture (green) pathways, and the methanol dehydrogenase (xoxF) itself and a maturation protein (red). Each of these pathways is represented by at least one known protein in the proteome (indicated by blue star). Gene name abbreviations: formaldehyde activating enzyme (fae); NAD(P)-dependent methylene-tetrahydromethanopterion dehydrogenase (mtdB); formyltransferase/hydroxylating complex (fhc); methenyl-tetrahydromethanopterin cyclohydrolase (mch).
Genome summary of methylotrophic organisms
Rows indicate draft genomes and columns indicate lists of individual genes, with the exception of PQQ biosynthesis where many subunits are represented in a single list.
Emergent self-organizing map of one soil sample
Image of emergent self-organizing map created from time series and coverage patterns of contigs from sample 10 – 20 cm 2 days after 2nd rain. 48 partial to near-complete genomes were defined (colors were assigned randomly).
Organism Table
a) Table listing genomes and megabins generated in the study. The summaries of genome characteristics are listed by sample and include genome completeness, size, GC content, and other genome bin quality information. b) Genomes recovered and the current NCBI and JGI IMG database of genomes for those phyla.
Proteomics Data
a) Proteomics data: Normalized spectral counts for proteins and their respective scaffolds and genome bins. b) Overview of protein identifications in each sample with the estimated protein false discovery rate. The false discovery rates for each sample are reported. Note: the false discovery rate was strictly controlled at the peptide level (1%).
Plant assemblage
2013 annual abundance survey and summary of plant species in the Angelo Coast Range Reserve meadow. Introduced species of summer flowering annual grasses and forbs were most abundant during this study.
Metabolite Information
Chemical formula and neutral mass for each compound in Figure 5 is listed along with LC-MS retention times and the ions associated with that metabolite. The primary ion used for analysis of the metabolite abundance is listed (selected based on detection limits and coefficient of variation across sample extractions) along with secondary ions and primary MS/MS fragments where available. For each ion entry the polarity (POS – positive ionization mode vs NEG – negative ionization mode), adduct (e.g., H+, H-, Na+) and m/z value are presented. Internal extraction and resuspension standards used are listed at the bottom of the table.
Metabolite Peak Areas
Peak areas of each metabolite used to generate Figure 5 and analyzed for significant changes (Table S7). Peak areas correspond to the ‘Ion (m/z) used for Sample Analyses’ column in Table S5.
Post-hoc Tukey tables
Post-hoc Tukey multiple comparison analysis of sugars (A) and nitrogenous compounds (B) demonstrating compounds with significant (α=0.05) differences in peak area across samples displayed in Figure 5.