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All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Untargeted, tandem mass spectrometry (LC/MS-MS) metaproteomes from soil samples in control and warming plots in Blodgett Forest, CA (2014-2021)

The pathways of carbon transport and loss through and from soils—soil organic matter (SOM) depolymerization to dissolved organic carbon and mineralization to carbon dioxide (CO2)—are fundamentally driven by microbial activity, which is strongly regulated by environmental conditions. As part of Lawrence Berkeley National Laboratory (LBNL) Terrestrial Ecosystem Science (TES) Belowground Biogeochemistry Science Focus Area (SFA), we have established a novel whole-soil long-term warming experiment at the University of California (UC) Blodgett Forest Research Station (Sierra Nevada) in 2014, where we study the role of biogeochemical, microbial and geochemical process interactions in SOM decomposition and stabilization. This package contains soil metaproteomics data in the context of site specific metagenomes from soil depth profiles in three paired control and warming plots from a temperate mixed forest in Northern California. Each paired plot had been subjected to experimental warming since June 2014 to simulate a predicted climate change scenario for northern California. These metaproteomes were collected in 2018 after 4.5 years of warming from five depth intervals (0-10 cm, 10-30 cm, 30-45 cm, 45-60 cm, 60-80 cm). For protein identification, the collected spectra were searched following a target-decoy search strategy against a database of metagenome predicted proteins (covering 96 samples from 2014 to 2021) representing the complete sequence diversity at the site. Data was searched with mass spectrometry database search tool (MS-GF+) using Pacific Northwest National Laboratory (PNNL)'s Data Management System (DMS) Processing pipeline. The metagenomes are published as part of another data package. Raw metaproteomic data and the data products from MS-GF+ are deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) database under accession no. MSV000097826. Here we present a dataset that includes spectral counts for the detected proteins across samples (EMSL50964_BrodieAllMAGs_Globals_SC.txt), the sequences of the detected proteins, and sample metadata file that contains site information for the soil metaproteome samples.

Belowground Biogeochemistry Science Focus Area↗
DOE OSTI2025

The microbiologist's guide to metaproteomics

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. By integrating metaproteomics with other omics disciplines, researchers can gain a comprehensive understanding of microbial ecology, interactions, and functional dynamics. This review, developed by the Metaproteomics Initiative (www.metaproteomics.org), serves as a practical guide for both microbiome and proteomics researchers, presenting key principles, state-of-the-art methodologies, and analytical workflows essential to metaproteomics. Topics covered include experimental design, sample preparation, mass spectrometry techniques, data analysis strategies, and statistical approaches.

bioinformatics↗
DOE OSTI2025

Activity-targeted metaproteomics uncovers rare syntrophic bacteria central to anaerobic community metabolism

Syntrophic microbial consortia can contribute significantly to the activity and function of anoxic ecosystems, yet are often too rare to study their in situ physiologies using traditional molecular methods. Here, in this study, we describe a technical innovation combining bioorthogonal non-canonical amino acid tagging (BONCAT), stable isotope probing, and metaproteomics to improve the recovery of proteins from active community members and track isotope incorporation. Both click chemistry-enabled cell-sorting and direct protein pulldown coupled to metaproteomics improved recovery of isotopically labeled proteins during acetate oxidation within a full-scale anaerobic digester. Resulting labeled protein expression profiles revealed elevated activity of a rare and uncharacterized syntrophic bacterium belonging to the family Natronincolaceae. BONCAT-based capture of newly translated proteins provided direct molecular evidence for the expression of a previously hypothesized oxidative glycine pathway for syntrophic acetate oxidation by this microorganism, showcasing the potential of targeted metaproteomics to characterize rare and active cells central to community metabolism in natural and engineered ecosystems.

Friedline, Skyler [Univ. of British Columbia, Vanc↗
DOE OSTI2026

The need for standardization and improved open (meta)data practices in metaproteomics

Metaproteomics enables functional insight into microbial communities by identifying and quantifying proteins in complex samples. Yet, heterogeneous analytical workflows and the lack of standardization across experimental and bioinformatics stages hinder reproducibility and comparability, limiting integration with other omics data. We here present a community-developed reporting checklist tailored to the specific needs of metaproteomics. We also outline current efforts to enable structured and interoperable metadata capture, drawing on standards from proteomics and microbiome research wherever possible. By promoting transparent reporting and advancing metadata practices, our recommendations aim to align metaproteomics more closely with FAIR principles and support reproducible and interoperable research practices.

Armengaud, Jean [Universite Paris-Saclay, France]↗
DOE OSTI2025

Syntrophic bacterial and host–microbe interactions in bacterial vaginosis

Bacterial vaginosis (BV) is a common, polymicrobial condition of the vaginal microbiota that is associated with symptoms such as malodor and excessive discharge, along with increased risk of various adverse sequelae. Host–bacteria and bacteria–bacteria interactions are thought to contribute to the condition, but many of these functions have yet to be elucidated. Using untargeted metaproteomics, we identified 1068 host and 1418 bacterial proteins in a set of cervicovaginal lavage samples collected from 20 participants with BV and 9 who were negative for the condition. We identified Dialister micraerophilus as a major producer of malodorous polyamines and identified a syntrophic interaction between this organism and Fannyhessea vaginae that leads to increased production of putrescine, a metabolite characteristic of BV. Although formate synthesis has not previously been noted in BV, we discovered diverse bacteria associated with the condition express pyruvate formate-lyase enzymes in vivo and confirm these organisms secrete formic acid in vitro. Sodium hypophosphite efficiently inhibited this function in multiple taxa. We also found that the fastidious organism Coriobacteriales bacterium DNF00809 can metabolize formic acid secreted by Gardnerella vaginalis, representing another syntrophic interaction. We noted an increased abundance of the host epithelial repair protein transglutaminase 3 in the metaproteomic data, which we confirmed by enzyme-linked immunosorbent assay. Other proteins identified in our samples implicate Finegoldia magna and Parvimonas micra in the production of malodorous trimethylamine. Some bacterial proteins identified represent novel targets for future therapeutics to disrupt BV communities and promote vaginal colonization by commensal lactobacilli.

bacterial vaginosis↗
DOE OSTI2026

Branched-chain amino acid specialization drove diversification within Calditenuaceae ( Caldarchaeia ) and enables their cultivation

Many thermophiles that are abundant in high-temperature geothermal systems have never been cultivated and are poorly understood, including deeply branching members of the archaeal phylum Thermoproteota. Here, we describe the genome-guided cultivation of one such organism, Calditenuis ramacidaminiphagus, and show that it has evolved a heterotrophic metabolism focused on branched-chain amino acids (BCAAs). Initially, fluorescence in situ hybridization and nanoscale secondary ion mass spectrometry (FISH-nanoSIMS) showed that Cal. ramacidaminiphagus assimilated amino acids rapidly in casamino acid-amended enrichment cultures. Metagenome and metaproteome analyses showed a high abundance and expression of BCAA transporter genes, suggesting a BCAA-focused metabolism. This inference was supported by the subsequent enrichment of Cal. ramacidaminiphagus in BCAA-fed cultures, reaching 2.66×10 6 cells/mL and 48.7% of the community, whereas it was outcompeted when polar amino acids were included. Metabolic reconstruction and metaproteomics suggest that BCAAs are channeled into the mevalonate pathway for lipid biosynthesis and fuel ATP production through the TCA cycle coupled with aerobic respiration and through production of branched-chain organic acids by overflow metabolism. Ancestral state reconstructions and phylogenetic analyses of 62 Caldarchaeales genomes revealed multiple horizontal transfers of BCAA transporters to the ancestor of the genus Calditenuis. Our study highlights the crucial role of BCAAs in the early evolution and niche of this genus, and suggests a high degree of resource partitioning even within low-diversity thermophilic communities.

Biological and medical sciences↗
DOE OSTI2025

Unraveling Bacterial Adaptation Strategies in the Microbiome Shaped by the Chemical Environment of the Plant Rhizosphere

The rhizosphere is a dynamic environment where rhizodeposits that include primary and secondary metabolites and mucilage serve as nutrient sources for soil microorganisms, attracting them toward plant roots. However, understanding how these microbes specifically respond to plant root chemical signals has been hindered by the challenges of disentangling physical and chemical interactions between the microbes and plant roots. To address this, we implemented an innovative filter-based experimental setup on plant roots that creates a physical barrier while facilitating the exchange of chemical signals. The proteomic analysis of 10 Populus root-associated bacterial strains grown in the presence or absence of a plant in either individual or mixed community conditions provided detailed insights into the functional responses of these strains to the root chemical environment. Additionally, this approach allowed us to discern the impact of root exudates on overall community dynamics. In particular, metaproteomic analyses revealed that each of these 10 microbial members responds uniquely to the presence of the plant, with Bacillus and Pantoea exhibiting the most dramatic favorable impact. Proteomic examination revealed the details of metabolism fine-tuning, including processes such as chemotaxis and ATP-binding cassette transporter proteins. This study demonstrates the application of a filter-based experimental setup to study microbial responses to plant chemicals and sheds light on adaptation strategies employed by various bacterial strains for survival in the rhizosphere.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

A bloom of a single bacterium shapes the microbiome during outdoor diatom cultivation collapse

Algae-dominated ecosystems are fundamentally influenced by their microbiome. We lack information on the identity and function of bacteria that specialize in consuming algal-derived dissolved organic matter in high algal density ecosystems such as outdoor algal ponds used for biofuel production. Here, we describe the metagenomic and metaproteomic signatures of a single bacterial strain that bloomed during a population-wide crash of the diatom, Phaeodactylum tricornutum, grown in outdoor ponds. 16S rRNA gene data indicated that a single Kordia sp. strain (family Flavobacteriaceae) contributed up to 93% of the bacterial community during P. tricornutum demise. Kordia sp. expressed proteins linked to microbial antagonism and biopolymer breakdown, which likely contributed to its dominance over other microbial taxa during diatom demise. Analysis of accompanying downstream microbiota (primarily of the Rhodobacteraceae family) provided evidence that cross-feeding may be a pathway supporting microbial diversity during diatom demise. In situ and laboratory data with a different strain suggested that Kordia was a primary degrader of biopolymers during algal demise, and co-occurring Rhodobacteraceae exploited degradation molecules for carbon. An analysis of 30 Rhodobacteraceae metagenome assembled genomes suggested that algal pond Rhodobacteraceae commonly harbored pathways to use diverse carbon and energy sources, including carbon monoxide, which may have contributed to the prevalence of this taxonomic group within the ponds. These observations further constrain the roles of functionally distinct heterotrophic bacteria in algal microbiomes, demonstrating how a single dominant bacterium, specialized in processing senescing or dead algal biomass, shapes the microbial community of outdoor algal biofuel ponds.

Kordia↗
DOE OSTI2025

Environmental matrix and moisture influence soil microbial phenotypes in a simplified porous media incubation

Soil moisture and porosity regulate microbial metabolism by influencing factors, such as system chemistry, substrate availability, and soil connectivity. However, accurately representing the soil environment and establishing a tractable microbial community that limits confounding variables is difficult. Here, we use a reduced-complexity microbial consortium grown in a glass bead porous media amended with chitin to test the effects of moisture and a structural matrix on microbial phenotypes. Leveraging metagenomes, metatranscriptomes, metaproteomes, and metabolomes, we saw that our porous media system significantly altered microbial phenotypes compared with the liquid incubations, denoting the importance of incorporating pores and surfaces for understanding microbial phenotypes in soils. These phenotypic shifts were mainly driven by differences in expression of Streptomyces and Ensifer, which included a significant decrease in overall chitin degradation between porous media and liquid. Our findings suggest that the success of Ensifer in porous media is likely related to its ability to repurpose carbon via the glyoxylate shunt amidst a lack of chitin degradation byproducts while potentially using polyhydroxyalkanoate granules as a C source. We also identified traits expressed by Ensifer and others, including motility, stress resistance, and carbon conservation, that likely influence the metabolic profiles observed across treatments. Together, these results demonstrate that porous media incubations promote structure-induced microbial phenotypes and are likely a better proxy for soil conditions than liquid culture systems. Furthermore, they emphasize that microbial phenotypes encompass not only the multi-enzyme pathways involved in metabolism but also include the complex interactions with the environment and other community members.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Meta-omics reveals role of photosynthesis in microbially induced carbonate precipitation at a CO2-rich geyser

Abstract Microbially induced carbonate precipitation (MICP) is a natural process with potential biotechnological applications to address both carbon sequestration and sustainable construction needs. However, our understanding of the microbial processes involved in MICP is limited to a few well-researched pathways such as ureolytic hydrolysis. To expand our knowledge of MICP, we conducted an omics-based study on sedimentary communities from travertine around the CO2-driven Crystal Geyser near Green River, Utah. Using metagenomics and metaproteomics, we identified the community members and potential metabolic pathways involved in MICP. We found variations in microbial community composition between the two sites we sampled, but Rhodobacterales were consistently the most abundant order, including both chemoheterotrophs and anoxygenic phototrophs. We also identified several highly abundant genera of Cyanobacteriales. The dominance of these community members across both sites and the abundant presence of photosynthesis-related proteins suggest that photosynthesis could play a role in MICP at Crystal Geyser. We also found abundant bacterial proteins involved in phosphorous starvation response at both sites suggesting that P-limitation shapes both composition and function of the microbial community driving MICP.

Violette, Marlene J.↗
DOE OSTI2026

Capacity of Arctic fjord sediments to degrade carbohydrates from permafrost active layer

The degradation of organic matter (OM) by microorganisms in thawing permafrost produces greenhouse gases. Terrestrial OM is transported into fjords through hydrological runoff, but it is unclear whether the microbial mechanisms of OM degradation on land persist after soils enter marine environments, which differ greatly in conditions and microbial communities. This question is particularly relevant for low-OM soils, which dominate Arctic landscapes and are more exposed to oxidants. Here, we compared OM-degrading capacity in permafrost-affected active layer soils and adjacent fjord sediments from Kongsfjorden, Svalbard, focusing on carbohydrate-active enzymes (CAZymes), which target some of the most abundant types of organic matter in soils. Using multi-omics approaches—metagenomics, metagenome-assembled genomes (MAGs), metabolomics, metatranscriptomics, and metaproteomics—we examined CAZyme presence, distribution, and activity. Despite environmental differences, both soils and sediments harbored diverse glycoside hydrolases and polysaccharide lyases, most of which showed evidence of activity. Verrucomicrobia expressed the highest number of CAZyme transcripts, indicating that they dominated active carbohydrate degradation in fjord sediments, while Acidobacteria and Actinobacteria were more active in soils. Notably, CAZymes in fjord sediments targeted primarily soil-derived OM, and the proportions of enzymes degrading terrestrial OM, marine OM, and microbial necromass—remnants of dead microbial cells were similar across both environments. These results suggest that microbial communities in both soils and fjord sediments are equipped to degrade carbohydrates, and that burial of terrestrial-derived OM in fjord sediments may not protect it from microbial breakdown under Arctic warming.

Chukwufumnanya, Abuah [University of Tennessee, Kn↗
DOE OSTI2025

Ranking Biological Features in Soil-Based Microbial Multi-Omics Data with Integration Modeling

Distinguishing the most important features (e.g. proteins, metabolites, etc.) per group (e.g. control and treatment) is a critical challenge in feature-rich multi-omics experiments, especially in soil data. Traditional feature identification and ranking approaches, such as differential expression, are based on single omics and thus not directly translatable to multi-omics experiments. Here, 5 multi-omics integration models (DIABLO, JACA, MOFA, MultiMLP, and SLIDE) that were not explicitly built for soil data applications were tested using a soil-based multi-omics experiment. The data were obtained from an experimental setup of an autoclaved soil system inoculated with 8 bacteria and using chitin as the carbon source and including samples collected at 0- (control), 4-, 8-, and 12-weeks post-inoculation. The omics data included metaproteomics, 16S rRNA sequencing, and LC-MS/MS metabolomics (in positive and negative mode). Each multi-omics integration model was implemented, and top features were compared to differential univariate statistics per omic type, demonstrating that integration approaches cut the potential number of top features from 2957 identified by differential statistics to 13-224 (a 99.6% to 92.4% reduction). Interestingly, most top features across integration models were not shared; though, scaling and averaging ranks across models shared similar patterns. This work highlights the usefulness of multi-omics integration models in soil-based microbial studies and the power of using multiple integration models together to interpret results.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

MARLOWE: An Untargeted Proteomics, Statistical Approach to Taxonomic Classification for Forensics

General proteomics research for fundamental science typically addresses laboratory- or patient-derived samples of known origin and composition. However, in a few research areas, such as environmental proteomics, clinical identification of infectious organisms, archeology, art/cultural history, and forensics, attributing the origin of a protein-containing sample to the organisms that produced it is a central focus. A small number of groups have approached this problem and developed software tools for taxonomic characterization and/or identification using bottom-up proteomics. Most such tools identify peptides via database search, and many rely on organism-specific peptides as markers. Our group recently introduced MARLOWE, a software tool for taxonomic characterization of unknown samples based on de novo peptide identification and signal-erosion-resistant strong peptides, which are shared peptides distributed in a taxonomy-dependent manner. In the current work, we further characterize the utility of MARLOWE using publicly available proteomics data from forensically-relevant samples. MARLOWE characterizes samples based on their protein profile, and returns ranked organism lists of potential contributors and taxonomic scores based on shared strong peptides between organisms. Overall, the correct characterization rate ranges between 44 and 100%, depending on the sample type and data acquisition parameters (with lower numbers associated with lower-quality data sets). MARLOWE demonstrates successful characterization of true contributors and close relatives, and provides sufficient specificity to distinguish certain microbial species. MARLOWE demonstrates its ability to provide insight into potential taxonomic sources for a wide range of sample types without prior assumptions about sample contents. As a result, this approach can find utility in forensic science and also broadly in bioanalytical applications that utilize proteomics approaches for taxonomic characterization.

Bacteria↗
DOE OSTI2025

Multi-omics of a model bacterial consortium deciphers details of chitin decomposition in soil

Soil microorganisms interact to carry out decomposition of complex organic carbon and nitrogen compounds, such as chitin, but the high diversity and complexity of the soil microbiome and habitat have posed a challenge to elucidating such interactions. Here, we sought to address this challenge by analysis of a model soil consortium (MSC-2) consisting of eight soil bacterial species. Our aim was to elucidate the specific roles of the member species during chitin metabolism. Samples were collected from MSC-2 incubated in chitin-enriched soil over 3 months. Multi-omics was used to understand how the community composition, transcripts, proteins, and chitin decomposition shifted over time. The data clearly and consistently revealed a temporal shift during chitin decomposition with defined contributions by individual species. A Streptomyces genus member (sp001905665) was a key player in early steps of chitin decomposition, with other MSC-2 members being central in carrying out later steps. These results illustrate how multi-omics applied to a defined consortium untangles the interactions between soil microorganisms.

chitin↗
DOE OSTI2025

Using culture‐independent methods to link active compound‐specific carbon degradation to greenhouse gas production and recycling in natural populations of permafrost microbes

Under this project we conducted one scouting field trip (2019) and two fieldworks (2021, 2022) in Ny-Alesund, Svalbard. The project supported seven PhD students, six undergraduate research assistants, three Postdoctoral research associates and one Research Professor. The project supported travel and fieldwork for three international students. This project has produced 49 conference presentations/posters, 18 peer-reviewed journal articles published at time of preparation of this report, while 6 journal articles are still at different stages of review process, 1 outreach website and 2 YouTube videos. In addition, the PIs gave many seminars to other universities and research institutions.

54 ENVIRONMENTAL SCIENCES↗