Stable isotope probing (SIP) enables tracking the nutrient flows from isotopically labeled substrates to specific microorganisms in microbial communities. In proteomic SIP, labeled proteins synthesized by the microbial consumers of labeled substrates are identified with a shotgun proteomics approach. Here, proteomic SIP was combined with targeted metagenomic binning to reconstruct metagenome-assembled genomes (MAGs) of the microorganisms producing labeled proteins. This approach was used to track carbon flows from ¹³CO₂ to the rhizosphere communities of Zea mays, Triticum aestivum, and Arabidopsis thaliana. Rhizosphere microorganisms that assimilated plant-derived ¹³C were capable of metabolic and signaling interactions with their plant hosts, as shown by their MAGs containing genes for phytohormone modulation, quorum sensing, and transport and metabolism of nutrients typical of those found in root exudates. XoxF-type methanol dehydrogenases were among the most abundant proteins identified in the rhizosphere metaproteomes. ¹³C-methanol proteomic SIP was used to test the hypothesis that XoxF was used to metabolize and assimilate methanol in the rhizosphere. We detected 7 ¹³C-labeled XoxF proteins and identified methylotrophic pathways in the MAGs of 8 ¹³C-labeled microorganisms, which supported the hypothesis. These two studies demonstrated the capability of proteomic SIP for functional characterization of active microorganisms in complex microbial communities.

Stable isotope probing (SIP) enables tracking the nutrient flows from isotopically labeled substrates to specific microorganisms in microbial communities. In proteomic SIP, labeled proteins synthesized by the microbial consumers of labeled substrates are identified with a shotgun proteomics approach. Here, proteomic SIP was combined with targeted metagenomic binning to reconstruct metagenome-assembled genomes (MAGs) of the microorganisms producing labeled proteins. This approach was used to track carbon flows from ¹³CO₂ to the rhizosphere communities of Zea mays, Triticum aestivum, and Arabidopsis thaliana. Rhizosphere microorganisms that assimilated plant-derived ¹³C were capable of metabolic and signaling interactions with their plant hosts, as shown by their MAGs containing genes for phytohormone modulation, quorum sensing, and transport and metabolism of nutrients typical of those found in root exudates. XoxF-type methanol dehydrogenases were among the most abundant proteins identified in the rhizosphere metaproteomes. ¹³C-methanol proteomic SIP was used to test the hypothesis that XoxF was used to metabolize and assimilate methanol in the rhizosphere. We detected 7 ¹³C-labeled XoxF proteins and identified methylotrophic pathways in the MAGs of 8 ¹³C-labeled microorganisms, which supported the hypothesis. These two studies demonstrated the capability of proteomic SIP for functional characterization of active microorganisms in complex microbial communities.

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.