Project description:Metagenome-assembled genomes (MAGs) have revealed the existence of novel bacterial and archaeal groups and provided insight into their genetic potential. However, metagenomics and even metatranscriptomics cannot resolve how the genetic potential translates into metabolic functions and physiological activity. Here, we present a novel approach for the quantitative and organism-specific assessment of the carbon flux through microbial communities with stable isotope probing-metaproteomics and integration of temporal dynamics in 13C incorporation by Stable Isotope Cluster Analysis (SIsCA). We used groundwater microcosms labeled with 13CO2 and D2O as model systems and stimulated them with reduced sulfur compounds to determine the ecosystem role of chemolithoautotrophic primary production. Raman microspectroscopy detected rapid deuterium incorporation in microbial cells from 12 days onwards, indicating activity of the groundwater organisms. SIsCA revealed that groundwater microorganisms fell into five distinct carbon assimilation strategies. Only one of these strategies, comprising less than 3.5% of the community, consisted of obligate autotrophs (Thiobacillus), with a 13C incorporation of approximately 95%. Instead, mixotrophic growth was the most successful strategy, and was represented by 12 of the 15 MAGs expressing pathways for autotrophic CO2 fixation, including Hydrogenophaga, Polaromonas and Dechloromonas, with varying 13C incorporation between 5% and 90%. Within 21 days, 43% of carbon in the community was replaced by 13C, increasing to 80% after 70 days. Of the 31 most abundant MAGs, 16 expressed pathways for sulfur oxidation, including strict heterotrophs. We concluded that chemolithoautotrophy drives the recycling of organic carbon and serves as a fill-up function in the groundwater. Mixotrophs preferred the uptake of organic carbon over the fixation of CO2, and heterotrophs oxidize inorganic compounds to preserve organic carbon. Our study showcases how next-generation physiology approach like SIsCA can move beyond metagenomics studies by providing information about expression of metabolic pathways and elucidating the role of MAGs in ecosystem functioning.

Project description:Background. Bacteria of the Candidate Phyla Radiation (CPR), constituting about 25% of the bacterial biodiversity, are characterized by small cell size and patchy genomes without complete key metabolic pathways suggesting symbiotic life styles. Gracilibacteria (BD1-5) are part of the CPR branch, they possess alternate coded genomes and have two cultivated members that were shown to be microbial predators. However, besides genomic sampling, little is known about the lifestyle of Gracilibacteria, their temporal dynamics, and activity in natural ecosystems, and particularly groundwater where they have initially been genomically resolved. The current study was set out with the aim of investigating the metaproteogenome of Gracilibacteria as a function of time in the cold-water geyser Wallender Born in the Volcanic Eifel region in Germany, to estimate their activity in situ and discern expressed genes involved in their lifestyle. Results. We coupled genome-resolved metagenomics and metaproteomics to investigate a microbial community enriched in Gracilibacteria across a 12-day time-series. Groundwater was collected and sequentially filtered onto 0.2-μm and 0.1-μm filters to fraction CPR and other bacteria. Based on 670 Gbps of metagenomic data, 1129 different ribosomal protein S3 marker genes and 751 high-quality genomes (123 population genomes after dereplication), we identified dominant bacteria belonging to Galionellales and Gracilibacteria along with keystone microbes, low in genomic abundance but substantially contributing to proteomic abundance. Seven high-quality Gracilibacteria genomes showed typical limitations in their central metabolism but no co-occurrence to potential hosts. Their genomes encoded for a high number of proteins related to a predatory lifestyle, whose expression was detected in the proteome and included subunits related to type IV and type II secretion systems, as well as features related to cell-cell interactions and cell motility. Conclusion. We present a highly resolved analysis coupling metagenomics to metaproteomics for elucidating microbial dynamics of Gracilibacteria in groundwater. We posit that Gracilibacteria are successful microbial predators in this ecosystem potentially aiding in population control of this highly disturbed microbial community from the deep biosphere.

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