Metaproteogenomics resolution of a high-CO2 aquifer community suggests an active symbiotic lifestyle of groundwater Gracilibacteria

Abstract

BackgroundBacteria 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 a symbiotic lifestyle. Gracilibacteria (BD1-5), which are part of the CPR branch, possess alternate coded genomes and have not yet been cultivated. However, besides genomic evidence, little is known about the lifestyle of Gracilibacteria, their temporal dynamics, and activity in natural ecosystems, particularly in groundwater, where they were initially been genomically resolved. Therefore, we here aimed to investigate Gracilibacteria activity in situ and to discern expressed genes involved in their lifestyle, using the metaproteogenome of Gracilibacteria as a function of time in the cold-water geyser Wallender Born in the Volcanic Eifel region in Germany. ResultsWe coupled genome-resolved metagenomics and metaproteomics to investigate a cold-water geyser microbial community enriched in Gracilibacteria across a 12-day time-series. Groundwater was collected and sequentially filtered 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, which were low in genomic abundance but substantially contributing to proteomic abundance. Seven high-quality Gracilibacteria genomes showed typical limitations, such as limited amino acid or nucleotide synthesis, in their central metabolism but no co-occurrence with potential hosts. The genomes of these Gracilibacteria encoded for a high number of proteins related to a symbiotic or even predatory lifestyle, e.g., type IV and type II secretion system subunits and features related to cell-cell interactions and cell motility, which were also detected on protein level. ConclusionsCoupling metagenomics to metaproteomics enabled us to identify microbial keystone taxa in a high-CO2 aquifer, and to reveal microbial dynamics of Gracilibacteria. We posit that Gracilibacteria might be successful microbial predators in this ecosystem, potentially aiding in population control of this highly perturbed microbial geyser community from the deep biosphere.