Enrichment of a parasitic member of Cand. Gracilibacteria along with its host from groundwater

The Candidate Phyla Radiation (CPR) superphylum harbors about 25% of the total bacterial diversity. The lifestyle and ecological role of its members are still poorly understood due to the lack of cultured representatives. We obtained a stable enrichment culture of a member of the Cand. Gracilibacteria (BD1-5) along with its host Azonexus sp. from groundwater of a sandstone aquifer. Cand. Gracilibacteria is a sister phylum of the Cand. Absconditabacteria, which is associated with a parasitic, host-associated lifestyle, raising the question if parasitism is a widespread trait among these lineages of the CPR. The culture was grown under anoxic, nitrate-reducing conditions with hydrogen or acetate as an electron donor. Each partner grew up to a density of about 10⁸ cells per ml, together accounting for 50 to 95% of the total community. Scanning electron microscopy revealed that Gracilibacteria strain FG1 is about 0.4 µm in size and lives epibiotically on its host. Observations of host cell degeneration suggest a parasitic nature of the interaction. The parasite divides by forming stacks of up to eight cells attached perpendicular to the host cell which might also play an important role in the infection process. When transferred to a fresh host culture, Gracilibacteria strain FG1 showed a rapid infection and growth response, resulting in a 50 times increase in cell number within three days. Metagenomic analysis revealed that strain Gracilibacteria FG1 lacks complete pathways for amino acid biosynthesis and central carbon metabolism, indicating strong dependence on the host cell for basic metabolism and cell division. The presence of genes encoding proteins related to type IV pili and type II secretion systems pointed to mechanisms of establishing cell-to-cell interactions. Relative abundances of Gracilibacteria strain FG1 and Azonexus sp. in the in situ groundwater community varied from 0.01 – 36% and 0.4 - 18%, respectively, over a 4-years observation period, suggesting a high relevance of this parasitic interaction for groundwater community dynamics. Our findings expand previous observations of a parasitic lifestyle of Cand. Absconditabacteria to the sister phylum Cand. Gracilibacteria, suggesting that cell sizes larger than 0.2 µm, stack formation resulting from host-attached cell division, and fatal parasitism are a typical feature of these groups, contrasting with smaller cell sizes and potentially non-predatory lifestyles of other CPR lineages.