Candidate Phyla Radiation bacteria in a groundwater enrichment showing genomic signatures of a free-living lifestyle

Bacteria of the widespread Candidate Phyla Radiation (CPR) often lack biosynthetic genes for essential building blocks like amino acids and nucleotides, causing dependence on other organisms. Available cultures of some CPR taxa, like Cand. Saccharimonadia and Cand. Absconditabacteriales, showed physical attachment of CPR to bacterial host cells. However, CPR taxa that typically dominate the planktonic community in groundwater habitats, like the Cand. Paceibacteria that can reach abundances of up to 45%, have not been obtained in culture so far. Recent studies have indicated that some members of these taxa might favor a free-living lifestyle.

Here, we present results of a metagenomics analysis on an aerobic groundwater-derived enrichment containing Cand. Paceibacteria that has been continuously maintained for more than two years. CPR constitute up to 25% of the enrichment community, based on coverage of metagenomic reads. Metagenome-assembled genomes (MAGs) affiliated with CPR in the enrichment were compared to CPR-MAGs previously obtained from a groundwater metagenomics campaign, with the aim to determine differences that might indicate a free-living lifestyle. In total, 14 MAGs affiliated with Cand. Paceibacteria, belonging to the order Cand. Kaiserbacteria, as well as 4 MAGs of CPR class ABY1, which is also common in groundwater, were recovered. The enriched CPR showed a significantly higher GC content and genome size than those found in groundwater CPR. Genes involved in nitrogen and sulfur cycling, like nitrite reductase nirK and sulfur dioxygenase sdo, as well as F-type ATPase genes, were more frequently present in enriched compared to groundwater MAGs, suggesting mechanisms for host-independent energy generation. Pathways for nucleotide biosynthesis were also more often present in enrichment versus groundwater MAGs, potentially enabling de-novo synthesis of these building blocks. Furthermore, CAZymes and peptidases that may provide access to organic carbon sources in the enrichment were abundant in the CPR-MAGs. Interestingly, the ATP-citrate lyase genes aclAB, key genes in the reverse TCA cycle for CO₂ fixation, were present in most Cand. Paceibacteria from the enrichment. The lack of a complete reverse TCA cycle, however, suggests a different function of these genes in CPR. Taken together, this shows that the specific, distinct functions in the enriched CPR-MAGs could benefit a host-independent lifestyle.