Unveiling plasmid diversity and functionality in pristine groundwater.

Background: Plasmids are key in creating a dynamic reservoir of genetic diversity, yet their impact on Earth's continental subsurface-an important microbial reservoir-remains unresolved. We analyzed 32 metagenomic samples from six groundwater wells within a hillslope aquifer system to assess the genetic and functional diversity of plasmids and to evaluate the role of these plasmids in horizontal gene transfer (HGT).

Results: Our results revealed 4,609 non-redundant mobile genetic elements (MGEs), with 14% (664) confidently classified as plasmids. These plasmids displayed well-specific populations, with fewer than 15% shared across wells. Plasmids were linked to diverse microbial phyla, including Pseudomonadota (42.17%), Nitrospirota (3.31%), Candidate Phyla Radiation (CPR) bacteria (2.56%), and Omnitrophota (2.11%). The presence of plasmids in the dominant CPR bacteria is significant, as this group remains underexplored in this context. Plasmid composition strongly correlated with well-specific microbial communities, suggesting local selection pressures. Functional analyses highlighted that conjugative plasmids carry genes crucial for metabolic processes, such as cobalamin biosynthesis and hydrocarbon degradation. Importantly, we found no evidence of high confidence emerging antibiotic resistance genes, contrasting with findings from sewage and polluted groundwater.

Conclusions: Overall, our study emphasizes the diversity, composition, and eco-evolutionary role of plasmids in the groundwater microbiome. The absence of known antibiotic resistance genes highlights the need to preserve groundwater in its pristine state to safeguard its unique genetic and functional landscape.