Virus-encoded auxiliary metabolic genes fuel the metabolism of organic pollutants in long-term contaminated groundwater.

Virus-encoded auxiliary metabolic genes (AMGs) are important for host metabolism, such as carbon and nitrogen cycling; however, whether and how viral AMGs affect bacterial adaptation to pollution stress in long-term contaminated groundwater remain unclear, significantly limiting our understanding of phage-host interactions on the transformation of organic pollutants in groundwater. Here, by leveraging viromics sequencing for virus-like particles (VLPs) and metagenomic sequencing for host-cell, we profiled comprehensive virome datasets (recovered 16,672 viral contigs) from sites contaminated for over 30 years with chlorinated hydrocarbons and benzene, toluene, ethylbenzene, and xylene (BTEX). We found that contaminated groundwater had higher viral abundance, higher diversity, a broader host spectrum, and more complex virus-host interactions than the control group. Through virus genome functional screening and global multi-habitat mapping analysis, we identified six AMGs involved in alleviating pollutant stress and degradation of xenobiotics, and verified the degradation activity of the AMGs (i.e., L-DEX) through heterologous gene expression experiments, revealing that virus-encoded AMGs have cross-habitat adaptive potential. For site-specific pollutants, further phylogenetic analysis and protein structure prediction of key metabolic genes L-DEX and pcaH were performed. The results showed that they exhibit high evolutionary conservation and functional integrity, suggesting the similar functions as bacterial homologous genes. Overall, these findings enhance our understanding of virus-host interactions in contaminated groundwater and reveal their ecological functions and roles in adaptive evolution.