Prophage diversity and metabolic signatures of viral induction in groundwater bacterial isolates

Viral lysis of bacterial hosts results in the release of metabolites and cell lysates that promote energy and material fluxes (viral shunt). Viruses can lyse their host immediately after infection (lytic cycle), or replicate their genome alongside the host genome (lysogenic cycle). The integrated viral genome (i.e. prophage) can be induced to enter the lytic cycle under certain conditions (e.g. environmental stress). While much is known about these processes in marine systems, the role of prophage diversity and virus-driven cell lysis in terrestrial food webs, particularly in groundwater environments, remains less understood.

Our study examines the effects of prophage-host interactions in groundwater, focusing on the release of dissolved organic matter (DOM) following prophage induction and identifying factors that influence prophage distribution and diversity. We used mitomycin C to induce prophages in 146 bacterial isolates from groundwater. Of these, 37 strains had inducible prophages and underwent further analysis. The genomes of inducible strains were sequenced using nanopore technology, while induced phages were quantified by fluorescence microscopy, and the DOM released during induction was analyzed by direct injection mass spectrometry.

We identified 120 prophages across the 37 inducible strains representing 15 bacterial clusters and 58 unique viral clusters. Notably, isolates within the same bacterial cluster did not consistently share phage clusters, suggesting a weak correlation between bacterial relatedness and prophage composition. Phage diversity varied widely, with a positive correlation between the total phage count and phage diversity per isolate. Mass spectrometric analysis of DOM showed consistent metabolic responses across many isolates after induction, revealing 993 distinct molecular species with significant changes. Approximately 39% of these changes were linked to species-specific factors, while 3% were attributable directly to phage induction, highlighting the combined influence of species identity and viral induction on the metabolic landscape. This pool of molecules provides a basis for identifying potential biomarkers of viral lysis and for advancing our understanding of the role of viruses in nutrient cycling and ecosystem dynamics in groundwater environments.