Extracellular vesicles are the main contributor to the non-viral protected extracellular sequence space

Marine environmental virus metagenomes, commonly referred to as ‘viromes’, are typically generated by physically separating virus-like particles (VLPs) from the microbial fraction based on their size and mass. However, most methods used to purify VLPs, enrich extracellular vesicles (EVs) and gene transfer agents (GTAs) simultaneously. Consequently, the sequence space traditionally referred to as a ‘virome’ contains host-associated sequences, transported via EVs or GTAs.

Researchers at the Max Planck Institute for Marine Microbiology propose to call the genetic material isolated from size-fractionated (0.22 um) and DNase-treated samples protected environmental DNA (peDNA). This sequence space contains viral genomes, DNA transduced by viruses and DNA transported in EVs and GTAs. Since there is no genetic signature for peDNA transported in EVs, GTAs and virus particles, the researchers rely on the successful removal of contaminating remaining cellular and free DNA when analyzing peDNA. Using marine samples collected from the North Sea, they generated a thoroughly purified peDNA dataset and developed a bioinformatic pipeline to determine the potential origin of the purified DNA. This pipeline was applied to their dataset as well as existing global marine ‘viromes’. Through this pipeline, the researchers identified known GTA and EV producers, as well as organisms with actively transducing proviruses as the source of the peDNA, thus confirming the reliability of their approach. Additionally, they identified novel and widespread EV producers, and found quantitative evidence suggesting that EV-mediated gene transfer plays a significant role in driving horizontal gene transfer (HGT) in the world’s oceans.

Conceptual composition of protected extracellular DNA

The top panel depicts microbial entities present in a water body: microbial cells, viruses containing viral and microbial genetic material, gene transfer agents and extracellular vesicles containing host DNA. After size filtration (0.22 μm) and DNase treatment and, if applicable, purification via density gradients, microbial cells and free DNA are removed (middle panel). The remaining DNA makes up the sequence space of protected extracellular DNA, peDNA (bottom panel).