Enrichment of Horizontally Transferred Gene Clusters in Bacterial Extracellular Vesicles via Non-Lytic Mechanisms

Bacterial extracellular vesicles are emerging as key mediators of horizontal gene transfer, enhancing microbial adaptability. A critical factor determining the effectiveness of horizontal gene transfer is the fraction of vesicles containing specific functional genes. However, the proportion of containing specific DNA fragments has not been adequately determined, which hinders the understanding of the conditions and mechanisms that facilitate the incorporation of specific genes into the vesicles and possible evolutionary roles of vesicle-derived DNA. Here, we demonstrate that enrichment of horizontally transferred genes into bacterial extracellular vesicles is driven by cellular processes by profiling the DNA content of hundreds of individual vesicles using a microdroplet-based sequencing technique. This approach revealed unique DNA profiles in vesicles from the oral pathogen Porphyromonas gingivalis, pinpointing genomic regions related to DNA reorganization such as CRISPR-Cas clusters. Comparative genomic and phylogenetic analyses of Porphyromonas genomes revealed traces of horizontal gene transfer in vesicle-enriched genes. Modulating vesicle-biogenesis routes, quantitative real-time PCR revealed that this selective enrichment was driven by blebbing-driven DNA packaging mechanisms rather than stress-induced lysis. Applied to dental plaque-derived bacterial extracellular vesicles, the droplet-based approach reveled O-antigen biosynthetic genes, key for host-bacterial interactions, were prevalent in the vesicles from Alcaligenes faecalis, suggesting the vesicles from this bacterium can modulate pathogenicity in oral biofilms through targeted DNA packaging. These findings suggest the prevalence of functionally relevant gene clusters in bacterial extracellular vesicles in oral microbiota and their evolutionary roles as DNA cargoes for modulating phage-bacterial and host-bacterial interactions via horizontal gene transfer.