| Summary: | Summary Gram‐positive bacterial extracellular membrane vesicles (EVs) have been drawing more attention in recent years. However, mechanistic insights are still lacking on how EVs are released through the cell walls in Gram‐positive bacteria. In this study, we characterized underlying mechanisms of EV production and provide evidence for a role of prophage activation in EV release using the Gram‐positive bacterium Lactococcus lactis as a model. By applying a standard EV isolation procedure, we observed the presence of EVs in the culture supernatant of a lysogenic L. lactis strain FM‐YL11, for which the prophage‐inducing condition led to an over 10‐fold increase in EV production in comparison with the non‐inducing condition. In contrast, the prophage‐encoded holin–lysin knockout mutant YL11ΔHLH and the prophage‐cured mutant FM‐YL12 produced constantly low levels of EVs. Under the prophage‐inducing condition, FM‐YL11 did not show massive cell lysis. Defective phage particles were found to be released in and associated with holin–lysin‐induced EVs from FM‐YL11, as demonstrated by transmission electron microscopic images, flow cytometry and proteomics analysis. Findings from this study further generalized the EV‐producing phenotype to Gram‐positive L. lactis, and provide additional insights into the EV production mechanism involving prophage‐encoded holin–lysin system. The knowledge on bacterial EV production can be applied to all Gram‐positive bacteria and other lactic acid bacteria with important roles in fermentations and probiotic formulations, to enable desired release and delivery of cellular components with nutritional values or probiotic effects.
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