Membrane Vesicle-Mediated Delivery of Antibacterial Lipopeptides by <i>Pseudoalteromonas piscicida</i>

Bacterial membrane vesicles (MVs) are natural delivery systems for biomolecules, such as enzymes and nucleic acids, but their role in transporting specialized metabolites is less understood. Many microbial metabolites are lipophilic and poorly water-soluble, raising questions about how they perform ecological functions in aquatic environments. Here, we demonstrate that <i>Pseudoalteromonas piscicida</i> JC3, a marine bacterium with probiotic potential, packages lipophilic depsipeptides known as bromoalterochromides (BACs) into outer membrane vesicles. Untargeted metabolomics and molecular networking identified six known and two previously unknown BACs, while targeted LC-MS/MS localized BACs to MVs and cells, with no detection in culture supernatants. Structure elucidation of a new analogue, bromoalterochromide E/E', was achieved through isolation and spectroscopic analysis, including modified Marfey's analysis to determine amino acid composition and chirality. Functional assays showed that BAC-loaded MVs exhibit antibacterial activity against <i>Staphylococcus aureus</i> and the marine pathogen <i>Vibrio anguillarum</i>, linking vesicle-mediated metabolite delivery to microbial competition. These findings highlight MVs as transporters of lipophilic natural products and suggest their potential as natural drug delivery vehicles in clinical and aquaculture settings.