A distinct subpopulation of membrane vesicles in Pseudomonas putida is enriched in enzymes for lignin catabolism.

Bacterial membrane vesicles (MVs) mediate diverse microbial processes and are emerging as powerful biomedical tools, but MV population heterogeneity remains an open question. Here, we separate, enumerate, and characterize two MV populations from the soil bacterium Pseudomonas putida during growth with or without lignin-derived carbon, a major carbon source from plant cells in the rhizosphere. Small MVs (MV-S, diameter ~100 nm) were produced from all cultures, whereas large MVs (MV-L, diameter ~300 nm) were observed during the late stationary phase of lignin cultivations. MV-S contained selectively packaged proteins with diverse physiological functions, whereas the MV-L proteome was smaller and largely enriched in outer membrane proteins. Interestingly, enzymes known to mediate the catabolism of lignin-derived aromatic compounds were enriched in MV-S. Overall, this study highlights the need for careful consideration of MV populations in microbial systems.IMPORTANCEMembrane vesicles (MVs) are extracellular lipid bodies that can be generated by single-cell microbes and contain biological cargo. Since their discovery, MVs have been shown to exhibit multiple functions, including nutrient acquisition, pathogenesis, and signaling. In soil, the breakdown of plants releases aromatic compounds from lignin, and it has been previously shown that a model bacterium that consumes aromatic compounds forms MVs with enzymes responsible for the consumption of aromatic compounds. Intriguingly, a small population and a large population of MVs were observed, and it was not known if they served the same or different functions. Here, MVs isolated from bacterial growth experiments on lignin were fractionated and characterized, revealing that distinct MV populations have distinct cargo and, thus, distinct functions.