Outer Membrane Vesicles, Lipidome, and Biofilm Formation in the Endophyte Enterobacter Cloacae SEA01 from Agave Tequilana

Bacterial outer-membrane vesicles (OMVs) mediate stress tolerance, biofilm formation, and interkingdom communication, but their role in beneficial endophytes remains underexplored. We isolated 11 non-redundant isolates associated with <i>Bacillus</i>, <i>Enterococcus</i>, <i>Kosakonia</i> and <i>Kocuria</i> from <i>Agave tequilana</i> seeds, identified by MALDI-TOF MS and 16S rRNA gene sequencing. We focused on the catalase-negative <i>Enterobacter cloacae</i> SEA01, which exhibits plant-promoting traits and support agave growth under nutrient-poor microcosms. In addition, this endophyte produces OMVs. Time-resolved SEM documented OMV release and cell aggregation within 9 h, followed by mature biofilms at 24 h with continued vesiculation. Purified OMVs (≈80-300 nm) contained extracellular DNA and were characterized by dynamic light scattering and UHPLC-ESI-QTOF-MS lipidomics. The OMV lipidome was dominated by phosphatidylethanolamine (~80%) and was enriched in monounsaturated fatty acids (16:1, 18:1), while the stress-associated cyclopropane fatty acids (17:1, 19:1) were comparatively retained in the whole-cell membranes; OMVs also exhibited reduced ubiquinone-8. SEA01 is catalase-negative, uncommon among plant-associated <i>Enterobacter</i>, suggesting a testable model in which oxidative factors modulate OMV output and biofilm assembly. These may have implications for recognition and redox signaling at the root interface. Future works should combine targeted proteomics/genomics with genetic or chemical disruption of catalase/OMV pathways.