Electron Microscopic Observation and Ultrastructural Analysis of Outer Membrane Vesicles Released by the Endophyte within Azolla microphylla

Previous studies have revealed that the free-living or purely cultured prokaryotes produce outer membrane vesicles (OMVs) which play a critical role in biological processes. In this study, the OMVs secreted by the symbiotic cyanobacterium and other endophytic bacteria (together referred to as endophyte) within a small water fern, Azolla-cyanobacterium association, were investigated by using scanning and transmission electron microscopies (EM). The results demonstrated that the vesiculation of both the cyanobacterium and the bacteria occurs not only at the stage of active vegetative growth, but their reproductive stages and even in dying process. Based on the ultrastructural characteristics, the OMVs discovered in the materials we used could be divided to five types:bilayer-couple with a central transparent area, outer-inner membranes, bilayer-couple with a core, bilayer-couple with multi-cores and bilayer-couple with multi-bulbs. Analyzing the changes of the cellular ultrastructure has led to a suggestion that peptidoglycan in periplasmic space is involved in formation and discharge of OMVs, which seems to have a universal significance to illuminate the mechanism for biogenesis of OMVs. In this paper, the interpretation of the EM images is also discussed, so as to avoid the misinterpretation caused by the artifacts due to the sample pretreatment.