Outer membrane vesicles (OMVs), produced by nonpathogenic Gram-negative bacteria, have potentially useful biotechnological applications in extraterrestrial extreme environments. However, their biological effects under the impact of various stressors have to be elucidated for safety reasons. In the spaceflight experiment, model biofilm kombucha microbial community (KMC) samples, in which Komagataeibacter intermedius was a dominant community-member, were exposed under simulated Martian factors (i.e., pressure, atmosphere, and UV-illumination) outside the International Space Station (ISS) for 1.5 years. In this study, we have determined that OMVs from post-flight K. intermedius displayed changes in membrane composition, depending on the location of the samples and some other factors. Membrane lipids such as sterols, fatty acids (FAs), and phospholipids (PLs) were modulated under the Mars-like stressors, and saturated FAs, as well as both short-chain saturated and trans FAs, appeared in the membranes of OMVs shed by both post-UV-illuminated and "dark" bacteria. The relative content of zwitterionic and anionic PLs changed, producing a change in surface properties of outer membranes, thereby resulting in a loss of interaction capability with polynucleotides. The changed composition of membranes promoted a bigger OMV size, which correlated with changes of OMV fitness. Biochemical characterization of the membrane-associated enzymes revealed an increase in their activity (DNAse, dehydrogenase) compared to wild type. Other functional membraneassociated capabilities of OMVs (e.g., proton accumulation, interaction with linear DNA, or synaptosomes) were also altered after exposure to the spaceflight stressors. Despite alterations in membranes, vesicles did not acquire endotoxicity, cytotoxicity, and neurotoxicity.
