Novosphingobium sp. PP1Y as a novel source of outer membrane vesicles

Outer membrane vesicles (OMVs) are nanostructures of 20– 200 nm diameter deriving from the surface of several Gram- negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and charac- terized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on sam- ples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular ma- trix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gra- dient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, pro- teomic and fatty acid analysis of purified OMVs revealed a specific biochemical “fingerprint”, suggesting interesting de- tails concerning their biogenesis and physiological role. More- over, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.