Biogenesis of Outer Membrane Vesicles in Serratia marcescens Is Thermoregulated and Can Be Induced by Activation of the Rcs

Outer membrane vesicles (OMVs) have been identified in a wide range of bacteria, yet little is known of their biogenesis. It has been proposed that OMVs can act as long-range toxin delivery vectors and as a novel stress response. We have found that the formation of OMVs in the Gram-negative opportunistic pathogen Serratia marcescens is thermoregulated, with a significant amount of OMVs produced at 22 or 30°C and negligible quantities formed at 37°C under laboratory conditions. Inactivation of the synthesis of the enterobacterial common antigen (ECA) resulted in a hypervesiculation phenotype, supporting the hypothe-sis that OMVs are produced in response to stress. We demonstrate that the phenotype can be reversed to wild-type (WT) levels upon the loss of the Rcs phosphorelay response regulator RcsB, but not RcsA, suggesting a role for the Rcs phosphorelay in the production of OMVs. MS fingerprinting of the OMVs provided evidence of cargo selection within wild-type cells, suggesting a possible role for SerratiaOMVs in toxin delivery. In addition, OMV-associated cargo proved toxic upon injection into the hae-mocoel ofGalleria mellonella larvae. These experiments demonstrate that OMVs are the result of a regulated process in Serratia and suggest that OMVs could play a role in virulence. Gram-negative bacteria, both pathogenic and nonpathogenic,release lipid bilayer vesicles from the outer membrane (OM) (3, 34). These outer membrane vesicles (OMVs), like the outer membrane from which they are generated, are composed of lipo-polysaccharide (LPS), phospholipids, and outer membrane pro