SummaryConditions that impair protein folding in the Gramnegative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.

It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over-or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the E cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.Release of outer membrane (OM) vesicles has been observed for all gram-negative bacteria studied to date (reviewed in references 5, 15, and 16). Native vesicles are rounded structures with lumenal periplasmic components bounded by an outer layer of outer membrane proteins (Omps) and lipids (16). For Escherichia coli, our laboratory has reported that strain DH5␣ releases 0.23% of Omps F and C and 0.14% of OmpA into vesicles (12); other investigators have found vesicles to account for 0.2 to 0.5% of bacterial culture material (9,20). Electron microscopy studies reveal bulging of the OM and subsequent fission of vesicles containing electron-dense material (16). These biochemical and microscopic observations suggest that OM vesicles are formed from protrusions that are pinched off from the OM in a manner that leads to the inclusion of periplasmic material.The wide variety of strains and diversity of environments for which vesiculation has been observed suggest an important role for vesicle production in gram-negative bacterial growth and survival (5,15,16). Vesicle production varies with growth phase and nutrient availability, and vesicle-associated enzymes may aid in nutrient scavenging. Vesicle-mediated transfer of toxic components to other bacteria can eliminate competing species. In addition, interactions between eukaryotic cells and vesicles from pathogenic bacteria suggest a role for vesicles in pathogenesis (13).We have conducted a screen to generate and identify mutants in E. coli that exhibit altered vesiculation levels. Both vesicle-overproducing and -underproducing mutants are of interest, ...

Significant duration-dependent reductions in ovarian cancer incidence in the general population are associated with OCP use.