Outer membrane vesicle production by Escherichia coli is independent of membrane instability

Abstract

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 sigma(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.

FIG. 1.

Representative data from the screen for vesiculation mutants. (A) Immunoblot with anti-LPS antibody of cell-free supernatants from cultures of insertion mutants. Representative high-vesiculation (circle) and low-vesiculation (square) mutant phenotypes are shown. Control wild-type (+) and washed wild-type (−) samples are at bottom right (rectangle). (B) Coomassie blue-stained SDS-PAGE of pelleted vesicles from cultures of wild-type DH5α (WT) and five Tn mutants demonstrates high vesiculation (mutants 2, 4, and 5) and low vesiculation (mutant 3) phenotypes. Molecular weight standard proteins (kDa) and the positions of Omps F, C, and A are labeled.

FIG. 2.

Relative increase (n-fold) in vesicle production of Tn mutants. Vesicle-overproducing mutants (A) and vesicle-underproducing mutants (B) are identified by protein- and lipid-based methods. Relative vesicle production/CFU compared to wild-type strain DH5α was calculated using Omp bands (black bars) or incorporation of the lipid probe FM4-64 (gray bars). Values are the average ± SEM (n ≥ 2).

FIG. 3.

Vesicle overproduction does not require increased Omp expression. CFP fluorescence measurements corresponding to ompC expression (relative fluorescence units [RFU]) for transductants containing vesicle overproduction mutations from MK7B29 (yieM::Tn5), MK11F26 (degP::Tn5), MK8A44 (nlpI::Tn5), and MK5B7 (rseA::Tn5). Values were corrected for culture optical density at an OD₆₀₀ of 0.4 and normalized to wild-type MDG131. Values are the average ± SEM (n ≥ 2).

FIG. 4.

Vesicles produced by strains with mutations in σ^E response pathway genes are released intact. (A) Electron micrographs of vesicles from the wild type and mutant strains MK6D31, MK5B7, and MK11F26. Size bar, 100 nm. (B) Equilibrium density gradients of vesicles from the wild type and mutant strains (listed in the panel A legend). Periplasm from DH5α was applied to the same gradient (bottom panel). Fraction 1 is the least dense; fraction 10 is the most dense. Prominent vesicle protein bands are visible in light fractions by Coomassie blue (CB)-stained SDS-PAGE; MBP was visualized by immunoblotting (α-MBP). rseA::Tn5 samples were concentrated by precipitation with 20% trichloroacetic acid.