Outer-Membrane Vesicles Released by Normally Growing Escherichia coli Contain Very Little Lipoprotein

Abstract

The lipoprotein content of the outer-membrane medium vesicles, which are released from Escherichia coli during normal growth, was compared to the lipoprotein content of the corresponding cellular outer membranes.It was found that the medium vesicles contained only 35% free lipoprotein and almost none of the bound lipoprotein when compared with cellular outer membranes. Medium vesicles also had reduced amounts of protein 11* and a protein V ( M , = 16000), while they contained large amounts of pore-forming proteins I and 1amB.A mechanism is proposed in which outer membrane vesicles are formed when the outer membrane expands faster than the underlying peptidoglycan layer. The lack or enrichment of individual proteins in medium vesicles may be determined by their interactions with the peptidoglycan-bound lipoprotein complex.The cell wall of gram-negative bacteria consists of a peptidoglycan layer and an outer membrane. These two layers are linked covalently via the bound form of the outer membrane lipoprotein [I -31.The lipoprotein is the most abundant protein of Escherichiu coli and many other gram-negative bacteria; there are of the order of 40000 such linkages per pm2 of bacterial cell surface, implying that the distance between neighbouring covalent murein-outer membrane connections averages S nm, which is less than a single membrane thickness [I].Despite this intimate and covalent contact between the outer membrane and the peptidoglycan layer, E. coli and other gram-negative bacteria release a significant portion of their outer membrane into the medium in the form of membrane vesicles during normal growth [4-81.Such vesicles have been observed under the electron microscope as outer-membrane 'blebs' that are released from the cell during the initial stages of septation [9,10]. Release of these vesicles is enhanced when protein synthesis is slowed down, for instance by growing a lysine auxotroph under lysine-limiting conditions or by growing E. coli in the presence of chloramphenicol [I 1,121. Adsorption of phage T4 to E. coli also results in a more pronounced release of outer-membrane vesicles into the medium [6,13].Extensive bleb formation has also been observed when a lipoprotein-lacking mutant was grown under conditions of magnesium starvation [14,15], while a mutant lacking the ompA protein a5 well as the lipoprotein showed extensive blebbing even in the presence of normal magnesium concentrations [16]. Comparable observations have been made in Salmonella typhimurium lkyD mutants which have a decreased amount of murein-bound lipoprotein [17-191. We have previously considered the possibility that, given the high average density of covalent lipoprotein-murein linkages in normal E. coli, outer-membrane vesicles may well be derived from envelope areas which are relatively poor in lipoprotein [4] ; the fact that lipoprotein-lacking mutants h z y m e . Lysozyme (EC 3.2.1.17) release considerably more outer membrane material than do normal cells supports this hypothesis.If the lack of (bound) lipoprotein in cert...

Deacylation of Lipopolysaccharide in Whole Escherichia coli during Destruction by Cellular and Extracellular Components of a Rabbit Peritoneal Inflammatory Exudate

“…3A), despite the ability of ascitic fluid to degrade purified LPS (5). LPS still associated with bacterial remnants or with bacterial outer membrane proteins or phospholipids (23)(24)(25) may be less subject to deacylation than purified LPS. Alternatively, because the dose of whole bacteria contained 5-10-fold more LPS than was presented previously in purified form (5), the deacylating capacity of AF may have been exceeded in the current experiments with whole bacteria.…”

Strong Decrease in Invasive Ability and Outer Membrane Vesicle Release in Crohn's Disease-Associated Adherent-Invasive Escherichia coli Strain LF82 with the yfgL Gene Deleted

“…OMVs arise from the surfaces of gram-negative bacteria and consist of outer membrane and entrapped periplasmic components. Two different mechanisms for the generation of OMVs have been proposed; one proposal is that OMVs are formed when the outer membrane expands faster than the underlying peptidoglycan layer (43) and another proposal is that the formation of OMVs is linked to turgor pressure of the cell envelope during bacterial growth (44). In the latter, the cell wall is excised and released from the peptidoglycan by lytic transglycosylases, and the accumulation of released muramyl peptides in the periplasmic space creates turgor pressure on the outer membrane and triggers OMV blebbing.…”

Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

“…Calculations of native OM vesicle production show that the vesicles represent a significant fraction of cellular material. For instance, vesicles produced by typical laboratory cultures of growing and dividing Pseudomonas aeruginosa and Escherichia coli cells account for ϳ1% of the OM material in the culture (8,43,139). In contrast, Neisseria meningitidis produces abundant numbers of vesicles, constituting 8 to 12% of radiolabeled protein and endotoxin in logphase cultures (24).…”