Molecular structure of membrane vesicles from Escherichia coli

Abstract

The molecular architecture of membrane vesicles prepared from Eseherichia coli ML 308-225 has been studied by using crossed immunoelectrophoresis, and a reference pattern of 52 discrete immunoprecipitates has been established. Progressive immunoadsorption experiments conducted with untreated control vesicles and with physically disrupted vesicles demonstrate that the membrane-associated immunogens fall into two categories: (i) those immunogens typified by ATPase (ATP phosphohydrolasei EC 3.6.1.3) and NADH dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3] whose expression is minimal unless the vesicles are disrupted; and (ih) immunogens such as Braun's lipoprotein that are expressed to similar extents in untreated an in isrted vesicles. A mathematical relationship between thepeak area subtended by an immunoprecipitate in the crossed immunelectrophoresis system and the quantity of vesicles used in the adsorption process has been derived. This relationship allows quantitation of the degree to which specific membrane immunogens partition between exposed and unexposed surfaces of the vesicle membrane. The results demonstrate conclusively that >95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same polarity as the intact cell.Moreover, the findings provide a strong indication that dislocation of immunogens from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 11%. Topologically sealed cytoplasmic membrane vesicles have provided a useful model system for the study of certain transport phenomena in bacteria (1-3). These vesicles are essentially devoid of cytoplasmic constituents and catalyze active transport by a respiration-dependent mechanism that does not involve the generation or utilization of ATP or other high-energy phosphate compounds. In addition, recent studies with this system (3-8) provide strong confirmation of the hypothesis that chemosmotic phenomena (9-12) play a central, obligatory role in this process.Despite a considerable body of evidence (see ref. 13 for a review) demonstrating that bacterial membrane vesicles prepared by osmotic lysis (14, 15) retain the same polarity as the intact cell, doubts apparently exist regarding the molecular structure of these vesicles. It has been argued, for example, that a significant percentage of these vesicles are everted (i.e., inside-out) and thus have a polarity that is opposite to that of the intact cell (16,17). Alternatively, it has been suggested that, during vesicle preparation, certain membrane proteins become detached from the inner surface of the membrane and are readsorbed onto the outer surface (18)(19)(20)(21)(22). This dislocation process, it is proposed, results in vesicles that are "mosaics" in the sense that some membrane proteins are located on both sides of the membrane in a nonrandom fashion (21,22). Much of the data on which these conclusions are based are derived from the use of two broad experimental approaches: (i) comp...

Type V Protein Secretion Pathway: the Autotransporter Story

“…7). It was in one such study of the E. coli cell envelope that the protein antigen 43 (Ag43) was discovered (371,372). The terminology for this protein arises from the fact that antigen 43 was the 43rd antigen in the crossed immunoelectrophoresis profile of membrane vesicles prepared from E. coli (Fig.…”

Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli

“…Although the available data are insufficient to establish a close evolutionary relationship between antigen 43 and fimbriae, it seems not inconceivable that antigen 43 functions as an adhesin. This possibility is now being investigated.…”

“…By use of subunit-specific antisera, it was shown that the purified a subunit was capable of reassociating with the 0i polypeptide. However, electron microscopic examination indicated that antigen 43 does not form a recognizable surface structure. The available evidence supports the view that antigen 43 is a complex consisting of a peripheral membrane protein (at) anchored to a subunit (Ii) that is integral to the outer membrane.…”

“…Extensive immunochemical studies have established a reference profile for Escherichia coli ML308-225 envelope components resolved by crossed immunoelectrophoresis (CIE; [37][38][39][40]43). Antigen 43 is often prominent in the array of precipitin arcs generated when outer membranes are analyzed by this technique.…”

Transport of multiple Escherichia coli strains in saturated porous media

“…Three decades ago, a major outer membrane protein termed Antigen 43 (Ag43) was discovered in Escherichia coli (E. coli) (Das Gracas de Luna et al, 2008;Owen and Kaback, 1978). Outer membrane proteins serve a variety of functions essential to survival of Gram-negative bacteria.…”