Interaction between Major Outer Membrane Protein (O‐8) and Lipopolysaccharide in <i>Escherichia coli</i> K12

Yamada, H.; Mizushima, Shôji

doi:10.1111/j.1432-1033.1980.tb04305.x

Cited by 60 publications

(51 citation statements)

References 39 publications

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“…Second, the LPS of the mutant Z61 reacted differently with the lipoprotein I as seen on polyacrylamide gels. An interaction of LPS with porin and a modulation of the conformation of porin by LPS has been demonstrated in E. coli by Yamada and Mizushima, using X-ray diffraction as a tool (39). Both the lipid and polysaccharide regions of LPS were thought to interact with the protein to determine the lattice conformation.…”

Section: Discussionmentioning

confidence: 99%

Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa

Kropinski¹,

Kuzio²,

Angus³

et al. 1982

Antimicrob Agents Chemother

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Lipopolysaccharides extracted from Pseudomonas aeruginosa strain K799 and its antibiotic-supersusceptible derivative Z61 were analyzed chemically and chromatographically. The side-chain polysaccharides purified by gel exclusion chromatography were compositionally identical, being composed of fucosamine (2-amino-2,6-dideoxygalactose), quinovosamine (2-amino-2,6-dideoxyglucose), and an unidentified amino sugar. In addition, low amounts of the core-specific components (glucose, rhamnose, alanine, and galactosamine) were found associated with the side chains from both strains. In the preceding paper (1), we provided evidence that the basis of the generally enhanced susceptibility to antibiotics of mutant Z61, when compared with its progenitor strain K799, was a more permeable outer membrane. In particular, the outer membrane of strain Z61 was more permeable to the hydrophilic antibiotic nitrocefin (1) and to other P-lactams (41), despite an apparently unaltered porin protein F, an outer membrane protein shown to form water-filled channels in model membrane systems (2a, 14). Indirect evidence was provided that mutant Z61 had an unspecified lipopolysaccharide (LPS) alteration (1). To gain further information regarding the mutation present in strain Z61, we characterized the LPS alteration in greater depth. Due to the rather complex method used to isolate mutant Z61 (41), we prepared a series of spontaneous revertants (1). Full revertants were isolated which were indistinguishable from the wild-type strain K799 in a wide variety of properties (1), including some LPS properties (see below). This suggests that Z61 and K799 differ by a single major mutation which is responsible for the antibiotic susceptibility and permeability differences between the strains as well as the

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Section: Discussionmentioning

confidence: 99%

Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa

Kropinski¹,

Kuzio²,

Angus³

et al. 1982

Antimicrob Agents Chemother

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“…They have similar molecular weights and amino acid sequences (22), the native proteins are very high in beta structure (25,32), both are tightly associated with the peptidoglycan (9,19,32), and they are very tightly associated with lipopolysaccharide (LPS) (10,12,36,40). Porins are known to form water-filled channels (2,33) and are unusually stable to a wide variety of perturbants such as sodium dodecyl sulfate (SDS) detergent (24,25,41), pH extremes from 2 to 12 (8), and temperatures up to 70°C (32).…”

mentioning

confidence: 99%

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12

Rocque¹,

Coughlin²,

McGroarty³

1987

J Bacteriol

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Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100°C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydratespecific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not aHl of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.The structure and interactions of the outer membrane porin proteins OmpF and OmpC from Escherichia coli are very similar. They have similar molecular weights and amino acid sequences (22), the native proteins are very high in beta structure (25, 32), both are tightly associated with the peptidoglycan (9, 19, 32), and they are very tightly associated with lipopolysaccharide (LPS) (10,12,36,40). Porins are known to form water-filled channels (2, 33) and are unusually stable to a wide variety of perturbants such as sodium dodecyl sulfate (SDS) detergent (24, 25, 41), pH extremes from 2 to 12 (8), and temperatures up to 70°C (32). Porins are also resistant to many proteases, perhaps because of their tight association with LPS (12); the proteins are thought to be functional only in the presence of LPS (12,20,33). Nakae et al. (24) have suggested that only the lipid component of LPS is essential for porin function. In contrast, Parr and co-workers (30) have reported that porin is functional in the absence of LPS. Thus, the importance of bound LPS for porin structure and function has yet to be resolved.The OmpF porin exists as a trimeric complex (24, 29) with three identical subunits with approximate molecular weights of 36,000 (13, 32). Low SDS binding coupled with the tight, compact shape of the trimer results in more rapid migration on polyacrylamide gels than the aggregate molecular weight would suggest. Investigator...

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“…We assume that it was this low LPS content which led to the strong tendency of the preparation to form clusters and regular 2D arrays. Yamada and Mizushima (50) showed that at least 1 LPS molecule per porin trimer was required for the formation of 2D crystalline arrays. The formation of crystals with p22 1 2 symmetry and alternating orientations of the porin trimers is reminiscent of three-dimensional crystals of other porins, for which alternating orientations of neighboring porin trimers also occurred along a certain crystal plane (12).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a porin from the outer membrane of Vibrio anguillarum

1996

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The outer membranes of the 10 serovars of Vibrio anguillarum showed a common major protein with a size of around 40 kDa. Antibodies against the major outer membrane protein (MOMP) of V. anguillarum AO18 (serovar O1) cross-reacted with the MOMPs of all the other serovars but not with the outer membrane proteins of Escherichia coli. The MOMP of V. anguillarum serovar O1 was isolated, reconstituted to two-dimensional crystals, and structurally characterized by electron microscopy and image processing. The unit cell structure of the crystalline MOMP, as well as the secondary structure composition of the protein with a high amount of ␤-structure, is strongly reminiscent of that of bacterial porins. The functional properties of the pores were investigated by conductance measurements with the MOMP reconstituted in planar lipid membranes. The V. anguillarum MOMP is characterized by a relatively weak cation selectivity and a moderate surface charge, and it shows voltage-dependent conductance effects. The MOMP is functionally similar to OmpF from E. coli, and it can be classified as a general diffusion porin.Vibrio anguillarum, a gram-negative bacterium, is the etiological agent of vibriosis, one of the most serious infection diseases of salmonid and marine fish species throughout the world (2). The diagnosis and control of V. anguillarum infections are of great economical importance in commercial fish farming.Since the first descriptions of V. anguillarum infections, many studies on the immunological characterization of this species and its pathogenicity have been performed. The molecular basis of these studies is closely related to the composition and function of the outer membrane (OM). Most studies of the V. anguillarum OM have been focused on the lipopolysaccharides (LPS) which are the major agents for antigenetic specificity (11). Several serotyping systems based on the slide agglutination method with thermostable O-antigens were described independently (29, 47). These serovars are considered an important epizootical characteristic. The three main serovars O1, O2, and O3 (according to the classification of Sør-ensen and Larsen [47]) include most of the strains isolated from fish infections. The remaining serovars are more commonly isolated from the natural environment and other aquatic species (47). Nevertheless, there is also an increasing interest in the OM proteins of V. anguillarum which may participate in mechanisms of pathogenicity directly. So, special attention has been paid the OM proteins involved in siderophore-dependent iron uptake (13,34). Several studies analyzed and compared the compositions of OM proteins from different serovars (1, 40). They describe the presence of usually one major OM protein (MOMP) in the molecular size range of about 30 to 40 kDa in all the strains tested. Recently, Suzuki et al. (48) suggested that this MOMP represents a porin because of its molecular weight.In the present work, we have studied the OM protein profiles of different serovars of V. anguillarum. The MOMP of one str...

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Interaction between Major Outer Membrane Protein (O‐8) and Lipopolysaccharide in Escherichia coli K12

Cited by 60 publications

References 39 publications

Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa

Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12

Characterization of a porin from the outer membrane of Vibrio anguillarum

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