Reconstitution of Model Membranes from Phospholipid and Outer Membrane Proteins of <i>Proteus mirabilis</i>

Nixdorff, Kathryn; Fitzer, Hildegard; Gmeiner, Jobst; Martin, Hans Herbert

doi:10.1111/j.1432-1033.1977.tb11927.x

Cited by 57 publications

(36 citation statements)

References 23 publications

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“…Several procedures were compared for the efficient and reproducible isolation of purified cell walls. The first procedure, a modification of one described by Nixdorff et al (1977), was used for the routine isolation of cell walls from P . syringue.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Partial Characterization of the Cell Wall of Pseudomonas syringae pv. syringae HS191: Comparison of Outer Membrane Proteins of HS191 with Those of Two Plasmidless Derivatives

Hurlbert

Gross

1983

Microbiology

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The outer membrane (OM) proteins of a Pseudomonas syringae pv. syringae strain (HS191), capable of causing holcus leaf spot of corn and other grasses, and two plasmidless derivatives of HS191, one avirulent (AO111) and one with reduced virulence (PSGlOO), were isolated by selective solubilization of the cytoplasmic membrane (CM) with 0.4% (w/v) SDS or by centrifugation on sucrose density gradients. OM preparations were enriched in hexosamine and 2-keto-3-deoxyoctonate and contained little of the CM enzymes NADH oxidase and malate dehydrogenase. Characterization of OM preparations by SDS-PAGE and isoelectric focusing indicated a minimum of 20 OM proteins; protein bands 8 and 9 quantitatively predominated. In addition, some of the proteins were modified by heat and others by 2-mercaptoethanol. Although protein 5 was absent in both plasmidless strains, A01 11 differed from PSG100 in lacking protein 5a and having two proteins (8b' and 8c') with different PI values. It is suggested that plasmid pCG 13 1 coded for protein 5 (mol. wt 68 000) and repressed the synthesis of proteins 4a (78000) and 5a (63000). The relationship of specific changes in OM protein composition to virulence is discussed. I NT,R 0 DUCT I 0 N Pseudomonas syringae pv. syringae is a plant-associated bacterium which, subject to environmental conditions and bacterial-plant compatibility, can establish diseases in at least 40 different plant genera (Stapp, 1961). Compatibility generally limits individual P. syringae strains to a few specific hosts. A number of investigators have proposed that the basic molecular mechanism(s) responsible for specificity in plant-bacterial disease relationships involves recognition of molecules present at the cell surfaces of the two organisms (Sequeira, 1978). Attention has centred on the involvement of bacterial surface components, such as capsule and lipopolysaccharide (LPS), in host-pathogen recognition phenomena and virulence. Comparatively little is known concerning the potential involvement of outer membrane (OM) proteins in bacterial phytopathogenicity and host recognition. Garrett et al. (1974) observed that selection for resistance to phage A7 resulted in attenuation of virulent cherry (Prunus avium) strains of Pseudomonas morsprunorum ( P . syringae pv. morsprunorum). Since resistance to phage often involves a change in one or more of the OM components (Osborn & Wu, 1980), it was proposed that at least one OM component functioned as a virulence determinant in P . rnorsprunorum. Although phage A7 was inactivated by purified LPS (Garrett et al., 1974), further studies (Hignett & Quirk, 1979;Quirk et al., 1976) suggested that an LPS-associated protein from P. morsprunorum contributed to virulence because of its phytotoxicity.

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Section: Methodsmentioning

confidence: 99%

Isolation and Partial Characterization of the Cell Wall of Pseudomonas syringae pv. syringae HS191: Comparison of Outer Membrane Proteins of HS191 with Those of Two Plasmidless Derivatives

Hurlbert

Gross

1983

Microbiology

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“…Nixdorff et al (22) reported that two major outer membrane proteins of Proteus mirabilis with apparent molecular weights of 36,000 and 39,000 formed hydrophilic pores in reconstituted membranes, and they claimed that the two kinds of pores have functionally similar properties, although the 39K protein preparation was assumed to contain a large amount of the 36K protein. Proteus mirabilis N-51, studied here, produces two major outer membrane proteins, i.e., 40K and 41K, and these two proteins may correspond to the 36K and 39K proteins, respectively, reported by Nixdorff et al (22). However, Proteus mirabilis N-51C1 lacking the 40K porin showed low permeation of hydrophilic cephalosporins, and the diffusion rate of the drugs through the outer membrane was independent of the hydrophilic property of the solute (Fig.…”

Section: Discussionmentioning

confidence: 99%

Identification of porins in outer membrane of Proteus, Morganella, and Providencia spp. and their role in outer membrane permeation of beta-lactams

Mitsuyama¹,

Hiruma²,

Yamaguchi³

et al. 1987

Antimicrob Agents Chemother

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Proteus mirabiis, Proteus vulgaris, MorganeUa morganii, Providencia rettgeri, and Providencia alcalifaciens, which were once classified into the same genus, Proteus, were studied. Cefoxitin-resistant mutants from these species were isolated, and it was confirmed that the resistance was attributed to the lack of an outer membrane protein, resulting in a significant decrease in the penetration of hydrophilic cephalosporins through the outer membrane. Comparison of the mutant strains with their parental strains in the diffusion rates of six monoanionic cephalosporins, a zwitterionic cephalosporin (cephaloridine), and a divalent anionic cephalosporin (cephalosporin C) suggested that each species had only one kind of porin protein, with molecular weights of 40,000 (Proteus mirabilis) or 37,000 (the other four species) and that the porins formed channels with cation selectivity, except for Proteus vulgaris. Porin proteins were purified from all the bacterial species except Providencia alcalifaciens, and the radius of the pores formed by the purified porins was estimated by the use of the liposome swelling assay. The pore radii were estimated to be approximately 0.59 nm (Proteus mirabilis), 0.63 nm (Proteus vulgaris), 0.58 nm (Providencia rettgeri), and 0.60 nm (M. morganiO), similar to the size of the pore radius of Escherichia coli porins.Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens were once classified into the same genus, Proteus (7), and, together with Serratia marcescens and Pseudomonas aeruginosa, are known as opportunistic pathogens. Except for Proteus mirabilis, these species show low susceptibility to many antibiotics. One of the reasons for such an intrinsic resistance may be the barrier effect of the outer membrane on antibiotic permeation. In the case of Pseudomonas aeruginosa, there is evidence suggesting that intrinsic resistance involves the outer membrane (1, 4, 32). However, the characterization of the outer membrane as the permeation route of antibiotics in the former Proteus species is incomplete. In our preliminary work (24), Proteus mirabilis N-51 was found to produce only a single major porinlike protein, with a molecular weight of 40,000. This 40K protein contributed to the bacterial susceptibility to cephalosporins and tetracycline. The present investigation was undertaken as an extension of the previous study to identify the porin proteins produced by Proteus vulgaris, M. morganii, Providencia rettgeri, and Providencia alcalifaciens and to evaluate the porin pores of these four species and of Proteus mirabilis as permeation routes for ,-lactam antibiotics. MATERIALS AND METHODSBacterial strains and P-lactamase production. Proteus mirabilis N-51, Proteus vulgaris K22-2, Providencia rettgeri RE-18, and Providencia alcalifaciens IN-06 are clinical isolates which barely produce 1-lactamase activity under usual growth conditions. M. morganii 1510/9, which is a mutant strain with lower 1-lactamase activity, was isolated from strain 151...

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“…This passive permeability is dependent to a large extent on a class of major outer membrane proteins [16,17] called matrix proteins [26], protein 1, protein I [11,30], or porins [16] The porins have been isolated from the outer membrane of Escherichia coli, Salmonella typhimurium, and Proteus mirabilis. They were shown to form permeability channels in vesicles reconstituted from phospholipids and lipopolysaccharide [16,17,23]. It was suggested that the active unit of porin aggregates is a trimer in E. coli and S. typhimurium [35,36].…”

Section: Summary the Three Types Of Porin (Matrix-proteins)mentioning

confidence: 99%

Determination of ion permeability through the channels made of porins from the outer membrane ofSalmonella typhimurium in lipid bilayer membranes

1980

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The three types of porin (matrix-proteins) from Salmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10)(-12)M), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1 M KC1. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39 K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins from Salmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1 M KC1 (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single conductance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.

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Reconstitution of Model Membranes from Phospholipid and Outer Membrane Proteins of Proteus mirabilis

Cited by 57 publications

References 23 publications

Isolation and Partial Characterization of the Cell Wall of Pseudomonas syringae pv. syringae HS191: Comparison of Outer Membrane Proteins of HS191 with Those of Two Plasmidless Derivatives

Isolation and Partial Characterization of the Cell Wall of Pseudomonas syringae pv. syringae HS191: Comparison of Outer Membrane Proteins of HS191 with Those of Two Plasmidless Derivatives

Identification of porins in outer membrane of Proteus, Morganella, and Providencia spp. and their role in outer membrane permeation of beta-lactams

Determination of ion permeability through the channels made of porins from the outer membrane ofSalmonella typhimurium in lipid bilayer membranes

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