The association on SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins of<i>Pseudomonas aeruginosa</i>as revealed by monoclonal antibodies and Western blotting

Poxton, Ian R.; Bell, Graham; Barclay, G. R.

doi:10.1111/j.1574-6968.1985.tb00676.x

Cited by 40 publications

(2 citation statements)

References 21 publications

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“…The OM of the algE-overexpressing strain E. coli K38(pGP1-2, pTR7-2) was isolated by the Sarkosyl method (25) and used to purify AlgE in large enough quantities for further characterizations. A homogeneous preparation of AlgE, as judged by SDS-PAGE, was obtained by immobilized metal ion affinity chromatography (14) followed by anionexchange chromatography (Fig.…”

Section: Resultsmentioning

confidence: 99%

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties

et al. 1994

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Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning P-strands, probably forming a P-barrel.Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCI and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.Pseudomonas aeruginosa is an opportunistic human pathogen which infects particularly persons who are suffering from cystic fibrosis. The initial infection occurs mostly with nonmucoid bacteria, which convert to mucoid strains in an early stage of infection (13,26). Mucoid bacteria produce copious amounts of alginate, which seems to be the most important virulence factor (12). The initial steps of the alginate biosynthesis, leading to the putative precursor GDP-mannuronic acid, are well-known (21). However, there is a lack of information about the final steps of the biosynthesis, i.e., the polymerization and export of alginate. Recently, the genes involved in acetylation (algF) and degradation (algL) of alginate were cloned and mapped in the alginate biosynthesis gene cluster (34 min) (10, 30). In addition, the gene product of algG (34 min) was purified and identified as an epimerase located in the periplasm that introduces guluronic acid residues into the alginate (9). We identified a 54-kDa protein (AlgE) in the outer membrane (OM) of P. aeruginosa which appears in mucoid strains only (15,26). This protein was purified and the N-terminal amino acid sequence was determined (14). At the same time...

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Section: Resultsmentioning

confidence: 99%

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties

et al. 1994

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“…Outer membrane proteins of P. aeruginosa PAO were prepared by the method of Poxton et al [16], and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by the method of Sawai et al [17].…”

Section: Characterization Of Outer Membrane Proteinsmentioning

confidence: 99%

Cloning and characterization of a DNA fragment that complements the nfxB mutation in Pseudomonas aeruginosa PAO

Okazaki¹,

Iyobe

Hashimoto

et al. 1991

FEMS Microbiology Letters

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The 2.2-kilobase DNA fragment that can complement the nfxB mutation in Pseudomonas aeruginosa PAO was cloned from chromosomal DNA of P. aeruginosa PAO1. The nfxB mutants show an increase in resistance to quinolones and hypersusceptibility to beta-lactam and aminoglycoside antibiotics. In the mutants, a 54 kDa outer membrane protein newly appeared. The phenotype was restored to the wild-type by transformation with plasmid carrying the 2.2-kb DNA fragment.

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Immunological Properties of Azospirillum Cell Surface: the Structure of Carbohydrate Antigens and Evaluation of their Involvement in Bacteria-Plant Contact Interactions

Matora

Solovova

Serebrennikova

et al. 1995

Azospirillum VI and Related Microorganisms

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Immunization of rabbits with glutar aldehyde-treated intact Azospirillum cells results in the production of antibodies specific mainly with regard to the cell O-antigens. EPS, CPS, and 0-specific polysaccharides of A. brasilense Sp 7 are antigenically identical. Pre-treatment of the wheat seedling roots and a carrot cell suspension with LPS of strain Sp 245 promotes significantly bacterial adhesion to wheat roots and Azospirillum-induced agglutination of carrot cells. It also leads to an increased synthesis of the major proteins in the wheat root apoplast, which is comparable with the plant response to inoculation with whole cells. This may be explained by an active effect of Azospirillum LPS on the receptor systems of plant cells.

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The association on SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins ofPseudomonas aeruginosaas revealed by monoclonal antibodies and Western blotting

Cited by 40 publications

References 21 publications

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties

Cloning and characterization of a DNA fragment that complements the nfxB mutation in Pseudomonas aeruginosa PAO

Immunological Properties of Azospirillum Cell Surface: the Structure of Carbohydrate Antigens and Evaluation of their Involvement in Bacteria-Plant Contact Interactions

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