Mapping and characterization of two mutations to antibiotic supersusceptibility in Pseudomonas aeruginosa.

Two mutations associated with antibiotic supersusceptibility in Pseudomonas aeruginosa strain Z61 were transferred separately into strain PAO222, using R68.45-mediated conjugation and phage F116L transduction. One mutation (absA) was 40% contransducible with pro-82 at 26 min on the P. aeruginosa chromosome and was associated with increased susceptibility to beta-lactams, gentamicin and hydrophobic agents. Strains carrying the absA mutation also displayed enhanced uptake of a hydrophobic fluorescent probe, 1-N-phenylnaphthylamine, and were found, by SDS-PAGE, to be altered in the pattern of lipopolysaccharide O-antigen distribution. The other mutation (absB), associated with increased susceptibility to beta-lactams and gentamicin but not to hydrophobic agents, was cotransducible with met-28 and proC at 20 min on the chromosome. The absB mutation caused a structurally undefined alteration in the physical interaction of EDTA and gentamicin with the outer membrane.

Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl.

Outer membrane porin proteins F, P, and D1 of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers.

Native oligomers of three Pseudomonas aeruginosa outer membrane porin proteins and one Escherichia coli porin were demonstrated by using a chemical cross-linking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was cross-linked to dimers in outer membrane and whole-cell cross-linking experiments. Purified preparations of P. aeruginosa proteins F, D1 (glucose induced), and P (phosphate starvation induced) and E. coli protein PhoE (Ic) were also cross-linked to reveal dimers and trimers upon two-dimensional sodium dodecyl sulfate-polyacrylamide electrophoretic analysis. Cross-linking of protein F was abolished by pretreatment of the protein with sodium dodecyl sulfate, indicating that the cross-linked products were due to native associations in the outer membrane.

Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant.

The Pseudomonas aeruginosa mutant Z61 has been shown to be highly supersusceptible to a wide range of antibiotics, including beta-lactams, aminoglycosides, rifampin, tetracycline, and chloramphenicol (W. Zimmerman, Int. J. Clin. Pharmacol. Biopharm. 17:131-134, 1979). Spontaneous revertants were isolated, using gentamicin or carbenicillin as selective agents, and shown to have two patterns of susceptibility to a group of 12 antibiotics. Partial revertants had 2- to 10-fold greater resistance to these antibiotics than mutant Z61, whereas full revertants had antibiotic susceptibilities indistinguishable from those of the wild-type strain K799, from which mutant Z61 had been derived. Uptake of a chromogenic beta-lactam nitrocefin was studied in both uninduced and induced cells of all strains by measuring the steady-state rate of nitrocefin hydrolysis by the inducible, periplasmic beta-lactamase in both whole and broken cells. This demonstrated that outer membrane permeability decreased as antibiotic resistance increased in the series mutant Z61, partial revertants, wild type, and full revertants. The data were consistent with the idea of low outer membrane permeability being caused by a low proportion of open functional porins in the outer membrane as the reason for the high natural antibiotic resistance of wild-type P, aeruginosa strains. In addition, it was observed that levels of benzylpenicillin below the minimal inhibitory concentration for mutant Z61 failed to induce beta-lactamase production. The possibility that this was related to the observed increase in outer membrane permeability is discussed. Preliminary evidence is presented that the pore-forming outer membrane porin protein F is not altered in mutant Z61.

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

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. An average molecular weight of 38,000 to 50,000 was calculated for this fraction based on the glucose and rhamnose levels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lipopolysaccharides from these two strains were microheterogeneous. Qualitative analysis of the lipopolysaccharide neutral sugars, using a series of single-step revertants of mutant Z61, demonstrated that full revertants showed patterns indistinguishable from those of the wild-type strain K799, whereas partial revertants had intermediate levels and mutant Z61 low levels of neutral sugars. Quantitative analysis revealed that the core oligosaccharide fraction from the wild-type strain had a glucose/rhamnose/galactosamine ratio of 4:1:1, whereas the core from Z61 exhibited major deficiencies in both glucose and rhamnose. The lipid A from both strains contained five fatty acids, namely, 3-hydroxydecanoate, dodecanoate, 2- and 3-hydroxydodecanoate, and hexadecanoate. Whereas the overall fatty acid content was equal, the mutant strain showed markedly lower levels of dodecanoate and hexadecanoate and increased levels of 2-hydroxydodecanoate. Results of whole-cell fatty acid analyses were consistent with this observation. Evidence for an additional alteration of the lipid A of strain Z61 was obtained from acid hydrolysis studies and infrared spectra of isolated lipid A, although the actual chemical basis could not be determined by a variety of techniques. It is suggested that the state of the lipopolysaccharide is able to influence the number of open functional protein F pores in the outer membrane of P. aeruginosa.