Pseudomonas aeruginosa outer membrane: peptidoglycan-associated proteins.

The Pseudomonas aeruginosa outer membrane was isolated with attached peptidoglycan and fractionated with Triton X-100, ethylenediaminetetraacetate, and lysozyme. The data suggest that major outer membrane proteins F, H2, and I are noncovalently associated with the peptidoglycan.     

Isolation of characterization of a major outer membrane protein of Pseudomonas aeruginosa. Evidence for the occurrence of a lipoprotein.

In the outer membrane of P. aeruginosa, a protein of apparent molecular weight 8,000 (protein I) is present as a major protein. Purification and chemical analysis of protein I were carried out. This protein was purified by essentially the same procedure as for the purification of the E. coli lipoprotein, which was developed by Inouye et al. (J. Bacteriol. (1976) 127, 555--563). The amino acid composition of protein I was determined. Protein I lacks proline, valine, isoleucine, phenylalanine, tryptophan, and half-cystine. Fatty acid analysis of the protein revealed that it contained 0.89 mol of fatty acids per mol of protein. Among the fatty acids hexadecanoic acid (C16:0) was predominant. In an in vivo labeling experiment, [2-3H]glycerol was incorporated into protein I. A protein with similar mobility to protein I on urea-SDS polyacrylamide gel electrophoresis was isolated from the purified peptidoglycan of P. aeruginosa by trypsin digestion. The amino acid composition of this protein was essentially the same as that of protein I. These results indicate that the outer membrane of P. aeruginosa contains a protein analogous to the E. coli lipoprotein, although considerable differences were observed in the amino acid composition and the fatty acid content.     

Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO.

Two independently derived, exotoxin A-deficient (Tox- phenotype), nitroso-guanidine-induced mutants of Pseudomonas aeruginosa PAO1 were isolated by using sensitive immunological assays. One mutant, designated PAOT10, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT10 and the parental strain. The mutation in PAOT10 (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.     

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.     

Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO.

Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the 11-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc- mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc- mutants but exhibited wild-type inducible expression.     

Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO.

Growth of Pseudomonas aeruginosa PAO1 at 15 to 45 degrees C in tryptic soy broth resulted in changes in the lipids, lipopolysaccharides (LPSs), and outer membrane proteins of the cells. Cells grown at 15 degrees C contained, relative to those cultivated at 45 degrees C, increased levels of the phospholipid fatty acids hexadecenoate and octadecenoate and reduced levels of the corresponding saturated fatty acids. Furthermore, the lipid A fatty acids also showed thermoadaptation with decreases in dodecanoic and hexadecanoic acids and increases in the level of 3-hydroxydecanoate and 2-hydroxdodecanoate as the growth temperature decreased. In addition, LPS extracted from cells cultivated at the lower temperatures contained a higher content of long-chain S-form molecules than that isolated from cells grown at higher temperatures. On the other hand, the percentage of LPS cores substituted with side-chain material decreased from 37.6 mol% at 45 degrees C to 19.3 mol% at 15 degrees C. The outer membrane protein profiles indicated that at low growth temperatures there was an increase in a polypeptide with an apparent molecular weight of 43,000 and decreases in the content of 21,000 (protein H1)- and 27,500-molecular-weight proteins.     

Isolation and characterization of Pseudomonas aeruginosa PAO mutant that produces altered elastase.

Pseudomonas aeruginosa PAO mutants defective in elastase were isolated by plate assays of nitrosoguanidine-mutagenized clones. A total of 75 elastase mutants were isolated from 43,000 mutagenized clones. One mutant (PAO-E64) was apparently identical to the parental strain except for its deficiency in elastase activity. This mutant produced an enzyme which was antigenically indistinguishable from parental elastase. Furthermore, equal levels of elastase antigen were produced by this mutant and its parental strain. The mutant elastase, however, had greatly reduced enzymatic activity. Mutant PAO-E64 is presumed to have a mutation in the structural gene for elastase. We have designated the genotype of the mutation in PAO-E64 as lasA1.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.     

Transformation of Pseudomonas aeruginosa strain PAO with bacteriophage and plasmid DNA.

A procedure has been developed which allows transformation of P. aeruginosa strain PAO with plasmid and bacteriophage DNA at a frequency of 10(-6) per recipient cell. The method is similar in outline to that developed for Escherichia coli. It involves growing the recipient cells to 3-5 x 10(8) per ml in nutrient broth, washing the cells with 0.1 M MgCl2, resuspending in 0.175 M CaCl2 for 20 min, exposing to DNA for 1 h and then heat pulsing at 42 degrees C for 1 min. Some plasmid markers are expressed immediately, whereas others require time for phenotypic expression.     

Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

Isolation and partial characterization of the major outer membrane protein of Chromatium vinosum.

The 42,000 major outer membrane protein of Chromatium vinosum was purified by a combination on ion-exchange chromatography, gel filtration, and isoelectric focusing. Upon isoelectric focusing, the final material produced four major hands. Three of the four bands were isolated and analyzed for similarity or differences. Protease peptide maps and cyanogen bromide maps of the three isoelectric species were identical. When the isolated isoelectric species were refocused, each produced multiple isoelectric species, suggesting that the procedure used was generating the multiple charged species. Protease treatment of the isolated outer membrane produced a 31,000 fragment from the 42,000 protein. This fragment was isolated by preparative sodium sulfate-polyacrylamide gel electrophoresis. Although the amino acid compositions of the 42,000 protein and its 31,000 trypsin fragment were different, their polarity index was the same (45%). The amino-terminal sequences of the 42,000 protein and 31,000 trypsin fragment were identical, and it concluded that the amino-terminal was buried in the membrane.     

Isolation and characterization of a bacteriophage specific for the lipopolysaccharide of rough derivatives of Pseudomonas aeruginosa strain PAO

A lipopolysaccharide (LPS)-defective (rough) mutant of Pseudomonas aeruginosa PAO was isolated by selection for resistance to the LPS-specific phage E79. The LPS of this mutant, AK-1012, lacked the O-antigenic side chain-specific amino sugar fucosamine as well as the core-specific sugars glucose and rhamnose. Using this strain, we isolated and characterized a phage, phi PLS27, which is specifically inactivated upon incubation with LPS extracted from rough mutants of P. aeruginosa PAO. phi PLS27 was found to be a Bradley type C phage and was very similar to coliphage T7 in a number of properties, including size, buoyant density, mass, and the number of structural proteins.     

The nature of Pseudomonas aeruginosa strain PAO bacteriophage receptors.

Receptors for phages specific to Pseudomonas aeruginosa strain PAO were studied. Phages 16, 44, 109, F8, and PBI are lipopolysaccharide (LPS) specific as shown by neutralization tests. The PhI50's of the LPS, adsorption rate constants with strain PAO and the plaque morphologies of these five phages were quite similar. Phages 1214 and 7 also appear to be LPS-specific on the basis of host-range studies. Phage 73 is pilus-specific, while phages 21 and 68 fall into a group which does not attach to pili, flagella, or LPS. A theoretical approach to the interpretation of phage-cell interactions is presented.     

Production and characterization of monoclonal antibodies to outer membrane proteins of Pseudomonas aeruginosa grown in iron-depleted media

The iron uptake systems of pathogenic bacteria provide potential targets for immunological intervention. We have partially purified the high molecular mass, iron-regulated outer membrane proteins (IROMPs) from Pseudomonas aeruginosa and used them to prepare a panel of monoclonal antibodies (mAbs). Five mAbs reacted with an 85 kDa IROMP separated by SDS-PAGE, but gave only low-level binding to whole cells by immunogold electron microscopy. However, iodination of whole cells indicated that the 85 kDa IROMP is surface-exposed. The mAbs were only cross-reactive with clinical isolates representing eight of the 17 International Antigenic Typing Scheme serotypes of P. aeruginosa, suggesting significant heterogeneity with respect to this IROMP.     

Isolation and characterization of a Pseudomonas aeruginosa PAO mutant defective in the structural gene for the LIVAT-binding protein.

A mutant of Pseudomonas aeruginosa PAO which has a defect in the structural gene for a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-binding protein) was isolated and characterized. DL-4-azaleucine was taken up via the high-affinity branched-chain amino acid transport system (LIV-I), but not via the low affinity system (LIV-II), and then inhibited the growth of P. aeruginosa cells. This finding enabled us to select mutants defective in the LIV-I transport system alone. Among such mutants, strain PAO3530 was found to produce an altered LIVAT-binding protein. The shock fluid of this strain contained a normal level of the protein which corresponded to the wild-type LIVAT-binding protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by an immunological test. However, the shock fluid showed almost no binding activity for branched-chain amino acids, suggesting that strain PAO3530 has a defect in the structural gene for the LIVAT-binding protein. The mutation locus (bra-310) was mapped in a region between cnu-9001 and oru-325 on the chromosome of P. aeruginosa PAO by conjugation mediated by plasmid FP5 or R68.45.     

Linker-insertion mutagenesis of Pseudomonas aeruginosa outer membrane protein OprF

The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb Hincll kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene. The kanamycin-resistance gene was then removed by Pstl digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full-length OprF variants. In addition, one permissive site-directed insertion, one non-permissive insertion and one carboxy-terminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF-specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2-mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high-molecular-weight core fragment in outer membranes and, where studied, to be accessible to indirect immunofluorescenee labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.     

Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.

Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.     

Isolation and characterization of major intrinsic microsomal membrane proteins.

Treatment of the membrane matrix derived from hepatic microsomes with buffered 1 M urea resulted in the selective extraction of a group of proteins together with a portion of the membrane lipid. Thorough chemical characterization of this fraction has been performed, and the proteins have been fractionated by two different procedures. The first of these, preparative polyacrylamide gel electrophoresis, has produced five highly homogeneous membrane proteins which have been characterized with regard to molecular weight, electrophoretic behavior in five different polyacrylamide systems, NH2 terminus, relative carbohydrate content, isoelectric point, and amino acid composition. The five proteins of this group fell in the molecular weight range of 54,000 to 96,000 and had isoelectric points ranging from pH 4.9 to pH 6.7. Further fractionation of the urea-soluble proteins by gel filtration in a sodium dodecyl sulfate-containing medium resulted in the isolation of four homogeneous molecular weight classes of proteins which have been characterized with respect to various physicochemical parameters. The major membrane glycoprotein (apparent molecular weight, 171,000) was isolated by this procedure and found to contain approximately equal amounts of NH2-terminal glycine and serine. suggesting the presence of at least two polypeptide chains in this molecular weight region. From the urea-insoluble fraction of the membrane comprising approximately 80% of the total protein, five intrinsic polypeptides designated S-5 through S-9 were isolated. S-5 (54,000) and S-6 (49,000) represent the most prominent components in the microsomal membrane, accounting for close to 30% of the total protein. Also isolated and characterized is the smallest membrane protein (S-9), a hydrophobic polypeptide of molecular weight 16,000. All of the urea-insoluble proteins are glycoproteins, and S-7 (35,000) gives the second most intense stain for carbohydrate of all proteins in the microsomal membrane.