Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

The chemical composition of the lipopolysaccharide from Pseudomonas aeruginosa strain PAO and a spontaneously derived rough mutant.

The chemical composition of the lipopolysaccharide (LPS) of the smooth strain Pseudomonas aeruginosa PAO 307 and a spontaneously derived rough mutant, obtained by selection for resistance to the LPS-specific phage E79, are compared. The rough LPS was shown to contain lipid A, heptose, 2-keto 3-deoxyoctonic acid, galactosamine, alanine and phosphate but lacked glucose, rhamnose and fucosamine which were important constituents, on a weight basis, of the smooth LPS. These results, and chromatographic analysis of the polysaccharide fraction indicate that the rough strain lacked side chain material and was defective in its inner core region. The chemical date obtained were consistent with a core in the PAO strain similar to that of strain NCTC 1999, enhancing the evidence for a common core polysaccharide in the LPS of P. aeruginosa strains.     

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.     

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.     

Isolation and characterization of a lipopolysaccharide-specific bacteriophage of Pseudomonas aeruginosa

Phage H22 was isolated from sewage using Pseudomonas aeruginosa NCTC 8505 (serotype 0:3) as the host. Although not O-specific, this phage was found to have lipopolysaccharide (LPS) as a receptor. The broad host-range and lack of O-specificity of the phage suggested that its receptor site was in the core region of the LPS. Phage H22 had a Bradley type A structure. It was unaffected by chloroform and diethyl ether, and was stable between pH 5 and 8 and in the temperature range 0 to 60 degrees C. The adsorption rate constant was 14.6 X 10(-9) ml min-1. The phage had a latent period of 43 min, with a rise time of 18 min and a burst size of 6. The adsorption of phage to whole cells and LPS occurred over a broad pH range. Maximum adsorption occurred at 50 degrees C and pH 7.5 in the presence of 0.001 M Ca2+.     

Identification of the cell wall receptor for bacteriophage E79 in Pseudomonas aeruginosa strain PAO.

Bacteriophage E79 was shown to interact with the lipopolysaccharide (LPS) of Pseudomonas aeruginosa strain PAO. LPS isolated from an E79-sensitive, smooth strain inactivated the phage, exhibiting a Phl50 value (concentration of LPS that caused a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) of 0.04 microgram/ml, whereas the LPS isolated from a rough mutant derived from the wild type showed no neutralizing activity towards E79. EDTA and sodium deoxycholate were demonstrated to abolish the neutralizing capacity of the smooth LPS. One E79 receptor site was shown to be equivalent to 10(-16) g of LPS.     

Studies on the Pseudomonas aeruginosa PAO1 bacteriophage receptors

Receptor for phage PIK specific for Pseudomonas aeruginosa strain PAO1 was studied. Phage PIK was strongly inactivated by lipopolysaccharide (LPS) in vitro, exhibiting a PhI50 of 4.8 micrograms/ml. Further it was noted that this inactivation by LPS was reduced to 50% by several mono- and disaccharides when tested in vitro. D-glucosamine, D-mannose and L-rhamnose were found to be most effective at the concentration of 0.045 M, 0.25 M and 0.35 M respectively. This suggests the possibility that phage PIK receptor in LPS contains D-mannose, L-rhamnose and D-glucosamine. Either one of the former two could be located at a terminal position alpha-linked to the adjacent residue or located internally in the polysaccharide chain linked through its C-4 position. A theoretical approach to the interpretation of phage cell interaction was also investigated.     

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 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.     

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.     

Isolation and characterization of a generalized transducing phage for Pseudomonas aeruginosa strains PAO1 and PA14

A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa. A clear-plaque variant of this bacteriophage was isolated. DMS3 is capable of mediating generalized transduction within and between P. aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P. aeruginosa.     

Isolation and characterization of Pseudomonas aeruginosa bacteriophage phikF77 mutants]

It was found that phage phi kF77 is resistant to all known Pseudomonas aeruginosa restriction systems. Three types of mutants (dc-) which were unable to grow on different restrictive strains were isolated. All of them belong to one complementation group. Some of these mutations affected also the number of nicks in phage phi kF77 DNA and increased phage resistance to temperature treatment. It may be supposed that genes responsible for antirestriction mechanisms and introduction of nicks into DNA are connected in definite way.     

Isolation and characterization of a Pseudomonas aeruginosa bacteriophage with a very limited host range

A Pseudomonas aeruginosa bacteriophage, phi PLS743, with extremely limited host range has been isolated. It belongs to the virus family Podoviridae, morphological type C1, and possesses a head diameter of 45 nm. The phage has a buoyant density in CsCl of 1.516 g/cm3, and its mass is 45 x 10(6) daltons. The phage particles are composed of double-stranded DNA (49.9 mol% G + C; 42.4 kilobase pairs) and 11 structural proteins (66% by weight). The major head protein, P5, has a Mr of 34,500. The DNA is not cut by SalI or XhoI restriction endonucleases, but is cut by PvuII (1 site), KpnI and BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), and HindIII (12 sites). A restriction endonuclease map is presented.     

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.     

The extraction and analysis of lipopolysaccharides from Pseudomonas aeruginosa strain PAO, and three rough mutants.

Three spontaneously arising rough mutants of Pseudomonas aeruginosa have been isolated by selection for resistance to virulent lipopolysaccharide (LPS) specific bacteriophages. In addition, the first phages specific for rough mutants of P. aeruginosa were isolated. Using these phage and autoagglutination patterns in 4% NaCl and acriflavine, these mutants could be clearly distinguished from the wild-type strain and each other. Chemical analysis of the LPS together with chromatographic resolution of the polysaccharide moieties showed alterations in both O-specific side chains and core regions.     

Isolation and characterization of major outer membrane proteins of Pseudomonas aeruginosa strain PAO with special reference to peptidoglycan-associated protein.

The outer membrane of Pseudomonas aeruginosa PAO contains six major proteins (proteins D, E, F, G,H, and I). Two of them (protein F and protein H) were found to be retained by the peptidoglycan layer when cell envelopes were extracted with 2% sodium dodecyl sulfate (SDS) solution at 35 degrees C. At higher temperature (greater than 55 degrees C), no proteins were retained by peptidoglycan. By making use of this property, purification of protein F and protein H was achieved. Three other major outer membrane proteins, D, E, and I were also isolated and characterized. Their amino acids compositions were determined. Circular dichroism spectra of these isolated proteins were measured in SDS solution. Protein F was rich in beta-structure, while protein I was rich in alpha-helix. When isolated protein F was heated (100 degrees C-15 min) in SDS solution, the circular dichroism spectrum changed significantly. In parallel with the conformational change, the electrophoretic mobility of protein F on urea-SDS polyacrylamide gel also changed. These results indicate that protein F is a so-called heat-modifiable protein.     

Specialized transduction of Pseudomonas aeruginosa PAO by bacteriophage D3.

D3, a temperate bacteriophage of Pseudomonas aeruginosa PAO, was found to specifically transduce the alleles met-49 and met-117. Induction of established lysogens with UV light was necessary for the production of transducing lysates. Transduced cells were immune to superinfection by phage D3 and could give rise to high-frequency transducing lysates. Cotransduction of these two alleles could not be demonstrated. met-117 was mapped to 26 min on the PAO genetic map. Complementation studies using the generalized transducing phage F116L indicated that met-49 is an allele of met-9011 which maps at 55 min. The integrated D3 prophage was shown to be coinherited with met-117 and with met-49.     

Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster

Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2–5 sugars which is the serotype-specific antigen. The gene clusters for A- and B-band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz ( rol ), wzy ( rfc ), and wbpA – wbpN . A further 3 ORFs not thought to be involved with LPS synthesis were identified ( hisH , hisF , and uvrB ). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis. A novel insertion sequence, IS 1209 , is present at the junction between the serogroup-specific and non-specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O-antigen biosynthesis.