Isolation and analysis of Pseudomonas aeruginosa PAO mutants resistant to nonlysogenic bacteriophages

Nonlysogenizing Pseudomonas aeruginosa PAO bacteriophages were studied. According to morphology of the plaques, they were distributed into three groups: phi k, phi m and phi mn. The mutants of P. aeruginosa PAO resistant to these bacteriophages were selected. On the basis of cris-cross resistance analysis of the mutants, a formal scheme of the receptor sites on the P. aeruginosa PAO bacterial cell surface is drawn. It is shown that bacteriophages phi k and phi m use different receptors for their adsorption. The receptors of phi m and phi mn phages are specifically interconnected. Thus, the receptor for phi k phages is connected with the receptor for phage phi 11. It appears that the receptor for bacteriophage E79 is identical to those of phi m phages. The phi m receptor is of a composite structure: it includes two different receptors used by phi mn phages.     

Glycollate inhibition of growth of Pseudomonas aeruginosa on lactate medium

Glycollate inhibited growth of Pseudomonas aeruginosa in media containing either pyruvate or lactate as carbon sources. Glycollamide, but not glyoxylate, showed similar effects. Spontaneous mutants (L/G strains) were isolated that were able to grow on lactate medium in the presence of glycollate: their growth in pyruvate medium was still inhibited by glycollate. Synthesis of membrane-bound NAD+-independent D(-)- and L(+)-lactate dehydrogenase (iLDHs) was inducible by D- or L-lactate in the parent strain but was constitutive in the L/G strains. Glycollate inhibited induction of the synthesis of iLDHs in the parent strain growing in succinate medium but had no effect under the same conditions on strain L/G1. Glycollate was a competitive inhibitor of L(+)-iLDH (Ki = 11 mM). No differences were found in the kinetic properties of L(+)-iLDH in cell-free extracts from strain L/G1 and the parent organism. Glycollate appears to inhibit growth on lactate medium predominantly through prevention of lactate induction of iLDH synthesis.     

Characterization of glutamine-requiring mutants of Pseudomonas aeruginosa.

Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation conferring glutamine auxotrophy was subsequently mapped and found to be located at about 15 min on the chromosomal map, close to and before hisII4. Furthermore, in transduction experiments, it appeared to be very closely linked to gln-2022, a suppressor mutation affecting nitrogen control. With immunological techniques, it could be demonstrated that the glutamine auxotrophs form an inactive glutamine synthetase protein which is regulated by glutamine or a product derived from it in a way similar to other nitrogen-controlled proteins.     

Isolation and characterization of multiflagellate mutants of Pseudomonas aeruginosa.

Multitrichously polar flagellated mutants were isolated from a monotrichously flagellated strain of Pseudomonas aeruginosa. The ability of the mutant cells to swarm in semisolid media at given gel strengths was increased by the multiflagellation. Observations of the mutant cells by electron microscopy revealed that the number of flagella produced per cell cycle was increased. F116 phage-mediated transduction showed that the multiflagellation occurred by a single mutation and that the mutation sites were linked to a fla cluster of this organism.     

Growth of Pseudomonas aeruginosa mutants lacking glutamate synthase activity.

Mutant strains SU1, SU4, and US1 lacking glutamate synthase (GOGAT) activity were isolated from strains of P. aeruginosa for which histidine is a growth rate-limiting source of nitrogen. Strains SU1 and SU4 were unable to grow when a low concentration of ammonia and a variety of compounds, including histidine, were supplied as sole sources of nitrogen. A revertant of strain SU1, strain 39, produced no GOGAT but high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase and had restored ability to grow on a limited number of nitrogen sources. Strain US1 grew at the same rate in histidine medium as did its parent; it was derepressed for glutamine synthase synthesis, and histidase was less sensitive to repression by ammonia than in the parent strain. We conclude that GOGAT is not essential for growth on histidine but high levels of glutamine synthase are required nd high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase can sustain growth at low concentrations of ammonia in the absence of GOGAT.     

Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants.

We isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up 14C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C12 to C19. However, growth on these alkanes and uptake of [14C]hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up [14C]hexadecane. The addition of small amounts of rhamnolipids restored growth on alkanes and [14C]hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P. aeruginosa.     

Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance.

Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P. aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.     

Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa

Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.     

Inability of Pseudomonas stutzeri denitrification mutants with the phenotype of Pseudomonas aeruginosa to grow in nitrous oxide.

Pseudomonas aeruginosa PAO1 reduced nitrous oxide to dinitrogen but did not grow anaerobically in nitrous oxide. Two transposon insertion Nos- mutants of Pseudomonas stutzeri exhibited the P. aeruginosa phenotype. Growth yield studies demonstrated that nitrous oxide produced in vivo was productively respired, but nitrous oxide supplied exogenously was not. The defect may be in electron transport or in nitrous oxide uptake.     

Antibody inhibition of ferripyochelin binding to Pseudomonas aeruginosa cell envelopes

A 14K molecular weight protein which has been shown to bind ferripyochelin has been purified from cell envelopes of Pseudomonas aeruginosa low iron grown cells. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to be free of contamination by lipopolysaccharide or carbohydrate. Antiserum to this protein was made in rabbits and was shown to react with the purified protein by immunoblot assay. The immunoglobulin G fraction of this antiserum blocked binding of [59Fe]pyochelin to isolated cell envelopes of P. aeruginosa in a dose-dependent fashion.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.     

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.