Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny.

Mutants of Pseudomonas aeruginosa with impaired ability to establish a lysogenic relationship with temperate bacteriophage (Les-) have been isolated. These les mutations map to two areas of the P. aeruginosa chromosomal map as determined by conjugational and transductional analyses. Two phenotypic classes of Les- mutants were identified. One class of mutations has pleiotropic effects on DNA metabolism. These mutants are unable to recombine genetic material acquired as a result of either conjugation or transduction (Rec-). In addition, the ability of these Les- Rec- mutants to repair UV-induced damage to bacteriophage is reduced (host-cell reactivation deficient, Hcr-). Mutants of the second class are Les-, Rec+, and Hcr+.     

Characterization of Pseudomonas aeruginosa derepressed R-plasmids.

A genetic study of conjugal transmissibility of two R-plasmids was undertaken in Pseudomonas aeruginosa. Conjugally derepressed mutants of the R-plasmids were isolated, and examination of 11 independent mutants revealed that 10 were recessive to the wild-type transfer repressor, whereas 1 mutant was cis dominant. Cross-repression was observed between the two R-plasmids, suggesting that they have functionally equivalent systems for regulating the expression of tra loci. The derepressed R-plasmid mutants exhibited several characteristics, in addition to derepressed transfer, that were not expressed by the parental plasmids. These included sensitivity to certain donor-specific phages, inhibition of multiplication of a transducing phage, and, in the one case examined, a high degree of entry exclusion. The coexpression of these different functions suggests that their respective genetic loci are controlled by the same regulatory system as that of tra, or else that they are part of the tra complex.     

New class of Bacillus subtilis glutamine-requiring mutants

By using genetic analysis, the mutations of eight glutamine-requiring mutants isolated from Bacillus subtilis 168 were all shown to be linked to the thyA marker. A three-factor transduction analysis performed with one of the gln mutations indicated that the gene order in this region of the B. subtilis chromosome was gltA-thyA-gln. On the basis of recombination index values, two closely linked groups were identified. The mutations belonging to one group were assigned to the structural gene for glutamine synthetase, and those belonging to the other group might impair a regulatory locus. The residual glutamine synthetase activities and the cross-reacting materials of the mutants from both recombination groups supported these conclusions.     

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.     

Characterization of two Pseudomonas aeruginosa mutants with defective secretion of extracellular proteins and comparison with other mutants

We have isolated 2 new pleiotropic mutants of Pseudomonas aeruginosa strain PAO with defective secretion of extracellular proteins (Xcp mutants). One of these mutants was compared to 2 different, previously isolated secretion mutants. All had similar phenotypes and were unable to release at least 4 exoproteins (lipase, elastase, alkaline phosphatase, and phospholipase C), whilst alkaline protease was still secreted. The exoproteins appeared to be blocked in the periplasmic space. No difference in molecular weight was detected between cell-bound forms of elastase and alkaline phosphatase in the different mutants and the corresponding extracellular forms from the wild-type strain. Genetic mapping showed that the mutations were located in the 55′ region of the chromosome.     

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.     

Characterization of Pseudomonas aeruginosa Bacteriophage 95

A new strain of bacteriophage, phage 95, specific to Pseudomonas aeruginosa and Ps. schuylkilliensis was isolated. It has been shown that the phage induces a lytic enzyme which hydrolyzes peptide bond between l-alanine and d-glutamic acid in the peptidoglycan of cell wall.

Here the characteristics of phage 95 are described. It possesses a hexagonal head of 650Å, a contractile tail of 1150Å and spike with fibers. Its DNA has a GC content of 48%, a density of 1.700 g · cm^?3 and Tm of 87.7°C. Photoreactivation was observed. Latent period is 20 min and burst size is 50. The phage is stable between pH 5 and 7.5, and unstable above 55°C. Culture condition and 300 liter scale cultivation are also presented.     

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.     

Catabolite repression of Pseudomonas aeruginosa amidase: isolation of promotor mutants.

Among mutants of Pseudomonas aeruginosa isolated from fluoroacetamide medium were some which synthesized amidase at about 5% of the rate of the parent constitutive strain, PAC101. Seven fluoroacetamide-resistant mutants with low amidase activity gave rise to secondary mutant strains on succinate+butyramide plates. One appeared to be an 'up-promotor' mutant and synthesized amidase at a high rate. This mutant, PAC433, was not stimulated by cyclic-AMP and was much less sensitive to catabolite repression by succinate. The mutation conferring resistance to catabolite repression was cotransduced at a frequency of 96% (26/27) with the amidase genes amiR, amiE. Five other revertants had catabolite repression-resistance mutations which were linked to the amidase genes and these also were probably promotor mutants. One strain had a mutation conferring resistance to catabolite repression which was unlinked to the amidase genes.     

Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa.

Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC1 and PC2, were selected by the swarm plate method after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were fully motile but incapable of swarming, suggesting that they had a defect in the intracellular signalling pathway. Computer-assisted capillary assays confirmed that they failed to show behavioral responses to chemical stimuli, including peptone, methyl thiocyanate, and phosphate. Two chemotaxis genes were cloned by phenotypic complementation of PC1 and PC2. From nucleotide sequence analysis, one gene was found to encode a putative polypeptide that was homologous to the enteric CheZ protein, while the other gene was cheY, which had been previously reported (M. N. Starnbach and S. Lory, Mol. Microbiol. 6:459-469, 1992). Deletion and complementation analysis showed that PC1 was a cheY mutant, whereas PC2 had a double mutation in the cheY and cheZ genes. A chromosomal cheZ mutant, constructed by inserting a kanamycin resistance gene cassette into the wild-type gene, changed its swimming direction much more frequently than did wild-type strain PAO1. In contrast, cheY mutants were found to rarely reverse their swimming directions.     

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.