Pleiotropic changes in the activities of extracellular toxins of Pseudomonas aeruginosa protease-deficient mutants

Protease-deficient mutants of Pseudomonas aeruginosa obtained by ethyl methane sulphonate treatment were found to show pleiotropic effect on various extracellular toxin activities. Of eight mutants five were completely deficient in lecithinase and four in collagenase activity. The decreased hemolytic activity was obtained in five mutants and enterotoxin activity in seven mutants as compared to parent strain.     

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.     

Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins.

We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme.     

Protective cross activity of the extracellular mucus of Pseudomonas aeruginosa

Extracellular slime was isolated from 15 P. aeruginosa typing strains of different O-serotypes (immunotypes). The isolated slime, partially purified by ethanol precipitation, was later referred to as crude slime. Glycolipoprotein was obtained from crude slime and lipopolysaccharide (LPS) was obtained from acetone-dried microbial cells by the method of aqueous-phenol extraction. All these antigenic preparations were studied in the active mouse cross-protection tests: immunized mice were challenged with 7 strains of different immunotypes, strain No. 170 019 or toxigenic strain PA-103. In experiments on mice the slime of different P. aeruginosa serotypes (immunotypes) was found to stimulate immunity to intraperitoneal infection with P. aeruginosa, both homologous or heterologous in respect to their immunotype, including toxigenic strains. Slime glycoprotein also stimulated active cross-immunity in mice, but the level of this immunity was higher than that of immunity stimulated by crude slime. LPS showed mostly weak protective activity in experiments on mice.     

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.     

Purification of extracellular lipase from Pseudomonas aeruginosa.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.     

6-Phosphogluconate dehydratase deficiency in pleiotropic carbohydrate-negative mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, strain PAO, have been isolated that are unable to grow on mannitol, glucose, gluconate, or 2-ketogluconate, cut that exhibit wild-type growth on pyruvate, lactate, citrate, succinate, or acetate. Although some of these mutants were also unable to grow on glycerol, the mutations formed a single linkage group by quantitative transductional analysis with phage F116 on glucose minimal agar medium. Cell extracts of all mutant strains were either lacking or severely deficient in 6-phosphogluconate dehydratase activity. Glu+ transductants derived from mutant strains that retained the wild-type ability for growth at the expense of glycerol also regained the ability to grow on all C-6 compounds. Although a role for the pentose phosphate pathway in the catabolism of C6 substrates was not found, a functional Entner-Doudoroff pathway appears to be essential for the catabolism of mannitol, glucose, gluconate, and 2-ketogluconate.     

Two malic enzymes in Pseudomonas aeruginosa

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) --> pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO(2)), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn(2+) being more effective than Mg(2+). The NADP-dependent enzyme is activated by K(+) and low concentrations of NH(4) (+). Both reactions are reversible, as shown by incubation with pyruvate, CO(2), NADH, or NADPH and Mn(2+). The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).     

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.     

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.     

Secretion of extracellular proteins by Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterial species of commercial value secreting numerous extracellular proteins, involved in pathogenesis. Most strains produce at least a lipase, a phospholipase, an alkaline phosphatase, an exotoxin and 2 proteases (elastase and alkaline protease). Various mechanisms for secretion of exoproteins appear to exist in P aeruginosa. Genetic analysis has led to the identification of 2 secretion pathways: i) a "general" secretion pathway, defined by the xcp mutations, which mediates secretion of most extracellular proteins, and; ii) an independent secretion pathway specific for alkaline protease. Our present knowledge on the pathways and components of the secretion machinery in P aeruginosa is reviewed in this article.     

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.     

Light-mediated changes in pigmentation of Pseudomonas aeruginosa cultures.

Cultures of Pseudomonas aeruginosa PAO grown under uninterrupted broad-spectrum light showed different pigmentation from dark-grown cultures. Whereas dark-grown bacteria produced pigments which resulted in blue-purple coloured agar, light-grown organisms produced red coloured plates. Extraction and quantification of pigments showed that both dark- and light-grown cultures produced similar concentrations of pyorubrin (red) and pyoverdin (yellow). In contrast, the concentration of pyocyanin (blue) was substantially reduced under certain lighting conditions. This decrease was dependent on both the light intensity and wavelength and occurred with light in the ultraviolet and violet region of the spectrum. After its release from bacteria, pyocyanin was rapidly and nonreversibly photoinactivated with first-order kinetics to produce colourless photoproduct(s).     

Regulation of amidase formation in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity

The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation between amidase and urease formation was observed. The results suggest that amidase formation in strain PAO is subject to nitrogen control and that glutamine or some compound derived from it mediates the nitrogen repression of amidase.     

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+.