Lysine decarboxylase in Pseudomonas aeruginosa]

The research of lysine, ornithine and arginine decarboxylases has been made for 50 strains of fluorescent Pseudomonas (P. aeruginosa, P. fluorescens, P. putida). By thin layer chromatography, all the strains of Pseudomonas aeruginosa and the fifth of the strains of P. putida had lysine decarboxylase activity at alcaline pH (optimal pH 8) ; Pseudomonas fluorescens did not produce this decarboxylase. Arginine and ornithine decarboxylase are absent for all the strains of fluorescent Pseudomonas.     

Molecular analysis of the Pseudomonas aeruginosa regulatory genes ptxR and ptxS.

We have previously described two Pseudomonas aeruginosa genes, ptxR, which enhances toxA and pvc (the pyoverdine chromophore operon) expression, and ptxS, the first gene of the kgu operon for the utilization of 2-ketogluconate by P. aeruginosa. ptxS interferes with the effect of ptxR on toxA expression. In this study, we have utilized DNA hybridization experiments to determine the presence of ptxR and ptxS homologous sequences in several gram-negative bacteria. ptxR homologous sequences were detected in P. aeruginosa strains only, while ptxS homologous sequences were detected in P. aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens. Using Northern blot hybridization experiments and a ptxS-lacZ fusion plasmid, we have shown that P. aeruginosa ptxR and ptxS are expressed in P. putida and P. fluorescens. Additional Northern blot hybridization experiments confirmed that ptxS is transcribed in P. putida and P. fluorescens strains that carried no plasmid. The presence of a PtxS homologue in these strains was examined by DNA-gel shift experiments. Specific gel shift bands were detected when the lysates of P. aeruginosa, P. putida, and P. fluorescens were incubated with the ptxS operator site as probe. kgu-hybridizing sequences were detected in P. putida and P. fluorescens. These results suggest that (i) ptxR is present in P. aeruginosa, while ptxS is present in P. aeruginosa, P. putida, and P. fluorescens; (ii) both ptxR and ptxS are expressed in P. putida and P fluorescens; and (iii) a PtxS homologue may exist in P. putida and P. fluorescens.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

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.     

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.     

Effect of plasmids from various incompatibility groups on the development of bacteriophages specific to Pseudomonas aeruginosa and Pseudomonas putida

The aim of this work was to study the effect of plasmids belonging to different incompatibility groups on the growth of bacteriophages in Pseudomonas aeruginosa and Pseudomonas putida strains. The growth of bacteriophages was shown to be limited most often due to the presence in cells of plasmids belonging to the P-2 incompatibility group. Plasmids of the Inc P-2 group differed from one another in the spectrum of bacteriophages whose growth they limited. Phages whose growth was suppressed in strains containing plasmids of the P-5, P-9 or P-10 incompatibility groups were found. Some plasmids showed no specific interaction with bacteriophages. The plasmids investigated differed in the studied trait in P. aeruginosa and P. putida cells. In contrast to P. aeruginosa PAO, P. putida PpGI plasmid containing cells did not maintain the growth of donor-specific bacteriophages and, to a lesser degree, limited the growth of phages specific for P. putida PpGI.     

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere.

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.     

L-arginine utilization by Pseudomonas species

The utilization of arginine was studied in several different Pseudomonas species. The arginine decarboxylase and agmatine deiminase pathways were found to be characteristic of Pseudomonas species of group I as defined by Palleroni et al. (1974). Pseudomonas putida strains had three distinct arginine catabolic pathways initiated by arginine decarboxylase, arginine deiminase and arginine oxidase, respectively. The two former routes were also present in P. fluorescens and P. mendocina and in P. aeruginosa which also used arginine by a further unknown pathway. None of these pathways occurred in P. cepacia strains; agmatine catabolism seemed to follow an unusual route involving guanidinobutyrate as intermediate.     

Plasmid control of the Pseudomonas aeruginosa and Pseudomonas putida phenotypes and of linalool and p-cymene oxidation.

Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P. aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P. aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observation implies that support of linalool oxidation effectively conceals the P. aeruginosa character.     

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa was characterized which could not grow anaerobically with nitrate as the terminal electron acceptor or with arginine as the sole energy source. In this anr mutant, nitrate reductase and arginine deiminase were not induced by oxygen limitation. The anr mutation was mapped in the 60-min region of the P. aeruginosa chromosome. A 1.3-kb chromosomal fragment from P. aeruginosa complemented the anr mutation and also restored anaerobic growth of an Escherichia coli fnr deletion mutant on nitrate medium, indicating that the 1.3-kb fragment specifies an FNR-like regulatory protein. The arcDABC operon, which encodes the arginine deiminase pathway enzymes of P. aeruginosa, was rendered virtually noninducible by a deletion or an insertion in the -40 region of the arc promoter. This -40 sequence (TTGAC....ATCAG) strongly resembled the consensus FNR-binding site (TTGAT....ATCAA) of E. coli. The cloned arc operon was expressed at low levels in E. coli; nevertheless, some FNR-dependent anaerobic induction could be observed. An FNR-dependent E. coli promoter containing the consensus FNR-binding site was expressed well in P. aeruginosa and was regulated by oxygen limitation. These findings suggest that P. aeruginosa and E. coli have similar mechanisms of anaerobic control.     

Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli

Anaerobic growth of Pseudomonas aeruginosa on nitrate or arginine requires the anr gene, which codes for a positive control element (ANR) capable of functionally complementing an fnr mutation in Escherichia coli. The anr gene was sequenced; it showed 51% identity with the fnr gene at the amino acid sequence level. Four cysteine residues known to be essential in the FNR protein are conserved in ANR. The anr gene product (deduced Mr 27,129) was visualized by the maxicell method and migrated like a 32 kDa protein in gel electrophoresis under denaturing conditions. An anr mutant of P. aeruginosa constructed by gene replacement was defective in nitrate respiration, arginine deiminase activity, and hydrogen cyanide biosynthesis, underscoring the diverse metabolic functions of ANR during oxygen limitation. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all had a functional analogue of ANR, indicating that similar anaerobic control mechanisms exist in these bacteria.     

Anaerobic transfer of antibiotic resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 x 10(-9) and 5.0 x 10(-8) cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 x 10(-7) and 6.5 x 10(-8) cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.     

Expression of the phospholipase C gene of Pseudomonas aeruginosa in Escherichia coli and Pseudomonas

The plc gene for phospholipase of Pseudomonas aeruginosa, able to be transcribed only from its own promoter, has been introduced into Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida cells in the recombinant plasmid pPMS21 of a wide host range. The expression of plc gene in all recipient cells has been shown to be phosphate regulated. The fact emphasizes the identity of pho-regulation systems in Escherichia coli and Pseudomonas cells. The level of phospholipase activity is similar in Pseudomonas putida and Pseudomonas aeruginosa under the conditions of the gene derepression, while in Escherichia coli cells the level does not exceed 10% of activity registered in Pseudomonas cells.     

The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.