Mapping of the loci involved in the catabolic oxidation of L-hydroxyproline in Pseudomonas aeruginosa PAO

Summary Genes specifying the oxidative utilization of hydroxyproline (Hyp) in P. aeruginosa PAO were located on the chromosome, around 19th minute by conjugation experiments. A map order of his-68-his-07-Hyp was assigned. Confirmation of this gene order was also demonstrated by transductional mapping studies. All the genes determining the enzymes of Hyp dissimiliatory pathway were closely linked.     

Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO

Summary Transposon insertion mutagenesis of the Pseudomonas aeruginosa PAO chromosome with Tn1 and Tn501 was carried out using a mutant plasmid of R68::Tn501 temperature-sensitive for replication and maintenance. This method consists of three steps. Firstly, the temperature-independent, drug-resistant clones were selected from the strain carrying this plasmid. In the temperature-indepent clones, the plasmid was integrated into the chromosome by Tn1- or Tn501-mediated cointegrate formation. Secondly, such clones were cultivated at a permissive temperature to provoke the excision of the integrated plasmid from the chromosome. Excision occurred by the reciprocal recombination between the two copies of Tn1 or Tn501 flanking the integrated plasmid, leaving one Tn1 or Tn501 insertion on the chromosome. Thirdly, the excised plasmid was cured by cultivating these isolates at a non-permissive temperature without selection for the drug resistance. Using this method, we isolated 1 Tn1-induced and 43 Tn501-induced auxotropic mutations in this organism. Genetic mapping allowed us to identify two new genes, pur-8001 and met-8003. The Tn501-induced auxotrophic mutations were distributed non-randomly among auxotrophic genes, and the reversion of the mutations by precise excision of the Tn501 insertion occurred very rarely.     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

Pseudomonas aeruginosa ExoS and ExoT

ExoS and ExoT are bi-functional type-III cytotoxins of Pseudomonas aeruginosa that share 76% primary amino acid homology and contain N-terminal RhoGAP domains and C-terminal ADP-ribosylation domains. The Rho GAP activities of ExoS and ExoT appear to be biochemically and biologically identical, targeting Rho, Rac, and Cdc42. Expression of the RhoGAP domain in mammalian cells results in the disruption of the actin cytoskeleton and interference of phagocytosis. Expression of the ADP-ribosyltransferase domain of ExoS elicits a cytotoxic phenotype in cultured cells, while expression of ExoT appears to interfere with host cell phagocytic activity. Recent studies showed that ExoS and ExoT ADP-ribosylate different substrates. While ExoS has poly-substrate specificity and can ADP-ribosylate numerous host proteins, ExoT ADP-ribosylates a more restricted subset of host proteins including the Crk proteins. Protein modeling predicts that electrostatic interactions contribute to the substrate specificity of the ADP-ribosyltransferase domains of ExoS and ExoT.     

Pseudomonas aeruginosa lipopolysaccharides and pathogenesis

Pseudomonas aeruginosa lipopolysaccharide (LPS) plays a key role in pathogenesis. In acute infections, a smooth LPS protects the organism from complement-mediated killing and, during chronic lung infections, an altered rough LPS helps the organism evade host defense mechanisms.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.     

PHAMCL biosynthesis systems in Pseudomonas aeruginosa and Pseudomonas putida strains show differences on monomer specificities

The production of PHA from plant oils by Pseudomonas species soil isolated from a sugarcane crop was evaluated. Out of 22 bacterial strains three were able to use efficiently plant oils to grow and to accumulate PHA. Pseudomonas putida and Pseudomonas aeruginosa strains produced PHA presenting differences on monomer composition compatible with variability on monomer specificity of their PHA biosynthesis system. The molar fraction of 3-hydroxydodecanoate detected in the PHA was linearly correlated to the oleic acid supplied. A non-linear relationship between the molar fractions of 3-hydroxy-6-dodecenoate (3HDdΔ₆) detected in PHA and the linoleic acid supplied was observed, compatible with saturation in the biosynthesis system capability to channel intermediate of β-oxidation to PHA synthesis. Although P. putida showed a higher 3HDdΔ₆ yield from linoleic acid when compared to P. aeruginosa, in both species it was less than 10% of the maximum theoretical value. These results contribute to the knowledge about the biosynthesis of PHA with a controlled composition from plant oils allowing in the future establishing the production of these polyesters as tailor-made polymers.     

Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

Background  

Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia.     

An anion-selective channel from the Pseudomonas aeruginosa outer membrane

Protein P from Pseudomonas aeruginosa outer membrane was reconstituted in lipid bilayer membranes from diphytanoylphosphatidylcholine. The reconstitution resulted in the formation of anion-selective channels with a conductance of 160 pS for 0.1 M chloride solution. The channels were at least 100-times more selective for anions than for cations as judged from zero-current membrane potentials. The single-channel conductance was dependent on the size of the different anions and saturated at higher salt concentrations suggesting single ion occupancy of the protein P channel.     

Association of the biocide 5-chloro-2-methyl-isothiazol-3-one with Pseudomonas aeruginosa and Pseudomonas fluorescens

The biocide 5-chloro-2-methyl-isothiazol-3-one (CMI) associated rapidly with cells of Pseudomonas aeruginosa and Pseudomonas fluorescens with association being nearly complete within 10–15 min. The cells serve as a sink for CMI, concentrating it up to 400-fold. Kinetics of association are very similar amongst the strains examined. Examination of the relation of CMI concentration to the rate of association indicates that there are two kinetically distinguishable processes, with the breakpoint occurring around the transition from inhibitory to suprainhibitory levels of CMI. This suggests the rapid onset of toxic effects at suprainhibitory CMI concentrations affects the associative process. Discharging the proton motive force by treatment with uncoupling agents or selectively depleting it by treatment with inhibitors reduces the amount of CMI which associates with the cells. Selective depletion of the ATP pool has no effect. These results suggest that either the proton motive force (pmf) is involved directly in CMI association in an active transport process, or that an intact pmf is required for some facet of the cells metabolism which maintains the cells as a sink for CMI. The nonchlorinated analogue 2-methyl-isothiazol-3-one is a poor competitor for CMI association.     

Algicidal activity of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa

The algicidal activity of the rhamnolipid biosurfactants (the mixture of Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀) produced by Pseudomonas aeruginosa was investigated in the present paper. The results indicated that the biosurfactants had potential algicidal effects on the harmful algal bloom (HAB) species, Heterosigma akashiwo. The growth of H. akashiwo was strongly inhibited in medium containing rhamnolipids (0.4–3.0 mg L⁻¹); moreover, the rhamnolipids showed strong lytic activity toward H. akashiwo at higher concentrations (≥4.0 mg L⁻¹). In addition, the effects of the rhamnolipids on the growth of Gymnodinium sp. and Prorocentrum dentatum, another two kinds of HAB species, were also studied. Compared with the dramatic algicidal effect on H. akashiwo, the cells of P. dentatum were inhibited or lysed at higher concentrations (1.0–10.0 mg L⁻¹), while the cells of Gymnodinium sp. were not suppressed with the same treatment, indicating the rhamnolipids had the potential for the selective control of HABs.Morphometric analysis at ultrastructural level by transmission electron micrographs indicated that the extent of ultrastructural damage of the alga was severe at high concentrations of rhamnolipids and during extended periods of contact. The first response occurred in the plasma membrane which partly disintegrated. The lack of membrane facilitated the rhamnolipid biosurfactants into the cells and allowed damage to other organelles, which resulted in the injury of chloroplast, vacuolization of mitochondria and deformation of the cristae, disruption of nuclear membrane and condensation of chromatin in nucleus, suggesting that the lytic activity of rhamnolipids was mainly due to their powerful surfactivity and their tendency to cohere on the surface of phospholipids bimolecular layer of the cells and further destroyed the layers, and then the structure of quasi-membrane configurations inside the cells was disintegrated, following by the irreversible damage to the ultrastructure and the loss of the function of organelles, consequently leading the cells to lyse.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Isolation and characterisation of deletion mutants involving the transfer genes of P-group plasmids in Pseudomonas aeruginosa

Summary The P-group plasmids RP1 and R26 are recovered at low frequency following conjugal transfer to B3-lysogens of P. aeruginosa PAO. The rare carbenicillin-resistant transcipients that do arise are usually transfer-defective (Tra^-) and may show the loss of other plasmid borne functions, namely kanamycin-resistance (Km^r) and reduced plating of phage GlOl (Spp⁺). The four phenotypic classes that occur among the Tra^- derivatives are respectively, Tra^- (69–81%), Tra^- Spp^- (12–30%), Tra^- Km^s and Tra^- Km^s Spp^- (0.2–1%), of which the latter three are due to plasmid deletions. This is seen from the sizes of the plasmids carried by these bacteria and from the transductional analysis of the R26-derivatives. Thus, although R26 (MW=52×10⁶ daltons) is too large to be transduced by phage F116L (MW=40×10⁶), this is possible for its Tra^- Km^s and Tra^- Km^s Spp^- derivatives. The phenotypes and frequencies of the various transcipient classes suggests that the gene order Km..Tra..Spp occurs in both RP1 and R26, and that Spp is more closely linked to Tra than is Km. These conclusions are supported by the sizes of the plasmid mutants since deletions spanning the loci Km Tra Spp, Km Tra, and Tra Spp involve the loss of DNA of MW 8-17×10⁶, 5-13×10⁶ and 1-9×10⁶ daltons respectively.Whilst all the transcipients displayed the incompatibility properties of the parent plasmids (Inc⁺), only some retained plasmid surface exclusion (Sfx⁺). Moreover, a strict correlation existed between the Sfx and Spp phenotypes such that the transcipients were either wild type, Sfx^- Spp^-, or displayed an intermediate phenotype for both characters. This suggests that these phenotypes are controlled by closely linked genes or are different manifestations of the same gene function. The deletion map of these various markers in both RP1 and R26 therefore seems to be Km..Tra..Sfx/Spp..Inc.     

The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa

Summary Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyltransferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.     

Cloning and sequencing of the Pseudomonas aeruginosa 1244 pilin structural gene

The pilin structural gene of Pseudomonas aeruginosa 1244 was cloned in both cosmids and lambda. Expression of the cloned gene was detected in P. aeruginosa strains PAO2003, PA103, and 653A by an immunoblot reaction utilizing monoclonal antibodies. Western blot analysis showed that pilin expressed from the cloned gene was slightly larger than native 1244 pilin when produced in strains PAO2003 and 653A, but distinctly smaller in PA103. Bacteriophages specific for the 1244 pilus did not lyse strain PAO2003 containing the cloned 1244 pilin gene, indicating that functional 1244 pili were not assembled in this recombinant strain. Nucleotide sequencing revealed a coding region which when translated would produce a 15,615 dalton peptide. The amino-terminal region of this peptide is identical with published pilin sequences. While the rest of the peptides are generally dissimilar, common residues are seen within potentially antigenic regions.     

Expression and activation of an esterase from Pseudomonas aeruginosa 1001 in Escherichia coli

An expression vector was constructed to overproduce a maltose binding protein (MBP)-esterase fusion protein in Escherichia coli. Soluble fusion protein was separated by centrifugation after cell disruption. The fusion protein was partially purified with amylose resin. The higher concentration of fusion protein (above 2 mg/ml) did not show any activity but about 0.3 mg/ml of fusion protein had the highest activity (142 U/ml). It is due to the difficulty of contact between substrate and active site of enzyme in compact form at high concentration. The fusion protein over-expressed could not be separated into MBP and esterase by the action of protease ‘Factor Xa’. The esterase could be cleaved from MBP fusion protein by the treatment of SDS with the Factor Xa, and the resulting esterase activity was increased to 34% after cleavage.