Wild-type Strain PAO1 Research Articles

Pyoverdin is essential for virulence of Pseudomonas aeruginosa

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.

Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA.

Conversion to a mucoid, exopolysaccharide alginate-overproducing phenotype in Pseudomonas aeruginosa is associated with chronic respiratory infections in cystic fibrosis. Mucoidy is caused by muc mutations that derepress the alternative sigma factor AlgU, which in turn activates alginate biosynthetic and ancillary regulatory genes. Here we report the molecular characterization of two newly identified genes, algW and mucD, that affect expression of mucoidy. The algW gene, mapping at 69 min, was isolated on the basis of its ability to suppress mucoidy and reduce transcription of the alginate biosynthetic gene algD. The predicted primary structure of AlgW displayed similarity to HtrA (DegP), a serine protease involved in proteolysis of abnormal proteins and required for resistance to oxidative and heat stress in enteric bacteria. Inactivation of algW on the chromosome of the wild-type nonmucoid strain PAO1 caused increased sensitivity to heat, H2O2, and paraquat, a redox cycling compound inducing intracellular levels of superoxide. This mutation also permitted significant induction of alginate production in the presence of subinhibitory concentrations of paraquat. Two new genes, mucC and mucD, were identified immediately downstream of the previously characterized portion (algU mucA mucB) of the gene cluster at 67.5 min encoding the alternative sigma factor AlgU and its regulators. Interestingly, the predicted gene product of mucD also showed similarities to HtrA. Inactivation of mucD on the PAO1 chromosome resulted in conversion to the mucoid phenotype. The mutation in mucD also caused increased sensitivity to H2O2 and heat killing. However, in contrast to algW mutants, no increase in susceptibility to paraquat was observed in mucD mutants. These findings indicate that algW and mucD play partially overlapping but distinct roles in P. aeruginosa resistance to reactive oxygen intermediates and heat. In addition, since mutations in mucD and algW cause conversion to mucoidy or lower the threshold for its induction by reactive oxygen intermediates, these factors may repress alginate synthesis either directly by acting on AlgU or its regulators or indirectly by removing physiological signals that may activate this stress response system.

Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000.

Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker.

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.