Cloning and nucleotide sequence of the pvdA gene encoding the pyoverdin biosynthetic enzyme L-ornithine N5-oxygenase in Pseudomonas aeruginosa.

The enzyme L-ornithine N5-oxygenase catalyzes the hydroxylation of L-ornithine (L-Orn), which represents an early step in the biosynthesis of the peptidic moiety of the fluorescent siderophore pyoverdin in Pseudomonas aeruginosa. A gene bank of DNA from P. aeruginosa PAO1 (ATCC 15692) was constructed in the broad-host-range cosmid pLAFR3 and mobilized into the L-Orn N5-oxygenase-defective (pvdA) P. aeruginosa mutant PALS124. Screening for fluorescent transconjugants made it possible to identify the trans-complementing cosmid pPV4, which was able to restore pyoverdin synthesis and L-Orn N5-oxygenase activity in the pvdA mutant PALS124. The 17-kb PAO1 DNA insert of pPV4 contained at least two genetic determinants involved in pyoverdin synthesis, i.e., pvdA and pvdC4, as shown by complementation analysis of a set of mutants blocked in different steps of the pyoverdin biosynthetic pathway. Deletion analysis, subcloning, and transposon mutagenesis enabled us to locate the pvdA gene in a minimum DNA fragment of 1.7 kb flanked by two SphI restriction sites. Complementation of the pvdA mutation was under stringent iron control; both pyoverdin synthesis and L-Orn N5-oxygenase activity were undetectable in cells of the trans-complemented mutant which had been grown in the presence of 100 microM FeCl3. The entire nucleotide sequence of the pvdA gene, from which the primary structure of the encoded polypeptide was deduced, was determined. The pvdA structural gene is 1,278 bp; the cloned DNA fragment contains at the 5' end of the gene a putative ribosome-binding site but apparently lacks known promoterlike sequences. The P. aeruginosa L-Orn N5-oxygenase gene codes for a 426-amino-acid peptide with a predicted molecular mass of 47.7 kDa and an isoelectric point of 8.1. The enzyme shows approximately 50% homology with functional analogs, i.e., L-lysine N6-hydroxylase of aerobactin-producing Escherichia coli and L-Orn N5-oxygenase of ferrichrome-producing Ustilago maydis. The pvdA gene was expressed in P. aeruginosa under the control of the T7 promoter. Induction of the T7 RNA polymerase system resulted in parallel increases of the L-Orn N5-oxygenase activity and of the amount of a 47.7-kDa polypeptide. We also constructed a site-specific pvdA mutant by insertion of a tetracycline-resistance cassette in the chromosomal pvdA gene of P. aeruginosa PAO1. Similarly to strain PALS124, the pvdA mutant obtained by gene disruption also disclosed no pyoverdin synthesis, lacked L-Orn N5-oxygenase activity, was complemented by the cloned pvdA gene, and produced pyoverdin at wild-type levels when fed with the biosynthetic precursor L-N5-OH-Orn. Southern blot analysis indicated that genes homologous to pvdA could be located within a 1.7-kb DNA fragment from SphI-digested genomic DNA of different hydroxamate-producing Pseudomonas spp. Our results suggest that omega-amino acid oxygenases have been conserved over a wide evolutionary range and probably evolved from a common ancestor.     

Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa

Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.     

Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1

The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in Pseudomonas is a condensation product of D-tyrosine and l-2,4-diaminobutyrate. Both pvdH and asd (encoding aspartate beta-semialdehyde dehydrogenase) knockout mutants of Pseudomonas aeruginosa PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of l-2,4-diaminobutyrate in the culture media. The pvdH gene was subcloned, and the gene product was hyperexpressed and purified from P. aeruginosa PAO1. PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate beta-semialdehyde and l-2,4-diaminobutyrate. Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for alpha-ketoglutarate. The specificity of the enzyme toward the smaller keto acid pyruvate is 41-fold lower. The enzyme has negligible activity toward other keto acids tested. Homologues of PvdH were present in the genomes of other Pseudomonas spp. These homologues were found in the DNA loci of the corresponding genomes that contain other pyoverdine synthesis genes. This suggests that there is a general mechanism of l-2,4-diaminobutyrate synthesis in Pseudomonas strains that produce the pyoverdine siderophore.     

Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation.

In addition to the two siderophores pyoverdine and pyochelin synthesized by Pseudomonas aeruginosa ATCC 15692 (strain PAO1), several siderophores produced by other bacteria or fungi, namely cepabactin, salicylic acid, desferriferrichrysin, desferriferricrocin, desferriferrioxamine B, desferriferrioxamine E and coprogen, were able to promote iron uptake with variable efficiencies into this bacterium. For most of these siderophores, these results were consistent with the growth stimulation produced by the same compounds in a plate bioassay. Desferriferrichrome A, enterobactin and desferriferrirubin, however, did not promote iron uptake, although enterobactin and desferriferrirubin stimulated bacterial growth. These paradoxical data are discussed in view of siderophore-inducible iron uptake systems, as demonstrated recently for enterobactin. Among the strains tested, including the wild-type PAO1, the pyoverdine-less mutant PAO6606 and the two porin-mutants P. aeruginosa H636 (oprF::omega) and P. aeruginosa H673 (oprD::Tn501), only for the porin-OprF mutant were fewer siderophores able to promote iron uptake compared to the other strains. Such results suggest that beside specific routes for iron uptake P. aeruginosa is also able to take up siderophore-liganded iron through OprF.     

Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of (59)Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Mapping of mutations affecting pyoverdine production in Pseudomonas aeruginosa

Abstract Pyoverdine, the yellow-green fluorescent pigment produced by Pseudomonas aeruginosa , is a highly efficient siderophore. Pyoverdine-deficient ( pvd ) mutants of P. aeruginosa PAO isolated after mutagenesis were non-fluorescent and unable to grow in the presence of 2.8 mM ethylenediamine-di-( o -hydroxyphenylacetate) (EDDHA). Addition of purified pyoverdine to media containing EDDHA restored growth of pvd mutants. 6 pvd mutations were mapped between catA and mtu -9002 (at 65–70 min on the chromosome map) by R68.45-mediated conjugation. 2 slightly leaky pvd mutations were localised between argC and strA (at 35 min) by transduction. Thus, we have identified at least 2 genes or gene clusters required for pyoverdine production in P. aeruginosa .     

铜绿假单胞菌Arr基因突变对生物膜和绿脓菌素合成的影响

为了研究铜绿假单胞菌Arr基因对生物膜和绿脓菌素合成的影响,采用抗庆大霉素基因序列(Gentamycin resistance cassette,aacC1)插入失活的策略构建了铜绿假单胞菌Arr基因突变株PA-AG,通过96孔板静止培养、结晶紫染色的方法检测其生物膜的形成量,利用抽提的方法检测绿脓菌素的合成量。结果在KMB或LB培养基中,突变株PA-AG形成生物膜的量均有所减少,野生株约是突变株的2倍,然而突变株合成绿脓菌素的能力却明显加强,约为野生株的2.5倍。由此推测,铜绿假单胞菌Arr基因在一定程度上促进了生物膜的形成,抑制了绿脓菌素的合成。     

Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa

Novel putative pyoverdine synthetase pvdIJK genes were found upstream of pvdD in the 6.2-Mb chromosome of Pseudomonas aerugilosa strain PAO1. These genes formed a locus implicated in pyoverdine biosynthesis. Sequence analysis showed that the product of these genes shared 43%, 60% and 57% identity with PvdD. PvdIJK are thought to be implicated in synthesis of pyoverdine, a siderophore chelating Fe3+. A pvdI mutant was obtained by gene disruption mutagenesis and confirmed by Southern hybridization. The pvdl mutant produced gave no significant growth on solid media supplemented with the iron chelator 2,2-dipyridyl; while the PvdI- phenotype abolished pyoverdine fluorescence. The role of PvdI in pathogenicity was tested by measuring the in vivo growth of P. aeruginosa wild-type and mutant strains in a chronic lung infection rat model, and by measuring the competitive infectivity index into a neutropenic mice model. The data obtained confirmed the importance of PvdI in virulence and iron uptake.     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

Substrate specificity of the nonribosomal peptide synthetase PvdD from Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdine(PAO), which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified. L-Threonine, one of the amino acid residues in pyoverdine(PAO), was an effective substrate for the recombinant protein in ATP-PP(i) exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including D-threonine, L-serine, and L-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases.     

Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis

Natriuretic peptides of various forms are present in animals and plants, and display structural similarities to cyclic antibacterial peptides. Pretreatment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide (BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced glial cell necrosis. In eukaryotes, natriuretic peptides act through receptors coupled to cyclases. We observed that stable analogs of cAMP (dibutyryl cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natriuretic peptide and CNP on bacteria. Further evidence for the involvement of bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacterial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS) extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and 8-bromo-cGMP induces higher levels of necrosis than LPS extracted from untreated bacteria. Capillary electrophoresis and MALDI-TOF MS analysis have shown that differences in LPS toxicity are due to specific differences in the structure of the macromolecule. Using a strain deleted in the vfr gene, we showed that the Vfr protein is essential for the effect of natriuretic peptides on P. aeruginosa PAO1 virulence. These data support the hypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriuretic peptide sensor system that may affect virulence by activating the expression of Vfr and LPS biosynthesis.     

Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa

The sigma factor RpoS (sigmaS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50 degrees C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.     

Pleiotropic effects of the mioC mutation on the physiology of Pseudomonas aeruginosa PAO1

Flavodoxin (Fld) is a bacterial electron-transfer protein that possesses flavin mononucleotide as a prosthetic group. In the genomes of the Pseudomonas species, the mioC gene is the sole gene, annotated Fld, but its function remains unclear. In this study, phenotype microarray analysis was performed using the wild-type and mioC mutant of pathogenic Pseudomonas aeruginosa PAO1. Our results showed that the mioC mutant is very resistant to oxidative stress. Different antibiotics and metals worked differently on the sensitivity of the mutant. Other pleiotropic effects of mutation in the mioC gene, such as biofilm formation, aggregation ability, motility and colony morphology, were observed under iron stress conditions. Most of the phenotypic and physiological changes could be recovered in the wild type by complementation. Mutation of the mioC gene also influenced the production of pigments. The mioC mutant and mioC over-expressed complementation cells, over-produced pyocyanin and pyoverdine, respectively. Various secreted chemicals were also changed in the mutant, which was confirmed by (1) H NMR analysis. Interestingly, physiological alterations of the mutant strain were restored by the cell-free supernatant of the wild type. The present study demonstrates that the mioC gene plays an important role in the physiology of P.?aeruginosa and might be considered as a suitable drug target candidate in pathogenic P.?aeruginosa.     

TOL plasmid in Pseudomonas aeruginosa PAO: thermosensitivity of self-maintenance and inhibition of host cell growth.

The TOL plasmid originally isolated in Pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of Pseudomonas, i.e., P. putida, P. fluorescens, and P. aeruginosa, except for a strain of P. aeruginosa, strain PAO. The same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. In addition, the transmissibility of the TOL plasmid from strain PAO to P. putida was low when the plasmid was modified by the donor. By using P. aeruginosa PAO carrying the TOL plasmid, the stability and genetic expression of the plasmid as well as its effect on the host cell growth were examined. Thus the self-maintenance of the plasmid was found to be thermosensitive. Furthermore, the TOL plasmid inhibited the growth of strain PAO at high temperature, accompanied by the formation of some filamentous cells. These thermosensitive properties of the TOL plasmid were host dependent and not exhibited in another strain of P. aeruginosa.     

Effect of superoxide dismutase gene inactivation on virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori

To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm, Bombyx mori, mutants of Pseudomonas aeruginosa PAO1 lacking manganese-SOD (PAO1sodM), iron-SOD (PAO1sodB), or both (PAO1sodMB) were generated. The mutants were injected into the hemocoel of B. mori. The virulence decreased in the order PAO1=PAO1sodM>PAO1sodB>PAO1sodMB. In particular, PAO1sodMB was avirulent at a dose of 10(5) cells or less. The sod double mutant PAO1sodMB was then complemented with either pSodM or pSodB in trans. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of P. aeruginosa against B. mori. The results of growth in B. mori hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in sod mutants. In conclusion, the SODs are required for full virulence of P. aeruginosa against B. mori and Fe-SOD is more important than Mn-SOD in the infection process.