Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand
Correspondence
Iain L Lamont
iain.lamont{at}stonebow.otago.ac.nz
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ABSTRACT |
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INTRODUCTION |
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The structural complexity of pyoverdinePAO suggests that the biosynthetic pathway will involve a number of enzymes in addition to those that have been identified to date. The sequence of the genome of P. aeruginosa strain PAO1 (Stover et al., 2000) provides a new approach for identifying pyoverdine synthesis genes. In this study we identified possible pyoverdine synthesis genes in the genome sequence, mutated them, and determined the effects of the mutations on pyoverdine production. In addition, we determined which genes are present in other pseudomonads and which are restricted to strains that, like P. aeruginosa PAO1, make type I pyoverdine.
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METHODS |
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Gene disruptions in P. aeruginosa.
PCR fragments (1·01·5 kb) corresponding to genes to be mutated were cloned into pGEM T-Easy, excised using restriction enzymes (usually HindIII and EcoRI) corresponding to sites that were incorporated into the PCR primers, and subcloned into pEX18Tc or pEX18Gm. Kanamycin-resistance cassettes were then cloned into restriction sites within the target genes unless the cloned fragment was internal to the gene to be mutated. pEX constructs were transferred into P. aeruginosa PAO1 by triparental conjugation using the helper plasmid pRK2013 as described previously (McMorran et al., 1996) with selection for transconjugants in which plasmid DNA had integrated into the chromosome of P. aeruginosa by homologous recombination. For heterodiploid strains in which conjugation gave rise to bacteria containing both a wild-type and a mutant (kanamycin-disrupted) gene, plasmid DNA containing the wild-type gene was cured from the bacteria by subculture in L-broth containing kanamycin, followed by sucrose-selection for plasmid-lacking strains (Hoang et al., 1998
). DNA from all recombinant P. aeruginosa strains was analysed by PCR and Southern blotting to ensure that the intended mutations had been generated.
Phenotypic analysis of bacteria.
P. aeruginosa strains were analysed for production of pyoverdine by growth on King's B agar and on agar supplemented with EDDA; EDDA prevents the growth of P. aeruginosa strains that are unable to make or take up (ferri)pyoverdine (Ankenbauer et al., 1986), and pyoverdine gives a yellow-green pigmentation around Pvd+ colonies (King et al., 1954
). Pyoverdine production was quantified by growing cultures of bacteria in King's B broth as described previously (McMorran et al., 2001
).
Computational analysis.
DNA sequences were obtained from the P. aeruginosa genome project (http://www.pseudomonas.com) and the P. aeruginosa genome database (http://pseudomonas.bit.uq.edu.au). Sequences were manipulated using Seqed (Devereux et al., 1984) and analysed using NLDNA and Codonuse as described previously (Merriman et al., 1995
). Database searches and analysis of the genomes of other fluorescent pseudomonads were carried out at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) with BLAST algorithms.
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RESULTS AND DISCUSSION |
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While this manuscript was being prepared, another study described mutations in many of the genes characterized in this study (Ochsner et al., 2002). The phenotypes obtained by these researchers are listed in Table 3
and are consistent with those described here. The role of pvdL in pyoverdine synthesis has also been described very recently (Mossialos et al., 2002
).
A total of 15 pvd genes have now been identified that are essential for pyoverdine synthesis in P. aeruginosa PAO1 and it is likely that most, if not all, of the genes that are essential for pyoverdine synthesis in this strain are now known. An early study mapped two mutations that affected pyoverdine synthesis to a locus at 23 min on the recalibrated genetic map of P. aeruginosa PAO1 (Hohnadel et al., 1986) but further study of these mutants has not been reported. The pvc genes lie at about 6670 min on the genetic map (Stintzi et al., 1996
), about 240 kb away from pvdS. All other pvd mutations, including all of the 24 mutations identified by Tsuda et al. (1995)
, have been mapped to the 47 min region of the P. aeruginosa chromosome that corresponds to the part of the genome represented in Fig. 2
. We have not mutated the 17 genes in this interval that did not meet our criteria for candidate pyoverdine synthesis genes and so cannot exclude the possibility that they contribute to pyoverdine synthesis. However, two of these genes (PA2403 and PA2407) were mutated by Ochsner et al. (2002)
and the mutant bacteria retained the ability to make pyoverdine. In addition the sequences of several of these genes suggest that they have functions other than pyoverdine synthesis. For example, PA2414 has 55 % sequence identity with L-sorbosone dehydrogenase from Acetobacter liquefaciens and PA2416 has 55 % identity with a periplasmic trehalase from E. coli (data not shown). A number of mutations in this part of the genome did not affect pyoverdine synthesis (Tsuda et al., 1995
).
Detection of pvd gene homologues in other Pseudomonas strains
The three different kinds of pyoverdines (types IIII) that are made by strains of P. aeruginosa all have the same dihydroxyquinoline component and are distinguished by the compositions of their peptides (Meyer et al., 1997; Meyer, 2000
) (Fig. 1
). It is likely that synthesis of the shared dihydroxyquinoline group, with its attached carboxylic acid or amide, has the same biosynthetic pathway in all strains and that the enzymes for this are encoded by orthologous genes in different strains. In contrast, type-specific genes probably direct synthesis of the peptides that distinguish the different pyoverdines.
Pyoverdine synthesis genes from P. aeruginosa PAO1 were used as hybridization probes with genomic DNA from other P. aeruginosa strains that produce type I, type II or type III pyoverdine (Fig. 3, Table 4
). Hybridizations were carried out under conditions of high stringency to ensure that only very similar DNA sequences would hybridize. Some of the probes hybridized with DNA from all of the strains tested, indicating that orthologous genes are present in all of these strains. Other genes were only present in type I strains.
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Genes that are present in only some strains (pvdA, pvdD, pvdF, pvdI and pvdJ in P. aeruginosa PAO1) most likely direct synthesis of the peptide components of pyoverdines. This has been demonstrated biochemically for the products of pvdA and pvdF that catalyse hydroxylation of ornithine and formylation of hydroxyornithine, respectively, to generate N5-formyl- N5-hydroxyornithine, which is present in the peptide of type I pyoverdine (McMorran et al., 2001; Visca et al., 1994
). Homologues of pvdA have been shown to be present in a strain of P. aeruginosa that makes type II pyoverdine and also in strains of P. fluorescens and Burkholderia cepacia, though not in a strain of P. putida (Visca et al., 1994
). A homologue of pvdA from Pseudomonas sp. B10 complemented a pvdA mutation in P. aeruginosa PAO1 (Ambrosi et al., 2000
), showing that it is an orthologue of pvdA. In this study, homologues of pvdA were present in all strains of P. aeruginosa that were tested, and also in P. fluorescens and P. putida, though not P. syringae (Table 4
). However homologues of pvdF were not found outside P. aeruginosa type I strains and synthesis of formylhydroxyornithine must involve a different biosynthetic process in other strains/species that incorporate this compound into pyoverdine or pseudobactin.
pvdD, pvdI and pvdJ were also detected by hybridization only in strains of P. aeruginosa making type I pyoverdine. These genes are thought to encode peptide synthetases (Lehoux et al., 2000; Merriman et al., 1995
), a family of proteins with many conserved sequence features (Marahiel et al., 1997
). PvdD directs incorporation of two L-threonine residues into pyoverdinePAO (Ackerley et al., 2003
) and pvdI and pvdJ are very likely to encode peptide synthetases that direct incorporation of the remaining six amino acids into the peptide of type I pyoverdines. Homologues of these gene products are present in the other fluorescent Pseudomonas species but the levels of sequence identity (4156 %) were no higher than those of paralogues of pvdD, pvdI and pvdJ in the P. aeruginosa genome (data not shown). The homologues in the other species are very likely to encode peptide synthetases that direct incorporation of different amino acids into pyoverdines/pseudobactins, or other secondary metabolites.
The pvdE gene product has all the characteristics of an ABC-type transporter protein (McMorran et al., 1996) although its substrate(s) has not been identified. Homologues were not detected by Southern blotting in other strains of P. aeruginosa, suggesting that the substrate is strain-specific although homologues are present in other fluorescent pseudomonads. ABC transporter proteins that have many shared sequence features may have different substrates (Higgins, 1992
, 2001
). It remains to be determined whether the PvdE homologues present in other species transport the same substrate as in P. aeruginosa PAO1, or whether they transport a different substrate with the sequence similarities reflecting shared structural features.
In conclusion, the research described here has identified eight previously undescribed genes that are required for synthesis of pyoverdine. Analysis of the distribution of pyoverdine synthesis genes amongst fluorescent pseudomonads, along with analysis of their sequences, indicates their possible roles in the biochemical pathway of pyoverdine synthesis. This will provide the basis for biochemical characterization of individual enzymes and a complete description of the pathway of pyoverdine synthesis.
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ACKNOWLEDGEMENTS |
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Received 31 October 2002;
revised 6 December 2002;
accepted 23 December 2002.