Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand1
Author for correspondence: Iain L. Lamont. Tel: +64 3 4797869. Fax: +64 3 4797866. e-mail: iain.lamont{at}stonebow.otago.ac.nz
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ABSTRACT |
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Keywords: pyoverdine, glycinamide ribonucleotide transformylase, secondary metabolite synthesis, evolution of biosynthetic pathways
Abbreviations: EDDA, ethylenediamine(o-hydroxy)phenylacetic acid; GART, glycinamide ribonucleotide transformylase
The GenBank accession number for the sequence reported in this paper is U07359.
a Present address: Centre for Molecular Biology and Biotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia.
b Present address: Louisiana State University Medical Center, Department of Biochemistry, 1901 Perdido Street, New Orleans, LA 70112, USA.
c Present address: Genesis Research and Development Corp. Ltd, PO Box 50, Auckland, New Zealand.
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INTRODUCTION |
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To better understand the molecular events involved in pyoverdine biosynthesis, we have now characterized a further gene, named pvdF, that is required for this process.
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METHODS |
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Measurement of pyoverdine.
To measure pyoverdine production, bacteria were grown in Kings B broth (King et al., 1954 ) to stationary phase and the absorbances of culture supernatants were measured between 350 and 500 nm. Pyoverdine has a characteristic absorbance spectrum in this range, with a peak at 403 nm (Hohnadel et al., 1986
).
Nucleic acid methodology.
Enzymes were purchased from Boehringer Mannheim, except for the Klenow fragment of DNA polymerase I (Amersham) and T4 polynucleotide kinase (New England Biolabs) and were used under the conditions recommended by the manufacturers. Preparation of plasmid DNA, treatment of DNA with enzymes, subcloning of DNA and transformation of plasmid constructs into E. coli were carried out using standard methods (Sambrook et al., 1989 ). DNA molecules were end-polished by using T4 DNA polymerase and the Klenow fragment (Sambrook et al., 1989
). Oligonucleotides were obtained from Otagoligos (Centre for Gene Research, Otago University, Dunedin, New Zealand) and Macromolecular Resources (Colorado State University, USA).
For DNA sequencing, plasmid DNA was prepared using the Wizard Plus Minipreps DNA Purification System (Promega). The sequences of both DNA strands were determined by using chemically synthesized oligonucleotides, and PstI and KpnI restriction fragments of pSOT3 (Rombel & Lamont, 1992 ), in conjunction with an automated sequencer. The sequence was determined across all restriction sites used in subcloning. The sequences were analysed by standard methods with version 8.1-UNIX of the Computer Genetics Group package (Devereux et al., 1984
) in conjunction with other programs as described previously (Merriman et al., 1995
); sequence alignments were performed using PILEUP (Devereux et al., 1984
) and residues were highlighted using BoxShade (http://www.ch.embnet.org/software/BOX_form.html). Southern analysis was done by standard methods (Sambrook et al., 1989
) with chromosomal DNA prepared by the method of Chen & Kuo (1993)
, and Northern (RNA) analysis was done as described previously (Rombel et al., 1995
).
Creating a mutation in pvdF.
A pvdF mutant strain of P. aeruginosa was constructed using a strategy similar to one we have used previously for mutating genes in P. aeruginosa (Cunliffe et al., 1995 ; Markie et al., 1986
). Plasmid pSOT3 DNA was treated with XhoI, which has a unique site within pvdF, and ligated to a kanamycin resistance cassette (kan) that had been purified from agarose following SalI treatment of pUC18-19Km (Markie et al., 1986
). The ligated DNA was transformed into E. coli MC1061 (Casabadan & Cohen, 1980
) with selection for Apr and Kmr bacteria. Plasmid DNA was prepared from one transformant, and restriction analysis confirmed that the kan gene had been inserted correctly into pvdF; the resulting plasmid was named pSOT3Km. Purified pSOT3Km DNA was treated with SalI to release a fragment containing the mutated pvdF gene. This fragment was end-polished and subcloned into pSUP202 (Simon et al., 1986
) which had been treated with EcoRI and end-polished. The DNA was transformed into E. coli MC1061 to give Apr Kmr Tcr transformants; restriction analysis confirmed that the plasmid DNA of one such transformant (pSUPFKm) consisted of pSUP202 carrying the mutated pvdF gene. This plasmid was transferred by triparental conjugation into P. aeruginosa PAO1 (Holloway, 1955
) in conjunction with helper plasmid pRK2013 (Figurski & Helinski, 1979
) as described previously (Merriman & Lamont, 1993
), with selection for Tcr Kmr bacteria which arose following recombination of the plasmid into the chromosome. A second recombination event was required for excision of the plasmid and loss of the wild-type pvdF gene, and bacteria in which this had occurred were identified as being Tcs Kmr.
Chemical determinations.
Wild-type P. aeruginosa PAO1 and the pvdF mutant strain were grown in succinate medium (Meyer & Abdallah, 1978 ) to early stationary phase (OD600 1·01·2). Cell lysates were prepared by sonication and samples were incubated at 20 °C for 1216 h after the addition of an equal volume (2 ml) of sodium phosphate buffer (100 mM, pH 7·0) containing 2 mM sodium pyruvate and 4 mM L-ornithine as described previously (Visca et al., 1994
). The amounts of hydroxylamine nitrogen were then determined using the Csaky test as modified by Gillam et al. (1971)
with hydroxylamine hydrochloride as a standard. The combined amounts of hydroxamate and hydroxylamine nitrogen were determined using a modification of the acid hydrolysis method of Gibson & McGrath (1970)
, which converts hydroxamate groups into hydroxylamines. Samples were heated at 130 °C for 30 min in the presence of 12 M HCl and neutralized by the addition of an excess of CaCO3. The amounts of hydroxylamine nitrogen were then assayed, with each assay being carried out in triplicate. Assays were also carried out with a P. aeruginosa PAO1 pvdS mutant that does not express genes involved in pyoverdine synthesis (Cunliffe et al., 1995
; Leoni et al., 1996
; Miyazaki et al., 1995
) in order to determine hydroxylamine nitrogen generated through other pathways. The levels of reactive material obtained with this strain (equivalent to approx 6·2 nmol hydroxylamine without hydrolysis, and 7·7 nmol hydroxylamine with hydrolysis) were subtracted from those obtained with the wild-type and pvdF mutants in order to determine the amounts of pyoverdine-related reactive material for these strains.
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RESULTS |
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To identify proteins similar to PvdF, the predicted amino acid sequence was compared to protein sequence databases using PSI-BLAST (Altschul et al., 1997 ). All of the most similar proteins were glycinamide ribonucleotide transformylase (GART; EC 2.1.2.2) enzymes from a very wide variety of species, including bacteria, fungi, plants and animals and an alignment of PvdF and some GART enzymes is shown in Fig. 3
. Pairwise alignments using GAP (Devereux et al., 1984
) showed that PvdF has 2327% identity and 4858% similarity with various GART enzymes including those shown in Fig. 3
. GART is involved in the de novo synthesis of purines, catalysing the formylation of glycinamide ribonucleotide to formylglycinamide ribonucleotide in conjunction with the cofactor N10-formyltetrahydrofolate (Buchanan & Hartman, 1959
). Several of the residues identified as being important for the function of the E. coli version of GART (Almassy et al., 1992
; Chen et al., 1992
; Warren et al., 1996
; Shim & Benkovic, 1999
; Greasley et al., 1999
) are present in the PvdF sequence (Fig. 3
). This suggests that PvdF possesses a similar catalytic activity.
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The pvdF coding sequence was disrupted in vitro by the insertion of a kanamycin resistance (kan) cassette derived from pUC18-19Km (Markie et al., 1986 ) into the XhoI site of the pvdF gene. The wild-type gene was then replaced with the mutant version as described in the Methods. Southern blotting, and also PCR with primers specific to pvdF and the kan cassette, followed by sequencing of the PCR product, confirmed that the expected recombination events had taken place (data not shown); the mutant strain was named PAO1pvdF.
The pvdF mutant was analysed for production of pyoverdine and the ability to make purines. Kings B medium (King et al., 1954 ) stimulates production of pyoverdine, observed as a yellow-green fluorescent compound. No pyoverdine synthesis was detectable when PAO1pvdF was grown on solid or in liquid Kings B, as observed previously with mutations in other pyoverdine genes (Cunliffe et al., 1995
; McMorran et al., 1996
; Merriman et al., 1995
), whereas the wild-type strain made large amounts of pyoverdine. In addition, the pvdF mutant strain was unable to grow on Kings B agar containing EDDA, an iron-chelating agent that prevents the growth of pyoverdine-deficient strains, whereas the isogenic wild-type strain grew on this medium. These data show that PAO1pvdF was unable to make pyoverdine. However, this strain was able to grow on EDDA-containing medium when pyoverdine was supplied from wild-type cells, indicating that its ability to take up and utilize ferri-pyoverdine was not affected. Reintroduction of the pvdF gene that had been cloned into plasmid pUCP22 (West et al., 1994
) restored the ability of PAO1pvdF to make pyoverdine, confirming the involvement of pvdF in pyoverdine synthesis.
To test the possible involvement of the pvdF gene product in purine synthesis, the mutant strain and the isogenic wild-type strain were inoculated onto minimal medium lacking purines. Both strains showed good growth, indicating that the PvdF protein is not essential for purine synthesis by P. aeruginosa. Analysis of the P. aeruginosa genome sequence (Stover et al., 2000 ) showed that a gene located at nt 10327131032048 in the genome encodes an enzyme that is 53% identical (72% similar) to GART from E. coli and is likely to be the GART enzyme of P. aeruginosa; this sequence was used in the alignment (Fig. 3
).
Transcriptional analysis of pvdF
Transcription of pvdF was analysed using Northern blotting (Fig. 4). A single hybridizing band was obtained with RNA extracted from iron-starved cells and no bands were detectable in RNA from cells grown in high-iron medium. This is consistent with previous results (Rombel et al., 1995
) and confirms that the level of iron regulates pvdF. The size of the hybridizing band, 0·95 kb, was less than that estimated previously (1·2 kb), although it corresponds well with the size of pvdF. The value determined here is likely to be more accurate as it was obtained by comparison with RNA molecular mass standards; previous estimates were made using the rRNA bands (Rombel et al., 1995
).
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For wild-type bacteria, cell extracts contained 3·3±2·0 nmol ml-1 (mean±SD) hydroxylamine nitrogen in the absence of acid hydrolysis. This increased to 10·1±3·8 nmol ml-1 following hydrolysis, showing that a significant amount of hydroxamate nitrogen was present. The hydroxylamine nitrogen detected in the absence of acid hydrolysis may reflect incomplete synthesis of N5-formyl-N5-hydroxyornithine from N5-hydroxyornithine. By contrast, the amount of hydroxylamine nitrogen in cell extracts from the pvdF mutant bacteria was 10·0±6·8 nmol ml-1 in the absence of acid hydrolysis and this did not change significantly following hydrolysis (11·6±7·0 nmol ml-1). This indicates that these bacteria were unable to form N5-formyl-N5-hydroxyornithine and consequently accumulated N5-hydroxyornithine. The amounts of hydroxylamine formed by the pvdF cell extracts were comparable to the amounts obtained with wild-type extracts after acid hydrolysis, showing that the mutant bacteria were not defective for hydroxylamine synthesis. These data support the hypothesis that PvdF catalyses the formation of N5-formyl-N5-hydroxyornithine from N5-hydroxyornithine.
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DISCUSSION |
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Micro-organisms produce a wide range of secondary metabolites and many of the genes that are involved are clearly members of multi-gene families that are specific to secondary metabolite synthesis. These include the peptide synthetase and polyketide synthetase families that are involved in very many pathways of secondary metabolism (Zuber & Marahiel, 1997 ; Khosla et al., 1999
). In contrast to these gene families, the pvdF gene has similarity to GART, an enzyme of primary metabolism. However, pvdF is required for synthesis of pyoverdine, a secondary metabolite, and not for purine synthesis. This indicates that an enzyme involved in primary metabolism has been co-opted into the biosynthetic pathway for production of a secondary metabolite. So far as we are aware, this is the first report of the involvement of a GART-like enyzme in synthesis of a secondary metabolite. The PvdF sequence has three residues that are known to be involved in catalysis in E. coli GART, and also a high level of similarity at sites that are likely to interact with the formyl tetrahydrofolate co-substrate (Fig. 3
), suggesting that PvdF may utilize the same co-substrate. A complete characterization of the reaction catalysed by PvdF will require purification of the protein. However, some of the differences between PvdF and GART enzymes presumably reflect the involvement of a different substrate. A number of inhibitors of GART enzymes have been identified (Kamen, 1997
; Takimoto, 1997
) and it will be of interest to determine whether any of these inhibit PvdF, or whether information on GARTinhibitor interactions can enable development of inhibitors of PvdF.
PvdF is only slightly more similar to the likely GART enzyme of P. aeruginosa than to the other GART enzymes shown in Fig. 3 so that either pvdF is the product of duplication of the GART-encoding gene followed by very extensive sequence divergence, or it was acquired by horizontal gene transfer. Siderophores produced by other species of bacteria, including ornibactin from Burkholderia cepacia (Stephan et al., 1993
) and exochelin MS from Mycobacterium smegmatis (Sharman et al., 1995
), also contain N5-formyl-N5-hydroxyornithine residues and it remains to be determined whether these bacteria contain enzymes with similarities to PvdF.
P. aeruginosa contains at least one other documented example of a gene of secondary metabolism with a clear homologue in a primary metabolic pathway. Synthesis of the phenazine pigment pyocyanin involves an anthranilate synthase encoded by the phnA and phnB genes, and this anthranilate synthase has a high level of similarity to enzymes involved in tryptophan synthesis in P. aeruginosa and other organisms (Essar et al. 1990 ); indeed, the phnAB gene pair was able to complement mutations in the corresponding trp genes in E. coli. Intriguingly, PhnA and B were more similar to tryptophan anthranilate synthase from E. coli than the corresponding enzyme from P. aeruginosa, suggesting that horizontal gene transfer may have played a role in the evolution of the pathway for synthesis of this secondary metabolite.
In summary, the data presented here provide further insights into the pathways of siderophore synthesis in P. aeruginosa, and also provide the first example of the involvement of a GART-like enzyme in secondary metabolite synthesis. Analysis of the complete sequences of microbial genomes is likely to provide other examples of enzymes from pathways of primary metabolism that have been incorporated into pathways of secondary metabolite synthesis.
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ACKNOWLEDGEMENTS |
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Received 18 October 2000;
revised 17 January 2001;
accepted 5 February 2001.