Dec 3, 1993 - 169:2352-2359. 23. Frank, D. W., D. G. Storey, M. S. Hindahl, and B. H. Iglewski. ... Moores, J. C., M. Magazin, G. S. Ditta, and J. Leong. 1984.
Vol. 176, No. 4
JOURNAL OF BACTERIOLOGY, Feb. 1994, p. 1128-1140
0021-9193/94/$04.00+0
Cloning and Nucleotide Sequence of the pvdA Gene Encoding the Pyoverdin Biosynthetic Enzyme L-Ornithine N5-Oxygenase in Pseudomonas aeruginosa PAOLO VISCA,* ALESSANDRA CIERVO, AND NICOLA ORSI Istituto di Microbiologia, Universita di Roma "La Sapienza", 00185 Rome, Italy Received 23 August 1993/Accepted 3 December 1993 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 PA01 (ATCC 15692) was constructed in the broad-hostrange 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 PA01 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 ,uM FeC13. 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 NV-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 501% homology with functional analogs, i.e., L-lysine N6-hydroxylase of aerobactinproducing 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 PA01. Similarly to strain PALS124, the pvdAl mutant obtained by gene disruption also disclosed no pyoverdin synthesis, lacked L-Orn N5-oxygenase activity, was complemented by the cloned pvd4 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 "-amino acid oxygenases have been conserved over a wide evolutionary range and probably evolved from a common ancestor.
dependent manner (for a review, see reference 1), with differences reflecting uptake specificities of the ferripyoverdin complexes (14). In P. aeruginosa PA01, an outer membrane protein of approximately 90 kDa has been shown to function in ferripyoverdin transport (41, 49, 50). Moreover, an 85-kDa Fe(III)-regulated protein which functions as the pyocin Sa receptor also appears to be involved in ferripyoverdin uptake (58). As a general rule, pyoverdins (or pseudobactins from plant-related isolates) contain one or two residues of L-N5-OH-
Siderophores are low-molecular-weight chelating agents synthetized and released by microorganisms in response to conditions of iron [Fe(III)] deficiency (44). Pseudomonas aeruginosa is an opportunistic human pathogen which is faced with stringent Fe(III) requirements for multiplication both in the environment and in the tissues of the infected host (11, 44). Like other members of the first genetic homology group of genus Pseudomonas (47), P. aeruginosa produces during Fe(III)-limited growth a water soluble, yellow-green fluorescent compound termed pyoverdin. This molecule is a hydroxamate siderophore composed of a 6,7-dihydroxyquinolinecontaining fluorescent chromophore joined to the N terminus of a partly cyclic octapeptide (D-Ser-L-Arg-D-Ser-L-N5-OHOrn-L-Lys-L-N5-OH-Orn-L-Thr-L-Thr, in P. aeruginosa PA01) (9). While the chromophore is conserved with minor structural variations in different pyoverdins from a number of fluorescent pseudomonads, the peptide composition varies in a strain-
Orn which participate in Fe(III) coordination with the quinoline hydroxyls of the chromophore. The close structural relationships among pyoverdins and pseudobactins are reflected at the DNA level. In fact, an extensive homology has been shown for genes involved in the biosynthesis of pyoverdin in different Pseudomonas spp., thus leading to the hypothesis of a possible evolution from a common ancestor (53). The genetic loci involved in pyoverdin synthesis have also been mapped in P. aeruginosa and found to be located at about 47 min of the PAO1 chromosome map (4, 29, 30, 65). Pyoverdin is endowed with a high affinity for Fe(III) (Kf, about 1032 at neutral pH) and promotes P. aeruginosa growth
* Corresponding author. Mailing address: Istituto di Microbiologia, Universita di Roma "La Sapienza", Piazzale Aldo Moro, 5, 00185 Rome, Italy. Phone: (6) 4991 4619. Fax: (6) 4991 4626.
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in the presence of human transferrin or serum (5). Therefore, a relevant role of pyoverdin in Fe(III) mobilization in vivo has also been postulated (5, 59). On the basis of previous information concerning the biosynthesis of hydroxamate siderophores (2, 3, 21, 27, 67), we assumed that hydroxylation of L-Orn constituted an early event in the generation of the peptidic moiety of pyoverdin. To substantiate this hypothesis, we have isolated and characterized P. aeruginosa PAO1 nonfluorescent (Flu -) mutants blocked in different steps of pyoverdin synthesis. Genetic and functional evidence proved that the pvdA (formerly known as pvd-I [65]) mutant strain PALS124 was impaired in the synthesis of L-N5-OH-Orn, i.e., lacked the L-Orn N5-oxygenase (hydroxylase) activity, and required L-N5-OH-Orn for pyoverdin synthesis. Thus, L-Orn and L-N5-OH-Orn are common precursors of pyoverdin, and the hydroxylation of L-Orn (catalyzed by the enzyme L-Orn N5-oxygenase) represents an essential step for the generation of the hydroxamate residues of the molecule (64, 65). In this paper, we report the localization and characterization of pvdA, a gene encoding the enzyme L-Orn N5-oxygenase, on cloned P. aeruginosa PAO1 chromosomal DNA. Structural features of both the nucleotide sequence and the translation product will be discussed with regard to the function in pyoverdin biosynthesis and with regard to phylogenetic relationships among members of the genus Pseudomonas.
MATERIALS AND METHODS Strains, plasmids, and media. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was routinely grown in LB medium (38). P. aeruginosa was grown in NYB (65) or SM9 (65) or on nutrient agar (NA; Difco) and cetrimide agar (Pseudosel; Becton Dickinson). Media were solidified with 1.2% agar N.1 (Unipath). Antibiotics were used in selective media at the following concentrations: tetracycline, 15 ,ug/ml for E. coli and 100 ,ug/ml for P. aeruginosa; chloramphenicol, 10 ,ug/ml for E. coli and 100 ,ug/ml for P. aeruginosa; ampicillin, 100 ,ug/ml for E. coli; carbenicillin, 500 ,ug/ml for P. aeruginosa; trimethoprim, 30 ,ug/ml for E. coli; and nalidixic acid, 25 ,ug/ml for E. coli. Construction of the P. aeruginosa PAO1 cosmid genomic library. High-molecular-weight genomic DNA from P. aeruginosa PAO1 was prepared by the procedure of Goldberg and Ohman (26). After partial digestion with Sau3A (0.025 U/,ug of genomic DNA for 1 h at 37°C) the DNA was size fractionated by ultracentrifugation (26,000 rpm for 24 h at 18°C in a Beckman SW28 rotor) on a linear sucrose gradient (10 to 40%) in TS buffer (1 M NaCl, 20 mM Tris-HCl, 5 mM EDTA [pH 8.0]). Fractions were collected from the bottom of the tube and analyzed for DNA size in a 0.7% agarose gel in Loening buffer (35) at 1 V/cm. Those fractions containing molecules predominantly of 15 to 30 kb were pooled, dialysed against TE (10 mM Tris-HCl, 1 mM EDTA [pH 7.2]) and precipitated with ethanol. The cosmid pLAFR3, a RK2-derived replicon (20, 60), was digested with BamHI to completion and treated with calf intestinal alkaline phosphatase. After phenol-chloroform extraction, the cosmid was precipitated and ligated in 1:3 molar ratio with P. aeruginosa PAO1 DNA partially digested with Sau3A, by using T4 DNA ligase for 24 h at 14°C and a further 24 h at 4°C. The ligation solution was packaged in vitro in lambda heads by using commercial extracts (Amersham). The bacteriophage particles were stored in chloroform-saturated phage dilution buffer (10 mM Tris-HCl, 10 mM MgSO4, 0.01% gelatin [pH 7.4]) and used to transduce E. coli S17.1 according to the supplier's protocol. Approximately 105 tetracycline-
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resistant colonies per ,ug of insert DNA were obtained. A total of 5,120 individual cosmid clones were stored at - 80°C in 96-well microtiter plates (Nunc) containing 200 ,ul of growth medium (24) supplemented with 15 ,ug of tetracycline per ml. E. coli and P. aeruginosa genetic techniques. The gene bank was screened by biparental E. coli S17.1-P. aeruginosa matings performed according to a modification of the procedure described in reference 36. Briefly, each storage plate containing 96 E. coli S17.1 clones harboring the gene bank was replicated in a microtiter plate containing (per well) 100 ,ul of NYB supplemented with 15 jig of tetracycline per ml, by using a 96-prong replicator. The plate was incubated at 37°C for 6 h and centrifuged (3,200 x g for 30 min at 37°C in a Heraeus 20RS thermostatic centrifuge equipped with a 2256 rotor), and the supernatant was discarded. The bacteria were washed once with saline (0.85% NaCl) and resuspended in 20 ,u1 of NYB. P. aeruginosa was grown to the stationary phase in NYB, washed once with saline, and resuspended in one-fifth of the original volume of NYB. Aliquots (20 ,ul) of the P. aeruginosa suspension were added to each well, and the bacterial mixtures (40 ,ul each) were plated on NA (Difco). After a 4-h mating at 37°C, the cells were removed from the NA plates, resuspended in saline, and plated on SM9 agar containing 100 ,ug of tetracycline per ml for single colonies. Two colonies of each transconjugant were transferred on cetrimide agar (a selective medium enhancing the Flu' phenotype) plates supplemented with 15 ,ug of tetracycline per ml. Plates were examined under UV light exposure after a 24- to 48-h incubation at 37°C to detect yellow-green, Flu' complemented exconjugants. The P. aeruginosa strains PAO1(pLAFR3) and PALS124(pLAFR3) were used as the positive (Flu') and negative (Flu-) controls, respectively. For complementation assays with selected clones of P. aeruginosa PAO1 DNA in E. coli S17.1 and for routine conjugal transfer of plasmids, the method described in reference 39 was used. Enzymatic assays and chemical determinations. The L-Orn N5-oxygenase activity was measured in cell lysates prepared as previously reported (43, 65). P. aeruginosa was grown to mid-logarithmic phase (A620, 0.30 to 0.40) in SM9 supplemented with either 150 ,uM nitrilotriacetic acid (to minimize protease activity and to reduce iron availability) or 100 ,uM FeCl3. Cells (approx. 40 mg dry weight) were collected by centrifugation (2,000 x g for 15 min at 4°C), washed once with saline, and resuspended in 1 ml of sodium phosphate buffer (pH 7.0) containing 2 mg of lysozyme per ml and 20 mM EDTA. After 15 min of incubation at 20°C, cells were poured in ice and subjected to ultrasonic disruption (two 30-s pulses, 16-p.m amplitude, in a MSE Soniprep 150 sonicator). The cell lysate was supplemented with 1 ml of 100 mM sodium phosphate buffer containing 40 p.g of DNase per ml, 100 ,ug of RNase per ml, 4 mM dithiothreitol, and 4 mM glutamine. After 30 min of incubation at 4°C, cell debris was removed by centrifugation (2,000 x g for 15 min at 4°C), and the resulting supernatant was immediately assayed for N5-hydroxylating activity. The enzyme assay was performed by mixing 2 ml of crude cell extract and 2 ml of 100 mM sodium phosphate buffer containing 2 mM sodium pyruvate and 4 mM L-Orn. Incubation was carried out at 37°C for up to 6 h in 10-ml tubes flushed with air (4 h was taken as the standard incubation time for most of the assays performed). The reaction was terminated by the addition of 10% trichloroacetic acid. The supernatant obtained after centrifugation (6,000 x g for 10 min at 20°C) was assayed for the presence of hydroxylamine nitrogen by iodine oxidation according to the Csaiky test (15), as modified by Gillam et al. (25). The assay system without any added
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VISCA ET AL.
J. BAcrERIOL. TABLE 1. Bacterial strains, phage, and plasmids
Strain, phage, or plasmid vector
E. coli DH5oxF'
71/18 S17.1 RU4420 P. aeruginosa
PAO1 (ATCC 15692) ATCC 27853 PALS124 PALS128 PALS106 PALS1 15 PALS125 PALS132 PALS147 PALS149 ADD1976 PAAC1 P. fluorescens CHAO P. fluorescens ATCC 13525 P. putida WCS358 Pseudomonas sp. BlO P. cepacia TVV75
Phage M13mpl8 and M13mpl9 Plasmid pLAFR3
pUCP18 and pUCP19 pEB16
Genotype or relevant characteristics
recAI endAl hsdRI 7 supE44 thi-l gyrA reLAI [+80 lacZAM15] A(lacZYAargF)U169, Nalr thi supE A(lac-proAB) F'[proAB+ lacIq lacZAM15] thi pro hsdR with chromosomally-integrated RP4-2-tet::Mu-Kan::Tn7, Tra+ Trir Strr thi-l endA I hsdR17 supE44 trp::Tn 1725, Cmr
Reference or source
34 18 57 63
Prototroph American Type Culture Collection Prototroph American Type Culture Collection pvdA (formerly designated pvd-J) 65 pvdB (formerly designated pvd-2) 65 pvdCl (formerly designated pvd-3A) 65 pvdC2 (formerly designated pvd-3B) 65 pvdC3 (formerly designated pvd-3C) 65 pvdC4 (formerly designated pvd-3D) 65 pvdC5 (formerly designated pvd-3E) 65 pvdC6 (formerly designated pvd-3F) 65 PAOI with chromosomally-integrated mini-D180, tetR tetA lacUVS 1acP T7 10 RNA polymerase, Tcr Site-specificpvdA mutant obtained by insertion of pPV2251 in PAO1 This study chromosome Prototroph 66 Prototroph 48 Prototroph 39 Prototroph 42 Prototroph 62
Single-stranded DNA cloning and sequencing vectors lacZot
69
Broad host range cosmid vector derived from IncPl plasmid pRK290; RK2 60 replicon Acos+ rix lacZot, Mob' Tra- Tcr E. coli-Pseudomonas shuttle vectors derived from pUC18 and pUC19; 55 pMB1, pRO1600 replicon lacZoa bla, Apr Cb' Broad host range expression vector with phage T7 gene 10 promoter (PT7) 10, 15a and terminators (T7+10); pMB1, pRO1600 replicon bla, Mob' Tra- Apr
Cbr
pME3087 pPV4 pPV AH pPV AX pPV AK pPV12 pPV6
pPV1 pPV15
pPVS pPV22 pPV221 pPV222 pPV223 pPV224 pPV225 HB
pEB16pvdA pPV2251
Suicide vector for gene disruption in P. aeruginosa; pMB1 replicon tetR tetA, Mob' Tra- Tcr 17-kb PAO1 DNA insert in pLAFR3 4.6-kb HindIII deletion of pPV4 13.5-kb XhoI deletion of pPV4 7.0-kb KpnI deletion of pPV4 4.0-kb KpnI fragment of pPV4 ligated to pUCP19 2.0-kb KpnI fragment of pPV4 ligated to pUCP19 1.0-kb KpnI fragment of pPV4 ligated to pUCP19 5.3-kb XhoI fragment of pPV4 ligated to pUCP19 8.2-kb XhoI fragment of pPV4 ligated to pUCP19 4.6-kb HindlIl fragment of pPV4 ligated to pUCP19 1.8-kb HindIII-SphI fragment of pPV22 ligated to pUCP19 1.4-kb HindIII-BamHI fragment of pPV22 ligated to pUCP19 2.8-kb XhoI-BglII fragment of pPV22 ligated to pUCP19 1.1-kb PstI fragment of pPV22 ligated to pUCP19 1.7-kb SphI fragment of pPV22 ligated to pUCP19. pPV225BH contains the same insert DNA in opposite orientation. 1.7-kb BamHI-HindIII fragment of pPV225BH ligated to pEB16 0.5-kb PstI fragment of pPV225HB ligated to pME3087
L-Orn served as the control. The activity values were corrected by subtracting the corresponding value of the control and expressed as nanomoles of hydroxylamine nitrogen produced per minute per gram (dry weight) of cells. An endogenous activity in PAO1 or PALS124 harboring the complementing plasmids was observed (approximately 10% of that in the L-Orn-supplemented sample), probably arising from a small
66
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study study study study study study study study study study study study study study study
(Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig. (Fig.
1) 1) 1) 1) 1) 1) 1) 1) 1) 1) 2B) 2B) 2B) 2B) 2B)
This study (Fig. SA) This study (Fig. 6A)
amount of L-Orn in the cell lysate. L-N5-OH-Orn was identified after ion-exchange chromatography and gel filtration. The hydroxylamine-positive material was loaded on a column (2 by 7 cm) of Dowex 50W-X8 (H+ form) resin and washed first with 100 ml of 0.1 N HCl and then with 50 ml of 6 N HCl. The latter fraction, containing all the hydroxylamine-positive material, was freeze-dried, resuspended in 2 ml of water, and loaded
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Sephadex G-10 column (2.5 by 52 cm, containing about 250 ml of resin) equilibrated and eluted with water. The hydroxylamine-positive material eluting at around 150 ml was concentrated by freeze-drying and assayed by iodine oxidation (15, 25). L-N5-OH-Orn was identified in an LKB Pharmacia 4400 amino acid analyzer as previously reported (65). L-N'5OH-Orn was obtained from acid hydrolysis of rhodotorulic acid (67). The estimation of pyoverdin in culture supernatants of P. aeruginosa strains was carried out by means of UV-visible and fluorescence spectroscopy, with a Beckman 25 spectrophotometer and a Jasco FP770 spectrofluorometer (1, 65). DNA manipulations. Small-scale (3- to 10-ml) preparations of plasmid DNA from E. coli and P. aeruginosa were carried out by the alkaline lysis method (38). Large-scale preparations of cosmids and plasmids from E. coli were performed with Qiagen Tip-500 kits (Diagen) according to the specifications of the manufacturer. The DNA was analyzed by agarose gel electrophoresis in Tris-borate buffer (38), and DNA bands were extracted with the Qiaex resin (Diagen) or by electroelution for fragments greater than 4 kb (38). E. coli was transformed with plasmid DNA by the standard CaCl2 method (38). P. aeruginosa cells were made competent according to a minor modification of the CaCl2 procedure. Briefly, an overnight culture of P. aeruginosa was diluted 100-fold in LB and
onto
a
incubated with orbital shaking (250 rpm) at 37°C until the A600 equal to 1.2. The culture was chilled in ice, centrifuged
was
(3,200 x g for 30 min at 4°C), and resuspended in 0.5 volume of ice-cold 0.1 M MgCl2. Bacteria were centrifuged, resuspended in 0.25 volume of ice-cold 0.1 M CaCl2, and left at 0°C for 30 min. After additional centrifugation, the cells were resuspended in 0.05 volume of 0.1 M CaCI2-10% glycerol and stored frozen at 80°C in 100-jil aliquots until used. Restriction endonucleases, T4 DNA ligase, and calf intestinal alkaline phosphatase were used under the conditions recommended by the manufacturer (U.S. Biochemicals). DNA was transferred onto nitrocellulose filters (Hybond-C extra; Amersham) by the method of Southern (38). Purified DNA fragments were labeled with [a-32P]dCTP (3,000 Ci/ nmol; Amersham) with a primer extension kit purchased from Pharmacia. Unincorporated nucleotides were removed by passage through a Sephadex G-50 spin column (38). DNA-DNA hybridization was carried out under high stringency. Filters were hybridized in a solution buffer containing 6 x SSC (0.9 M NaCl, 0.09 M sodium citrate [pH 8.0]), 0.01 M EDTA, 0.5% sodium dodecyl sulfate (SDS), 0.1% polyvinylpyrrolidone, 0.1% Ficoll, and 0.1% bovine serum albumin at 68°C for 20 h and washed at 68°C by serial passages in 2 x SSC-0.5% SDS, 2 x SSC-0.1% SDS, and 0.1 x SSC-0.5% SDS for 30, 30, and 60 min, respectively. Transposon Tn1725 mutagenesis. A modification of the procedure described by Magazin et al. (37) was used. E. coli RU4420 harboring a chromosomal insertion of Tnl 725 (63) was transformed with pPV22, a pUCP19-derived plasmid carrying genomic P. aeruginosa PAO1 DNA complementing the pvdA mutation. Transformants were selected on NA containing 50 jig of chloramphenicol and 100 jig of ampicillin per ml. Individual transformants were grown at 30°C to the stationary phase in NYB supplemented with 50 jig of chloramphenicol and 100 jig of ampicillin per ml. Aliquots of 100 jil from each culture were plated on NA chloramphenicol gradient plates (0 to 1,000 jig of chloramphenicol per ml) in the presence of 100 jig of ampicillin per ml). Since at least 100 copies of plasmids pUCP18 and pUCP19 are present in each E. coli cell (55), transposon Tnl725 insertions in pPV22 were selected for increased chloramphenicol resistance. Colonies -
(pvdA)
GENE OF P. AERUGINOSA
1131
showing the highest levels of chloramphenicol resistance were isolated on NA containing 500 ,ug of chloramphenicol and 100 ,ug of ampicillin per ml and further analyzed to determine the location of Tnl 725 insertions by restriction mapping. Plasmids containing Tn]725 insertions in the cloned genomic DNA fragment from P. aeruginosa PAO1 were used to transform P. aeruginosa PALS124 and tested for complementation of the pvdA mutation. DNA sequencing and analysis. Overlapping DNA fragments generated after digestion of pPV225HB with different restriction enzymes were cloned at compatible sites of the replicative forms of M13mpl8 and M13mpl9 phages (69). The inserts of single-stranded forms of the M13 constructions were sequenced by the dideoxy chain termination method (38) with T7 DNA polymerase (Sequenase version 2.0; U.S. Biochemicals) and o-35S-dATP for labeling. In some cases, internal restriction fragments from pPV225HB were subcloned in pUCP18 and pUCP19 and sequenced on both strands with universal and reverse pUC primers (U.S. Biochemicals). To resolve band compressions due to high G+C content of P. aeruginosa DNA, sequencing reactions were performed by using both dGTP and 7-deaza-dGTP (54). Multiple sequence alignments were made by comparison with the GENEMBL and SWISSPIR data banks by using the programs within the Genetics Computer Group (University of Wisconsin) package (version 7.2). Bacteriophage T7 RNA polymerase-controlled expression of the pvd4 gene. The two-component T7 system developed by Brunschwig and Darzins (10) for the expression of cloned genes in P. aeruginosa was used. The BamHI-HindIII fragment from pPV225BH was ligated to BamHI-HindIII-digested pEB16. This expression vector contains the phage T7 gene 10 promoter upstream from the multiple cloning site and two T7 transcriptional terminators following the multiple cloning site (1Sa). The resulting construct, pEB16pvdA, was used to transform the P. aeruginosa strain ADD1976 carrying the T7 RNA polymerase gene under the control of the lacUV5 promoter stably integrated in the PAO1 chromosome (10). To monitor the expression of the pvdA gene, strain ADD1976 (pEB16pvdA) and the control strain ADD1976 (pEB16) were grown at 37°C in 50 ml of SM9 containing 100 ,ug of tetracycline and 500 ,ug of carbenicillin per ml until the A600 was 0.5. Cultures were induced with 2 mM isopropyl-f-D-thiogalactopyranoside (IPTG), and incubation was carried out for 1 h to allow expression of the T7 RNA polymerase gene. Rifampin was then added to a final concentration of 200 ,ug/ml to shut off P. aeruginosa RNA polymerase transcription. After a 2-h incubation at 37°C, cells were harvested by centrifugation (4,500 x g for 15 min at 4°C), resuspended in 200 ,ul of TE buffer supplemented with 1 mM phenylmethylsulfonyl fluoride, and disrupted by sonication. The protein content of the lysates was determined according to Bradford (7), and approximately 50 jig of total bacterial proteins was suspended in gel loading buffer (0.25 M Tris-HCl, 2% SDS, 10% 2-mercaptoethanol, 20% glycerol), heated at 100°C for 5 min, and loaded in a 0.1% SDS-12.5% polyacrylamide gel according to Laemmli (33). Electrophoresis was carried out at 10 V/cm in Tris-glycine buffer (25 mM Tris-HCl [pH 8.3], 192 mM glycine, 1% SDS). After electrophoresis, gels were stained with Coomassie brilliant blue, destained, and photographed. Construction of pvd4 mutants by gene disruption. A 0.5-kb PstI fragment internal to the pvdA gene was cloned from pPV225HB in the suicide mobilizable vector pME3087 (66), giving pPV2251. This recombinant plasmid, which cannot replicate in P. aeruginosa, was conjugated from E. coli S17.1 to wild-type P. aeruginosa PA01. P. aeruginosa clones in which
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VISCA ET AL.
homologous recombination between the pvdA internal fragment of pPV2251 and the chromosomal pvdA gene had occurred were selected for the tetracycline resistance marker of the plasmid. The disruption of thepvdA gene by insertion of pPV2251 was assessed by lack of L-Orn N5-hydroxylase activity and Flu- phenotype and confirmed by hybridization analysis of the BglII- and Sall-digested chromosomal DNA of the putative mutant with the 1.2-kb SphlI-SalT DNA probe from pPV225HB, encompassing most of the pvdA gene and part of the 3' flanking region. Homology of the pvd4 gene with chromosomal DNA from hydroxamate-producing Pseudomonas spp. The DNA-DNA homology between pvdA and putative L-Orn N5-oxygenase genes from different pseudomonads was also assessed. All the Pseudomonas strains tested were characterized by the production of siderophores containing one or two residues of L-N5OH-Orn in their peptidic moiety. The total DNAs from P. aeruginosa ATCC 27853 (14), Pseudomonasfluorescens CHAO (66), P. fluorescens ATCC13525 (48), Pseudomonas putida WCS358 (39), Pseudomonas sp. B10 (42), and Pseudomonas cepacia TVV75 (62) were prepared as detailed above for P. aeruginosa PAO1. The fragments generated after complete SphlI digestion of 50 ,ug of total DNA were separated by electrophoresis on a 1.0% agarose gel in Tris-borate buffer (38), transferred onto a nitrocellulose filter (Hybond-C extra, Amersham), and hybridized with the 1.2-kb SphlI-SalT DNA probe from pPV225HB. Nucleotide sequence accession number. The nucleotide sequence data reported here have been submitted to the GenBank-EMBL data base under accession number Z25465.
RESULTS Isolation of the recombinant cosmid pPV4 complementing the pvd4 mutation in the pyoverdin-deficient P. aeruginosa strain PALS124. A genomic library of P. aeruginosa PAO1 DNA was constructed in the broad-host-range cosmid pLAFR3 and maintained in E. coli S17.1. The average length of cloned PAO1 DNA inserts was 19.2 kb, as determined after EcoRI-HindITI restriction analysis of 48 randomly selected recombinant clones. In all cases, a fragment of approximately 22 kb comigrated with the EcoRI-digested vector (data not shown). The E. coli clones harboring the gene bank were individually tested for complementation of the pvd4 mutation by matings with the L-Orn N5-oxygenase-defective mutant PALS124. Exconjugants, which appeared at a frequency of approximately 10- per donor cell, were selected on tetracycline-supplemented SM9. The scoring of the Flu' phenotype on this medium was rendered difficult by the highly fluorescent background due to the presence of 100 ,ug of tetracycline per ml. To overcome this problem, two colonies of each exconjugant were replicated on cetrimide agar plates supplemented with tetracycline at a lower concentration. Only one cosmid clone out of 5,120 tested was able to complement the defect in fluorescence of PALS124. Unlike PALS124, the complemented pvdA mutant was able to grow at wild-type levels on SM9 agar plates supplemented with the chelator 2,2'-dipyridyl at 500 ,uM. The complementing cosmid, designated pPV4, was conjugated from E. coli S17.1 to cells of pvdA, of pvdB, and of different pvdC mutants. Restoration of the Flu' phenotype by complementation of pvd mutations was demonstrated not only in the pvdA mutant PALS124 but also in the pvdC4 mutant PALS132. Restriction analysis of cosmid pPV4 showed a DNA insert of approximately 17 kb. Combined digestions with two or three restriction enzymes enabled us to construct a preliminary physical map of pPV4 (Fig. 1). To determine the approximate
J.
BACrERIOL.
location of the pvdA gene, complementation tests were conducted with various derivatives of pPV4 obtained either by deletion of internal fragments (after cleavage with restriction enzymes and subsequent religation) or by subcloning restriction fragments in the multicopy vector pUCP19 (Fig. 1). pPV22 is a complementing plasmid containing a minimum 4.6-kb Hindlll fragment from pPV4, able to restore the Flu' phenotype and the L-Orn N5-oxygenase activity in the pvdA mutant PALS124. Synthesis of pyoverdin and L-Orn N5-oxygenase activity in PALS124 complemented with plasmids pPV4, pPVAK, pPV5, and pPV22 were under stringent Fe(III) control, being undetectable in cells which had been grown in SM9 supplemented with 100 puM FeCl3 (data not shown). Location of the pvd4 gene in pPV22 by Tnl 725 mutagenesis and subcloning. Clone pPV22, which complements the pvdA mutation, was characterized in more detail. A restriction of the 4.6-kb P. aeruginosa DNA was constructed, and map the plasmid was introduced in E. coli RU4420 for Tnl725 mutagenesis (63). Tn)725 is an 8.9-kb-long transposable element with two EcoRI sites located in the 32-bp inverted repeats from Tnl721 and two HindIll sites flanking the internal 3.3-kb Cmr cassette; it has no sites for BamHT and BglTI (63). Spontaneous transposition events of Tnl725 in the high-copy-number plasmid pPV22 were selected for increased chloramphenicol resistance on 0 to 1000 pug of chloramphenicol per ml gradient plates. A total of 50 clones which were able to grow in the presence of 500 ,ug or more of chloramphenicol per ml were selected and analyzed by restriction mapping to determine the location of Tn1725 insertions. Tn.l725 insertions in pPV22 were shown for 32 clones, and 13 were found to be located within the 4.6-kb DNA fragment from PAO1. Four insertions (designated 7, 8, 20, and 24) caused no pyoverdin synthesis in complementation assays with the pvdA mutant PALS124 and completely abolished L-Orn N5-oxygenase activity (Fig. 2A). The active insertions were all located in a 1.7-kb SphT fragment. Further subcloning in pUCP19 of internal DNA fragments generated after digestion of pPV22 with various restriction enzymes supported the location of the pvdA gene in the 1.7-kb SphlI fragment (Fig. 2B). In fact, complementation of the pvdA mutation was shown by plasmids pPV225BH and pPV225HB, which contain the entire 1.7-kb SphI fragment from pPV22 cloned in opposite orientations. Also, in the case of PALS124 (pPV225), synthesis of pyoverdin and L-Orn N5-oxygenase activity were completely repressed by the presence of 100 p.M FeCl3 in the cell growth medium (data not
shown).
Sequence of the pvd4 gene. Figure 2C shows the collection of inserts in M13 clones which were used for sequencing the 1.7-kb PAO1 DNA fragment from pPV225HB. The entire region was sequenced for a minimum of three times by using both dGTP and 7-deaza-dGTP. Examination of the sequence showed one open reading frame (ORF) of significant length
(Fig. 2C and 3). The putative structural gene was 1,278 bp long, contained an ATG translation start codon located 7 bp downstream from an AGGA sequence which might form part of a ribosome-binding site (56). The G+C content of the entire region was 65%. The regions flanking this ORF were examined. The putative start site for transcription has yet to be determined, but no evident promoterlike sequences, i.e., the - 35 and - 10 consensus sequences, were detected, nor was it possible to identify regions of significant homology with known promoter sequences from other fluorescent pseudomonads (19, 23, 46, 51). Moreover, sequences of dyad symmetry representing putative transcriptional regulators were absent from both sides of the ORF. The predicted L-Orn N5-oxygenase (PvdA) protein consists of 426 amino acids (42.0% hydro-
L-ORNITHINE N5-OXYGENASE
VOL. 176, 1994
(pvdA)
GENE OF P. AERUGINOSA
Plasmid Vector 1 kb HBg X BSm
pPV4
pPV'H pPVAX
| L HI II _p
lpH IpUCP191
pPV6 pPVll
pUCPl9
pPV15
pPV5 pPV22
KX
Sm
KSm
KX
Sm
KSm II
I I
6m-
HBg X
IpLAFR 1 1
pPV12
11 HBg
L-I-
HBg ' _BSm
pPVAK
Bg HBg
Bg
-1 11 1
KX
HBg X BSm
PUCPl91 H
BSm
HBg BgII Bg
H
Bg
H
XK
KSSm K X
KX
I'l
sm
Sm
Relative fluorescence hydroxylase activity (%) 88.8 86.3 0.0
0.0
0.0
0.0
83.1
92.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0 89.7
0.0 102.9
98.2
177.7
Sm
Sm
K
Sm
sm
I XII BSm
1l 1111Ii
--_______I
KSm
pUCPl9~ lpucplgl lp1CP191 I IpUCP19
li i XKSSmK
XKSSm K -1 1 1 1I K
K
HBg
XKSSm K
1133
K Smu I
X I
FIG. 1. Physical map of pPV4 and derivative plasmids and complementation analysis in P. aeruginosa PALS124 (pvdA). Plasmid designations Restriction and vectors employed are shown on the left side of the figure. Broken lines indicate deletions of internal fragments of the pPV4 insert. the sites used for deletions and subcloning are indicated. Relative fluorescence and hydroxylase activity of mutant PALS124 (pvdA) harboring the different plasmids are reported on the right side of the figure and expressed as percent of the value of the control PAO1 harboring corresponding vector. Abbreviations: B, BamHI; Bg, Bglll; H, Hindlll; K, KpnI; S, Sacl; Sm, SmaI; X, XhoI. Production of yellow-green, fluids from fluorescent pigment was detected by fluorometric determinations (emission at 455 nm after excitation at 405 nm) of supernatantL-Orn as the late-exponential cultures (A6, = 1.0) in SM9 diluted 1:2 in 200 mM Tris-HCI, pH 11. The hydroxylase activity was determined with min substrate (65). The hydroxylase activities of PAO1(pLAFR3) and PAO1(pUCP19) were 12.2 and 11.9 nmol of hydroxylamine nitrogen per respectively. of cells, weight) per g (dry
phobic, 32.1% polar uncharged, 14.6% acid, and 11.3% basic residues) with a deduced isoelectric point (pl) of 8.1 and a molecular mass of 47.7 kDa. A FASTA comparison of the deduced amino acid sequence of PvdA with the SWISSPIR data bank revealed extensive similarity to two functional analogs, i.e., to L-Orn N5-oxygenase encoded by the Ustilago maydis sidl gene (40) and to L-lysine N6-hydroxylase encoded by the E. coli iucD gene (28). The optimal alignment of PvdA, Sidl, and IucD sequences is shown in Fig. 4. PvdA and Sidl are 38.5% identical, with 58% sequence similarity if conservative substitutions are included. A lower homology was observed between PvdA and IucD; the two proteins are 31% identical, with a similarity approaching 50%. It is worth noting that both prokaryotic enzymes, i.e., PvdA and IucD, contain a putative flavin-binding motif (sequence IGVG[T/F]GP) located 13 and 7 amino acids downstream from the N terminus, which was absent in the U. maydis Sidl protein (40). The hydropathicity patterns according to Kyte and Doolittle (32) of the three functional analogs through their entire sequence were analyzed. Both PvdA and IucD showed a highly hydrophobic region approximately located in the first 25 residues from the N terminus (data not shown). This hydrophobic region, which was absent in the eukaryotic Sidl protein, is characterized by an amino acid composition resembling that of signal peptides and has been involved in the anchoring of the IucD protein to the inner side of the cytoplasmic membrane (28). Isolation and identification of the pvd4 gene product. The pvdA gene was expressed in P. aeruginosa by using a twocomponent bacteriophage T7-derived system; this approach was adopted in order to circumvent problems concerned with the poor expression of P. aeruginosa genes in heterologous systems (10, 54). The 1.7-kb BamHI-HindIII DNA fragment
encompassing the entire pvdA gene was cloned from pPV225BH in the expression vector pEB16, under the control of the bacteriophage T7 gene 10 promoter. The resulting construct, designated pEB16pvdA, was used to transform P. aeruginosa ADD1976, a PA01 derivative containing the lacUV5/lacIq-regulated T7 RNA polymerase gene and a Tcr cassette stably integrated in the chromosome as a bacteriophage D3112-derived element (54) (Fig. 5A). Strain ADD1976 (pEB16pvdA) was grown in SM9 and, during the mid-log phase, induced with IPTG to allow expression of T7 RNA polymerase. One hour after induction, rifampin was added to inhibit P. aeruginosa RNA polymerase, thus allowing selective transcription of the pvdA gene under the control of the T7 promoter. Strain ADD1976(pEB16) was used as control. Upon IPTG induction, strain ADD1976(pEB16pvdA) overproduced a 47.7-kDa protein which was expressed at a lower level in the absence of the inducer (Fig. SB). Expression of the 47.7-kDa protein was not enhanced after IPTG induction of the control strain ADD1976(pEB16). In line with these results, an approximately fourfold increase in L-Orn N5-oxygenase activity was observed after IPTG induction of ADD1976(pEB16pvdA) (data not shown). Thus, the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) molecular mass estimate of the putative pvdA gene product is in agreement with the predicted value of 47.7 kDa. As a control, the 1.7-kb HindIII-BamHI DNA fragment from pPV225HB was also cloned in the compatible sites of pEB16. The resulting plasmid, containing the pvdA gene in opposite orientation with respect to the T7 promoter, was employed in the T7-controlled expression assay. SDSPAGE from lysates of IPTG-induced cells did not allow the detection of any polypeptide encoded by the cloned fragment (data not shown).
1134
VISCA ET AL.
Plasmid
J. BACFERIOL.
200_bp
248
A HBg
pPV22
B pPV221
.N
H
l
Bg
A
X AAB
l
Bg A
SA
A: P
ll $7
11 ll
XPAAB .m I
H
pPV222
A
SA
P A HE II
III
NU S/X PA AB
S ASh
ASh
P
SB/Bg
P
I
H P
Bg A
XEP E
Si,
P
SaPEg-Sa--
Sa
Sa Sa SaPEg
I II
Sa
S
Bg III
P
200 bp
PvI[
177.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
I117.3
279.3
E
P
pPV224 pPV225 IHB
98.2
ESh
III-
pPV223
ShC
I l
XPAAB E H
C
S
Relative fluorescence hydroxylase (%) activity (%)
Sa
SPv Bg
I
11
Sa
H
Sh
-
Sa
-
Sh
--
Sa Sa pvdA
FIG. 2. (A) Physical map of the 4.6-kb HindlIl fragment of pPV22 and the locations Tnl725 insertions. Numbered triangles designate the locations of Tn]725 insertions in the PAO1 DNA fragment of pPV22. V and V ofrefer to the ability or inability, respectively, of the Tn]725-mutagenized fragment to complement P. aeruginosa PALS124 (pvdA) for pyoverdin synthesis and hydroxylase activity. (B) Complementation analysis in P. aeruginosa PALS124 (pvdA) of plasmids derived from pPV22. fluorescence and hydroxylase activity shown by mutant PALS124 (pvdA) harboring the different plasmids are reported on the right sideRelative of the (C) Detailed physical map of the 1.7-kb SphI fragment from pPV225HB and strategy for sequencing the pvdA gene. The large open arrowfigure. shows the proposed location and 5'-to-3' orientation of the pvdA gene. Thin arrows indicate the locations and orientations of inserts cloned in M13 The asterisk and corresponding arrow show the location and 5'-to-3' orientation of a synthetic or pUCP for sequencing with universal primers. oligonucleotide (20-mer) used for sequencing a defined region of the M13 insert. *, pUCP19 vector; multiple cloning site of pUCP19; O, fragment the multiple cloning site of pLAFR3. Abbreviations: A, AvaI; B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; Eg, EagI; H, Hindlll; P, PstI; Pv, PvuI;from Sm, SmaI; X, XhoI. Production of yellow-green, fluorescent pigment was detected by fluorometric PvII, PvuII; S, Sall; Sa, Sau3A; Sh, SphI; determinations (emission at 455 nm after excitation at 405 nm) of supernatant fluids from late-exponential cultures (A6(0 1.0) in SM9 diluted 1:2 in 200 mM Tris-HCl, pH 11. The hydroxylase activity was determined with L-Orn as the substrate (65). The hydroxylase activities and 11.9 nmol of hydroxylamine nitrogen per min per g (dry weight) of cells, respectively. of PAO1(pLAFR3) and PAO1 (pUCP19) were 12.2 l,
=
Site-directed mutagenesis of the pvd4 gene and homology with chromosomal DNA from hydroxamate-producing pseudomonads. In order to prove unambiguously that the product of the pvdA gene is in fact the L-Orn N5-oxygenase enzyme, we constructed apvdA mutant by gene disruption. The 0.5-kb Pstl fragment internal to the pvd4 gene was ligated in the Pstl site of the suicide vector pME3087 (66), giving pPV2251 (Fig. 6A). This plasmid was conjugated from E. coli S17.1 to wild-type P. aeruginosa PAOI. Since pPV2251, as a pMB1-derived replicon, cannot replicate in P. aeruginosa, selection for the Tcr marker carried by the vector gave rise to the recovery of clones in which homologous recombination between the pvdA fragment of pPV2251 and the chromosomal pvdA gene occurred. By mating approximately 1010 donor and recipient cells, 281 Tcr P. aeruginosa exconjugants were selected on SM9 supplemented with 100 ,ug of tetracycline per ml, all of which disclosed no fluorescence on either SM9 or cetrimide agar plates. One of these clones, designated PAAC1, was characterized in detail. Unlike wild-type PAO1, but similarly to the pvdA4 mutant PALS124, PAAC1 did not produce pyoverdin in SM9, grew poorly in SM9-agar plates supplemented with the chelator 2,2'-dipyridyl at 500 ,uM, and lacked L-Orn N5oxygenase activity, but produced pyoverdin at wild-type levels when grown in SM9 supplemented with 400 ,uM L-N5-OH-Orn.
Moreover, pyoverdin synthesis in P. aeruginosa PAAC1 was restored after transformation with plasmid pP225HB. Comparative Southern analysis of BglII- or Sall-digested genomic DNA from PAOI, PAAC1, and PALS124 with the 1.2-kb SphI-Sall probe from pPV225HB confirmed the expected recombination event and allowed the definition of the location and orientation of pPV2251 insertion in the pvdA gene (Fig. 6B). We next decided to investigate the homology of the cloned pvdA gene with putative genes encoding L-Orn N5-oxygenases from a variety of Pseudomonas spp. We selected strains which are known to synthetize L-N5-OH-Orn-containing siderophores, including P. aeruginosa ATCC 27853 (producing pyoverdinPA27853 [14]), P. fluorescens CHAO (producing pyoverdinCHAO [66]), P. fluorescens ATCC 13525 (producing pyoverdinATCC13525 and ferribactin [1, 48]), P. putida WCS358 (producing pseudobactinWCS358 [1, 39]), Pseudomonas sp. BlO (producing pseudobactinBlO [1, 42]), and P. cepacia TVV75 (producing an ornibactinlike siderophore [61, 62]). The genomic DNA from these strains was digested with SphI and Southern analyzed with the 1.2-kb SphI-SalI DNA probe from pPV225HB. Hybridization was carried out under high stringency, so that only DNA fragments with relatively high homology with pvdA were detected. The results obtained (Fig. 7)
VOL. 176, 1994
L-ORNITHINE NW-OXYGENASE (pvdA) GENE OF P. AERUGINOSA
TTCCTGWTa)GTTAGTCAACAGA
OC
T
GTGCCCrAM
1135
CCAGAGGAACJ>GAA
100 ATG ACT CAG GCA ACT GCA ACC GCC GTG GTT CAC GAT CTC ATC GOT GTC GGC TTC GGC Thr Gln Ala Thr Ala Thr Ala Val Val His Asp Lou Ile Gly Val Gly Phe Gly 200 GCC Cr0 GCG ATT GCC CTC CAG GAA COGG GCC CAG GCG CAG GOC GCC CTG GAA GTG CTM Ala Leu Ala Ile Ala Leu Gln Glu Arg Ala Gln Ala Gln Gly Ala Lou Glu Val Lou
Met
CCT TCC AAT ATC Pro Ser Asn Ile>
TTC CTM GAC AAG Phe Lou Asp Lys>
300 CAG GGC GAC TAC COGC TOG CAC GOC AAC ACC CTG GTG TCG CAG AGC GAG TTG CAG ATC TCC TTC CTC AAG Gln Gly Asp Tyr Arg Trp His Gly Asn Thr Leu Val Ser Gln Ser Glu Leu Gln Ile Ser Phe Leu Lys>
GAC CrG GTG TCC CT0 CGC AAC CCC ACC AGT CCG TAT TCC TTC GTC AAC TAC CAC AAG CAC GAT CGT Asp Lou Val Ser Leu Arg Asn Pro Thr Ser Pro Tyr Ser Phe Val Asn Tyr CTO Lou His Lys His Asp Arg> 400
CTG GTC GAC TTC ATC AAC CTG GGC ACC TTC TAT CCC Lou Val Asp Phe Ile Asn Lou Gly Thr Phe Tyr Pro
TOC CGG Cys Arg
GTC GCC AGC CAC TTC CAG GAG CAG AGC COGC TAC Val Ala Ser His Phe Gln Glu Gln Ser Arg Tyr
GAA GAG GTC Glu Glu Val
ATG GAG TTC AAC GAC TAC CTG CGC TGG Met Glu Phe Asn Asp Tyr Leu Arg Trp> 500
GOC Gly
CTO
Lou
CGC ATC GAG CCG ATG CTG AGC Arg Ile Glu Pro Met Leu Ser>
GCC GGC CAG GTC GAG GCG CTG COG GTG ATC TCG CGC AAC GCC GAC GGC GAG GAG Cr0 GOT CGC ACC ACC Ala Gly Gln Val Glu Ala Leu Arg Val Ile Ser Arg Asn Ala Asp Gly Glu Glu Leu Val Arg Thr Thr> 600 CGC GCC CTG GMT GTC AGT CCC GGC GCC ACC CCG CGT ATC CCG CAG GTG TTC CGT GCG CTC AAG GGC GAC Arg Ala Leu Val Val Ser Pro Gly Gly Thr Pro Arg Ile Pro Gln Val Phe Arg Ala Leu Lys Gly Asp> 700 GGC CGG GTM TTC CAC CAC AGC CAG TAC CTG GAA CAC ATG GCC AAG CAG CCC TGC AGT GGC AAG CCOG Gly Arg Val Ph. His His Ser Gln [yr Lou Glu His Met Ala Lys Gln Pro Cys AGC Ser Ser Gly Lys Pro>
ATG AAG ATC GCC ATT ATC GGC GGC GGG CAG AGC GCG GOCG GAG GCC ATC GAC CTC AAC GAC AGC TAC Met Lys Ile Ala Ile Ie Gly Gly Gly Gln Ser Ala Ala Glu Ala TTC Phe Ile Asp Leu Asn Asp Ser Tyr> 800 CCG TCG GTG CAG GCC GAC ATM ATC Cr0 COT GCC TCG GCG CTC AAG CCOG GCG GAC GAT AGC Pro Ser Val Gln Ala Asp Met Ile Lou Arg Ala Ser Ala Lou Lys Pro Ala Asp Asp Ser CCG TTiC GTC Pro Phe Val>
900 AAC GAA GTG TTC GCG CCG AAG TTC ACC GAT CTC ATC TAC A0C COC GAM CAT GCC GAA CGC GAG CGT TTG Asn Glu Val Phe Ala Pro Lys Phe Thr Asp Lou Ile Tyr Ser Arg Glu His Ala Glu Arg Glu Arg Leu>
CTG COC GMA TAC CAC AAC ACC AAC TAT TCG GOT GTG GAT ACC GAC CTG ATC GAG CGC ATC TAC GGC GTC [yr Ser Val Val Asp Thr Asp Lou Ile Glu Arg Ile Tyr Gly Val> 1000
Lou Arg Glu Tyr His Asn Thr Asn
TTC TAC COC CAG AAA GTC TCC GGC ATC CCOG CC CAC GCC TTC CGT TGC ATG ACC ACC G0G GAG CGC GCG Phe Tyr Arg Gln Lys Val Ser Gly Ile Pro Arg His Ala Phe Arg Cys Met Thr Thr Val Glu Arg Alai 1100 ACC GCC ACC GCC CAG GGC ATC GAG CTG GCG TTG CGC GAC GCC GOT AGC GGC GAG CTA AGC Thr Ala Thr Ala Gln Gly Ile Glu Lou Ala Lou Arg Asp Ala Gly Ser Gly Glu Leu Ser GTA GAG ACC Val Glu Thr> TAC GAC GCA GTG ATC CTG GCC ACC GGC TAT GAG CGC CAG CAC CGC CAA CTG CTC GAA CCG CTG GCG Tyr Asp Ala Val Ile Lou Ala Thr Gly Tyr Glu Arg Gln TTG Leu His Arg Gln Leu Leu Glu Pro Loeu Ala> 1200 GAG TAC CrC GGC GAC CAC GAG ATC GGC CGC GAC TAC COGC CAG CCG ACG AGC GCr GCA AGG Glu Tyr Lou Gly Asp His Glu Ile Gly Arg Asp Tyr Arg CTG Lou Gln Pro Thr Ser Ala Ala Arg 1300 TCT ACG CGC AGG GCT TCA GCC AGG CCA GCC ATG GCC TCA GCG ACA CCC TGC TOT COG TGC TGC Ser Thr Arg Arg Ala Ser Ala Arg Pro Ala Met Ala Ser Ala Thr Pro Cys Cys Arg Cys Cys
TGG CGA Trp Arg>
CGG TGC Arg Cys>
1400 GTG CCG AGG AAA TCT CCG GCT CCC TOT ACC AGC ACC TOA Val Pro Arg Lys Ser Pro Ala Pro Cys Thr Ser Thr ***> AGCCGTACTGOGGcCCGoCCTGCACGAGCACGCCCTG
1500
GCCACCNATCGGCCGCCGTACGCCCTrGTCCCCGTTTCCOGCCAGGGATG
CCCGGCAC G
TT
WTCMGCCC
FIG. 3. Nucleotide sequence of pvdA. The amino acid sequence of the predicted product is shown below the nucleotide sequence. The numbers above each line refer to the nucleotide sequence, and the asterisks translation define of 50 nucleotides. Nucleotide number 1 was arbitrarily located at position - 93 with respect to the ATG initiation codon. The potential blocks ribosome-binding site (56) is underlined.
J. BACTERIOL.
VISCA ET AL.
1136
PvdA .
.
.
.
.
.
.
.
.
.
Sidl MSAPTLDVES IucD
_
PVdA V Sid1lA
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R
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A.G SEE NERNAK H D QER
VET Y ET I R F V E LES
...
FO .
... . .
........
DPTEKQAG M LRKPRNI H R
HE
I
G
RG
EL_
FTL*WSGPKE
NN
PvdA ........ . . Sidl RWL.AKIRERT
. T P C C R 3RC YPKPRS_W A
VPEKSP
IuCD
IQWRSG
FVVNASM PCE
ATUQRTICR
.........
D I
G S G
184
......
240
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A
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225 299 200
331
418 309
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162
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..
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WDEQQRAPHS
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EVIERRREML
FDEKRRLFLV
133
R
EV
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.
13 6
..
180
..
DAAAQLDISI
E E L Q
X
RQ
..
DAEQDTVA.VR
ESNLRRPELEG
K V S G I
.. .... ... ..........
N
C VK H M
Y_Q
......
-
.K. .W.X...T.K..UV.
.
F
...... .............
LALASASP
*Y CGTQ
T
V
SAADLR . DYTY I S
H R
M
93
1280 KW 83
V-RIL A-Q-A
SP
SUAV
KAS P
L
_D
60 28
G
K
A-V
VISETG* AAA NL
Sidl VSKSID-EEE TFKURUD IucD
LFDLSMPUAL
S R _ EE ELLR _ VS_QD-IS
Y
LH-AASLRHP
_
VT _F INWSRN N
*T V
34
EE
A L S HI
S
F
_N D A V A-
I* V
LY3H HU
CLYQVR
D
IGWHHCPI
E
E
RR
.TQSCFHAS
RGEWGEAE
IUCDL
DHH
............
.N D S Y E
..
GNT SQ E *A LG-Q-
_ _
T
- -AQR AQ - SUR-S S
I
AMNMVSSHTT .
L E H-A-Q P C S GEV FEH S Q LV-SGFF IPS-L-LEPE
PvdA A Sidl T
PvdA
.
.................
Sidl IucD
.
P_S_ GMD
LG T FY K E Q GV
....
IucD PvdA
.
DR_ QA R E H
IUCD Y_l!ST S R L R T
PVdA Sid1
T TAVVV VUKDEII MKK S .M.............
.
.. ..........
PvdA El. Q GA-E V Sidl EN E T NK AH IucD .. -D C
IucD
.
.
PLAASTSSLR
.IN
. .
.
478 354
AEAEA
405
P
K
538
VUG R D
409
T426
V C570 ..
426
FIG. 4. Alignment of PvdA, Sidl, and IucD amino acid sequences. The deduced sequence of PvdA was employed in data base searches, and the best matching sequences, all representing w-amino acid oxygenases (or hydroxylases), were aligned with PILEUP and PRETTY programs by using the default settings. Gaps are indicated by dots. Amino acid residues that are identical in two or all three of the sequences are printed inis white on black. Letters on grey background indicate conservative amino acid replacements. The putative flavin-binding site of lucD (28, 40) underlined.
show that the pvdA probe hybridizes to an approximately 1.7-kb SphI fragment from genomic DNA of Pseudomonas strains ATCC 27853, CHAO, ATCC 13525, B10, and TVV75. No apparent homology between the pvdA DNA and the putative counterpart of P. putida WCS358 was shown, in spite of the fact that this plant-related isolate produces a fluorescent siderophore containing one residue of 3-amino-1-hydroxy-2piperidone (cyclic L-N5-OH-Orn). Moreover, an additional hybridization band at approximately 0.8 kb was visible in the genomic digest of P. aeruginosa ATCC 27853, which could reflect a certain heterogeneity of the pvdA locus in this species.
DISCUSSION The genetic organization of Fe(III) transport systems mediated by fluorescent siderophores in Pseudomonas spp. is under investigation in several laboratories (36, 37, 39, 42, 45, 50, 53). Reports concerning the structure and function of genes encoding ferripseudobactin and ferripyoverdin uptake proteins (6, 37, 50) and regulatory elements affecting siderophore gene expression (46, 51) have been published, but little or no information is available on pyoverdin and pseudobactin biosynthetic genes. Due to the structural complexity of fluorescent siderophores, it is likely that several enzymes are required for their biosynthesis. It has been proposed that the biosynthesis of the peptide backbone of pyoverdins and pseudobactins is nonribosomal (42), as has also been shown for other amino
acid-containing siderophores and for peptide antibiotics (31). Thus, an isomerase, an oxygenase, and a formyltransferase are likely to be involved in the generation of precursors required for pyoverdin synthesis in P. aeruginosa PAO1. Utilizing a gene bank of P. aeruginosa PAO1 DNA, we have identified the recombinant cosmid pPV4, able to complement in trans both pvdA and pvdC4 mutations. The complementing loci were located within a 17-kb DNA fragment. Deletion analysis, subcloning, and transposon mutagenesis enabled us to locate the DNA region complementing the pvdA mutation in a 1.7-kb DNA fragment flanked by two SphI restriction sites. Enzyme assays performed with lysates of the mutant strain PALS124 trans complemented with the cloned pvdA gene proved the restoration of the L-Orn N5-oxygenase activity. Furthermore, complementation of the pvdA mutation by the cloned DNA was confirmed by the restoration of the pyoverdin-proficient phenotype and by the ability of the complemented mutant to multiply at wild-type levels under conditions of Fe(III) deficiency. It should be stressed that trans complementation of the pvdA mutation with different recombinant plasmids tested was under stringent Fe(III)-control, as was also L-Orn N5-oxygenase activity. We determined the complete nucleotide sequence of the 1.7-kb SphI fragment and identified an ORF of 1,278 bp whose translation product, deduced by computer analysis, consisted in a 426-amino-acid sequence, corresponding to a 47.7-kDa polypeptide. The putative pvdA gene product revealed extensive homology with the enzymes
VOL. 176, 1994
L-ORNITHINE N5-OXYGENASE (pvdA) GENE OF P. AERUGINOSA
1137
T7 RNA attL
polimerase
lac UV5 kcIq
tetR
tetA
attR 1
Mini-D 180 element (9.9kb) chromosomally integrated in P. aeruginosa ADD 1976
2
3
4
5
kDa
- 47.7
A
B
FIG. 5. Expression of the pvd-l gene by the two-component T7 RNA polymerase system in P. aeruginosa ADD1976. (A) The upper part shows the genetic map of the mini-D180 element in P. aeruginosa ADD1976 as deduced from references and 54. The lower part shows the second component of the T7 system, constructed as follows. The 1.7 BamHI-HindIII insert of pPV225BH, 10 encompassing the pvdA gene, was ligated to the compatible sites of pEB16 (10, 15a), giving pEB16pvdA. Plasmids pEB16 and pEB16pvdA were introduced in P. aeruginosa ADD1976 either by transformation or by conjugation from E. coli S17.1 (57). (B) SDS-PAGE of proteins 50 (approximately in p.g each lane) from lysates of strain ADD1976 harboring plasmids pEB16 (lanes 2 and 3) and pEB16pvdA (lanes 4 and 5). Lane 1, molecular mass standards including rabbit muscle phosphorilase (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg-white ovalbumin (42.7 kDa), bovine carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and hen egg-white lysozyme (14.4 kDa); lanes 2 and 4, uninduced lanes 3 and 5, cells induced with 2 mM IPTG. Abbreviations: attL and attR, mini-D3112 left and right termini, respectively (54); lacUV5, cells; lac promoter carrying UV5 mutation; lacl4, gene encoding the repressor protein of the lac operon; tetR and tet;A, tetracycline resistance genes; bla, 1-lactamase gene; PT7, bacteriophage T7 gene 10 promoter; T+, T7 terminators; B, BamHI; H, HindlIl. Physical maps are not in actual proportions.
L-Orn N5-oxygenase encoded by the sidi gene of ferrichromeproducing U. maydis (40) and L-lysine N6-hydroxylase encoded by the iucD gene of aerobactin-producing E. coli (28). Both enzymes are close functional analogs of PvdA. The homology of PvdA with its functional analogs was extended through the whole amino acid sequence. It was more significant with Sidl than IucD, probably as a consequence of the different substrate specificity of the two prokaryotic enzymes. However, PvdA showed at the N terminus the motif IGVGFGP which, except for a T--F substitution, corresponds to the putative FADbinding motif described in IucD (40). pvdA, sid], and iucD showed low homology in analysis of the DNA sequences; a FASTA comparison of the three sequences revealed only one region of significant homology (60.3% identity) in a 389-bp overlap between nucleotides 109 to 498 of pvdA and 94 to 483 of sidl (40), corresponding to the N-terminal regions of encoded peptides. The G+C content of the pvdA gene was consistent with that reported for the P. aeruginosa genome. Codon usage analysis indicates that P. aeruginosa optimal codons (68) were frequently used throughout the coding sequence, thus probably enhancing the rate of translation. Moreover, a relatively high thermodynamic stability for the 1,278-base transcript of the pvdA coding sequence could be predicted by computer analysis (a AG of approximately - 490 kcal/mol in comparison with -335 kcal/mol of the 1,278-base transcript of the iucD coding sequence). Sequence analysis did not allow the detection of transcription start and stop signals flanking the ORF, nor was it possible to demonstrate significant homology of the DNA region preceding the translation start codon with positively-regulated or Fe(III)-regulated promoters known in P. aeruginosa and other Pseudomonas spp.
(19, 23, 46). Although a Fur-like protein and Fur-box-like sequences have recently been shown in P. aeruginosa (12, 17, 46, 51, 52), the latter were absent in the DNA region preceding the translation start codon of pvdA. Furthermore, transcriptional terminators were apparently lacking from the DNA region downstream of the 3' end of the pvdA coding sequence. These observations lead us to hypothesize thatpvd4, like many siderophore biosynthetic genes from different bacterial species, could be translated from a polycistronic messenger (8). Indeed, the presence in cosmid pPV4 of DNA regions complementing two distinct but closely associated mutations, i.e.,pvd4 and pvdC4, points to a clustering of pyoverdin genes, as already proposed for other fluorescent pseudomonads 39, 42). Nevertheless, the strict negative regulation exerted(36, Fe(III) on the expression of L-Orn N5-oxygenase activity by in PALS124 trans complemented with pPV225 suggests that the DNA region preceding the pvdA start codon in pPV225 may contain negative regulatory elements, e.g., repressor-binding sequences. Clearly, further experiments are required to clarify this discrepancy and localize the transcription start site and regulatory DNA elements involved in Fe(III)-dependent expression of the pvdA gene. Hydrophilicity pattern comparison of the PvdA, Sidl, and IucD proteins revealed that both prokaryotic enzymes were characterized by a conserved, highly hydrophobic region at their N terminus. This region was absent in the eukaryotic Sidi protein. Recent studies have demonstrated that inner membrane-spanning domains can be generated by short stretches of hydrophobic or neutral amino acids at the N-terminal region of membrane-associated peptides in gram-negative bacteria (13, 16, 22). In IucD, the N-terminal hydrophobic domain displays consensus
1138
J. BACTERIOL.
VlSCA ET AL.
A
0.5 kb oriV
B
mob
o,lr
1
Inegato P. aeruginosa PAO1
3
4
5
6
kb
I
pPV2251 (7.4 kb)
2
-11.1
3.7
chromosome I
2.0 Bg
P SSh Sh P P I I
bff SI *etA,
mob oriT
oraV s
P SBgShPS
LiJLJAL
P. aeruginosa PAAC1 chromosome 4.9 kb
3.7 kb
0.8 kb
-.8
___
11.1
kb-
FIG. 6. Construction of a P. aeruginosa pvdA mutant by gene disruption. (A) The 0.5-kb PstI internal fragment of pvdAl was ligated to pME3087 (66) which had been cut with PstI, giving pPV2251. This construct was introduced into E. coli S17.1 (57) by transformation and then conjugated into wild-type PAO1. Selection for tetracycline resistance carried by the plasmid resulted in the recovery of clone PAAC1, carrying pPV2251 integrated in the pvdA gene by homologous recombination. (B) Southern analysis of P. aeruginosa strains PAO1 (lanes 1 and 4), PAAC1 (lanes 2 and 5) and PALS124 (lanes 3 and 6) with the pvdA probe. The chromosomal DNA (approximately 50 p.g) was digested with BglII (lanes 1 to 3) and Sall (lanes 4 to 6), run on an agarose gel, transferred to nitrocellulose, and hybridized with the 1.2-kb SphI-SalI fragment from pPV225HB, A encompassing most of the pvdA gene and part of the 3' flanking region. The sizes of hybridization bands are indicated at the bottom of panelfor and shown on the right side of the autoradiogram in panel B. Abbreviations: tetR and tetA, tetracycline resistance genes; mob, genes mobilization; oriT, origin of transfer; oriV, origin of replication; Bg, BglII; P, PstI; S, Sall; Sh, SphI.
several features resembling those of signal peptides and has been involved in the anchoring of the enzyme to the inner side of the cytoplasmic membrane (28). Thus, a similar location can also be speculated for the PvdA enzyme.
1
2
3
4
5
6
7
kb
-1.7
-0.8 FIG. 7. Southern analysis of genomic DNA from different PseudoThe genomic DNAs (approximately 50 ,ug) from P. aeruginosa PAOI (lane 1), P. aeruginosa ATCC 27853 (lane 2), P. fluorescens CHAO (lane 3), P. fluorescens ATCC 13525 (lane 4), P. putida WCS358 (lane 5), Pseudomonas sp. B10 (lane 6), and P. cepacia TVV75 (lane 7) were digested with SphI, electrophoresed, and transferred to nitrocellulose. The digested genomes were probed with the 1.2-kb SphI-Sall fragment from pPV225HB. The sizes of hybridization bands are shown on the right side of the autoradiogram. monas spp.
The pvdA gene product was expressed in P. aeruginosa by using a bacteriophage T7 RNA polymerase-controlled expression system (10). The molecular mass of the expressed protein determined by SDS-PAGE was 47.7 kDa, consistent with the value predicted from sequence analysis. Moreover, there was an approximately fourfold increase in both L-Orn N5-oxygenase activity and the level of the 47.7-kDa protein upon induction of the T7 transcription system. We have also constructed a pvdA insertion mutant by homologous recombination between an internal fragment of the pvdA gene cloned in a suicide plasmid and the wild-type allele. Like the pvdA mutant PALS124, the site-specific insertion mutant PAAC1 grew poorly in minimal medium supplemented with the chelator 2,2'-dipyridyl, disclosed no fluorescence, lacked L-Orn N5-oxygenase activity, and was biochemically complemented by the pyoverdin precursor L-N5-OH-Orn. Moreover, transformation of the site-specific mutant with a plasmid harboring the pvdA gene restored the pyoverdinproficient phenotype. Taken together, these results unambiguously indicate that the cloned gene encodes the L-Orn N5-oxygenase enzyme. The clonedpvdA gene was also used to search for functional analogs in some hydroxamate-producing strains belonging to genus Pseudomonas. Interestingly, an SphI fragment of 1.7 kb hybridized with the pvdA probe in all the strains examined except P. putida WCS358. This observation suggests that L-Orn N5-oxygenase genes could display a fair degree of homology between different members of genus Pseudomonas, in agree-
L-ORNITHINE N5-OXYGENASE
VOL. 176, 1994
ment with results previously reported for pyoverdin biosynthetic genes in fluorescent pseudomonads (53). Hydroxylation of an w-amino acid and its further acylation are primary steps in the generation of the Fe(III)-chelating group of several hydroxamate-like siderophores, including ferrichromes (21, 40, 67), rhodotorulic acid (2, 3), and aerobactin (8, 27, 43). In this report, we have demonstrated that the enzyme catalyzing the N5-hydroxylation of L-Orn in P. aeruginosa PAO1 shares features with functional analogs found in other bacterial and fungal species. Thus, w-amino acid oxygenases appear to be relatively conserved over a wide evolutionary range. These enzymes catalyze a unique microbial reaction, not known to occur in animal and plant cells. A better understanding of their mechanism of action could allow the development of drugs (inhibitors and analogs) of relevant therapeutic value because of a capability of interfering with in vivo Fe(III) uptake by pathogenic bacteria. ACKNOWLEDGMENTS We are indebted to A. Darzins (Ohio State University, Columbus, Ohio), D. Haas (University of Lausanne, Lausanne, Switzerland), J. B. Neilands (University of California, Berkeley, Calif.), H. P. Schweizer (University of Calgary Health Sciences Center, Calgary, Alberta, Canada), and V. Venturi (State University, Utrecht, The Netherlands) for the generous gift of strains, plasmids, and reagents. We are also grateful to A. Crisanti and E. Zennaro for encouraging advice.
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