Abstract
Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers. DNA hybridization analysis using B. bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B. bronchiseptica mutants. Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B. pertussis genomic DNA region. Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases. Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis. Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity. Enzyme assays confirmed that group III B. bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin. Siderophore production by an analogous mutant of B. pertussis constructed by allelic exchange was undetectable. We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production. Putrescine is an essential precursor of alcaligin in Bordetella spp.
Nutritional iron limitation, mediated primarily by specific host iron-binding glycoproteins, is a front-line host defense against disease-causing infectious agents. Strategies aimed at defeating host iron restriction may involve the action of lowmolecular-mass, high-affinity, ferric iron-specific chelators of microbial origin, termed siderophores, that are synthesized coordinately with their cognate surface receptors and transport machinery in response to iron starvation (28). The role of siderophores in microbial pathogenesis is well established (29, 53, 54) .
Bordetella pertussis, the causative agent of human whooping cough or pertussis, and Bordetella bronchiseptica, the agent of swine atrophic rhinitis and kennel cough in dogs, are mucosal pathogens that colonize the upper respiratory tracts of their mammalian hosts. In the first reported molecular genetic studies of Bordetella iron acquisition systems, Armstrong and Clements (3) described the isolation of B. bronchiseptica mutants deficient in siderophore activity following transposon mutagenesis. DNA hybridization analysis using DNA probe sequences flanking the transposon insertions established the existence of homologs of B. bronchiseptica siderophore genes in B. pertussis. Reciprocal cross-feeding experiments provided additional support for the hypothesis that the hydroxamate siderophores produced and utilized by B. pertussis and B. bronchiseptica were structurally similar or identical. In a subsequent report, complementation of deregulated iron transport system mutants of B. bronchiseptica by the fur gene of B. pertussis (9) confirmed the involvement of common regulatory determinants governing iron transport in the two related species.
Recent purification and spectroscopic analysis of Bordetella siderophores (10, 33) found that the iron chelators produced by both B. pertussis and B. bronchiseptica were identical to the potent macrocyclic dihydroxamate siderophore alcaligin, 1,8(S), 11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12, 15-tetrone (molecular formula, C 16 H 28 N 4 O 8 ; molecular weight, 404), previously isolated from the taxonomically related bacterial species Alcaligenes denitrificans subsp. xylosoxydans (35, 36) . Evidence for biological activity of the purified siderophores was gathered with growth stimulation and 55 Fealcaligin transport assays of Bordetella spp. (10). Uptake rates and saturability of iron uptake observed in 55 Fe transport assays were indicative of a high-affinity ferric alcaligin transport system and provided the first direct evidence, beyond simple growth stimulation assays, of alcaligin-mediated Bordetella iron transport.
We report the identification of a gene encoding an alcaligin siderophore biosynthetic activity defined by previously reported transposon mutations abrogating alcaligin production in B. bronchiseptica (3) as an ornithine decarboxylase (Odc) gene, indicating that putrescine is an essential precursor for alcaligin siderophore biosynthesis in Bordetella spp.
Bacterial strains and plasmids. B. bronchiseptica B013N, a nalidixic acidresistant derivative of strain B013, and the isolation of siderophore biosynthetic mutants BRM3 and BRM5 by mini-Tn5/lacZ1 (16) insertional inactivation have been described previously (3). Virulent-phase B. pertussis UT25 has also been described previously (18 

Nutritional iron limitation, mediated primarily by specific host iron-binding glycoproteins, is a front-line host defense against disease-causing infectious agents. Strategies aimed at defeating host iron restriction may involve the action of lowmolecular-mass, high-affinity, ferric iron-specific chelators of microbial origin, termed siderophores, that are synthesized coordinately with their cognate surface receptors and transport machinery in response to iron starvation (28) . The role of siderophores in microbial pathogenesis is well established (29, 53, 54) .
Bordetella pertussis, the causative agent of human whooping cough or pertussis, and Bordetella bronchiseptica, the agent of swine atrophic rhinitis and kennel cough in dogs, are mucosal pathogens that colonize the upper respiratory tracts of their mammalian hosts. In the first reported molecular genetic studies of Bordetella iron acquisition systems, Armstrong and Clements (3) described the isolation of B. bronchiseptica mutants deficient in siderophore activity following transposon mutagenesis. DNA hybridization analysis using DNA probe sequences flanking the transposon insertions established the existence of homologs of B. bronchiseptica siderophore genes in B. pertussis. Reciprocal cross-feeding experiments provided additional support for the hypothesis that the hydroxamate siderophores produced and utilized by B. pertussis and B. bronchiseptica were structurally similar or identical. In a subsequent report, complementation of deregulated iron transport system mutants of B. bronchiseptica by the fur gene of B. pertussis (9) confirmed the involvement of common regulatory determinants governing iron transport in the two related species.
Recent purification and spectroscopic analysis of Bordetella siderophores (10, 33) found that the iron chelators produced by both B. pertussis and B. bronchiseptica were identical to the potent macrocyclic dihydroxamate siderophore alcaligin, 1,8(S), 11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12, 15-tetrone (molecular formula, C 16 H 28 N 4 O 8 ; molecular weight, 404), previously isolated from the taxonomically related bacterial species Alcaligenes denitrificans subsp. xylosoxydans (35, 36) . Evidence for biological activity of the purified siderophores was gathered with growth stimulation and 55 Fealcaligin transport assays of Bordetella spp. (10) . Uptake rates and saturability of iron uptake observed in 55 Fe transport assays were indicative of a high-affinity ferric alcaligin transport system and provided the first direct evidence, beyond simple growth stimulation assays, of alcaligin-mediated Bordetella iron transport.
We report the identification of a gene encoding an alcaligin siderophore biosynthetic activity defined by previously reported transposon mutations abrogating alcaligin production in B. bronchiseptica (3) as an ornithine decarboxylase (Odc) gene, indicating that putrescine is an essential precursor for alcaligin siderophore biosynthesis in Bordetella spp.
MATERIALS AND METHODS
Bacterial strains and plasmids. B. bronchiseptica B013N, a nalidixic acidresistant derivative of strain B013, and the isolation of siderophore biosynthetic mutants BRM3 and BRM5 by mini-Tn5/lacZ1 (16) insertional inactivation have been described previously (3) . Virulent-phase B. pertussis UT25 has also been described previously (18) .
(Bethesda Research Laboratories, Gaithersburg, Md.) was used as the host strain for routine plasmid construction and propagation and as the donor strain in conjugal transfer of plasmids to Bordetella strains. Plasmid pRK/3.2P, carrying the speC constitutive ornithine decarboxylase gene of E. coli, was constructed by subcloning the 3.2-kb E. coli PstI insert DNA fragment of plasmid pODC1 (8) into the broad-host-range plasmid vector pRK415 (26) . DH5␣ (pRK2013) was used as the source of plasmid-encoded mobilization functions (19) in triparental matings to transfer the cosmid pCP13-based gene bank of B. pertussis UT25 (11) as well as plasmid vector pRK415 derivatives to Bordetella strains. E. coli K38 (pGP1-2) (46) was used as the host strain in Bordetella protein expression studies. Growth conditions. B. pertussis was maintained on Bordet-Gengou agar plates (7), and B. bronchiseptica was maintained on blood agar plates or standard Luria-Bertani agar plates. Modified Stainer-Scholte medium (39, 41) (SS) was used for broth cultures of Bordetella spp. Iron-replete and iron-depleted SS culture conditions were achieved by the methods of Armstrong and Clements (3); media and supplements were deferrated with Chelex-100 resin (Bio-Rad Laboratories, Richmond, Calif.). Tetracycline was used at 15 g/ml to select for pCP13-based cosmids and pRK415 plasmid derivatives. Kanamycin was used at 50 g/ml for maintenance of pRK2013 and pGP1-2 and for selection of kanamycin cassette insertion markers in allelic exchange procedures, while ampicillin was used at 100 g/ml for maintenance of other plasmids in E. coli. Culture media were supplemented as required with cadaverine, agmatine, putrescine, ornithine, arginine, or spermidine at a 50-g/ml final concentration, or with 1,4-diamino-2-butanone dihydrochloride (DAB) (Sigma Chemical Co., St. Louis, Mo.) at concentrations ranging from 0 to 2 mM. All glassware was acid cleaned and rinsed repeatedly in distilled deionized water prior to use. Optical densities of SS cultures were monitored with a Klett-Summerson colorimeter fitted with a no. 54 filter (Klett Manufacturing Co., Long Island City, N.Y.).
Conjugation. Conjugal transfer of pCP13 and pRK415 derivatives to Bordetella strains was accomplished by triparental matings with E. coli DH5␣ as the plasmid donor strain and DH5␣ (pRK2013) as the source of mobilization functions. Suspensions of donor, helper, and recipient strains in SS plus 10 mM MgSO 4 were combined at an estimated 1:1:3 cell ratio, spotted onto Luria-Bertani or blood agar plates (for B. bronchiseptica recipients) or Bordet-Gengou agar plates (for B. pertussis recipients), and incubated at 37ЊC for 3 to 5 h. Bacteria were streaked from mating spots directly onto agar plates containing the appropriate selective antibiotics and crude colicin B (3, 12) . Plates were incubated at 37ЊC for 1 to 4 days, at which time transconjugant colonies were apparent.
Routine DNA procedures. DNA cloning and hybridization analysis were performed as described previously (38) , and transformation of E. coli was by the CaCl 2 method of Cohen and coworkers (14) .
Radiochemicals. Conditional expression of plasmid-encoded Bordetella proteins in E. coli. Exclusive expression of Bordetella proteins in E. coli used the temperature-sensitive T7 RNA polymerase-promoter system of Tabor and Richardson (46) . Recombinant plasmid pT7/odc carries an approximately 2.8-kb odc ϩ DNA fragment of B. pertussis UT25 in the T7 promoter plasmid vector pGEM4Z (Promega Corporation, Madison, Wis.). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of radiolabeled bacterial cell proteins was performed essentially as described previously (27) .
Nucleotide sequencing. Nucleotide sequencing was performed by the dideoxy chain termination method with a Sequenase version 2.0 kit (United States Biochemical Corp., Cleveland, Ohio). Double-stranded plasmid templates were isolated by the alkaline lysis procedure (6) and further purified by precipitation in the presence of 500 mM sodium chloride and 6.5% polyethylene glycol 8000. Nucleotide sequence data were derived from plasmid subclones of odc ϩ B. pertussis cosmids, as well as from a collection of deletion derivatives generated by treatment of odc ϩ plasmid subclones with exonuclease III (21) by using a commercially available kit (Erase-a-Base; Promega Corporation) or from deletion derivatives resulting from in vivo intramolecular transposition of transposon ␥␦ (52) with the Deletion Factory System version 2.0 (Life Technologies, Inc., Gaithersburg, Md.). Nucleotide sequence data management and analysis used DNA Strider 1.2 software (Service de Biochimie et de Génétique Moléculaire, Gif sur Yvette, France) for the Macintosh computer. Database searches and data retrievals used the BLAST (1) and RETRIEVE electronic mail servers developed by the National Center for Biotechnology Information at the National Library of Medicine. For protein database BLASTP searches, Bordetella DNA sequences were translated in all six possible reading frames, and the resulting amino acid sequences were transmitted to the National Center for Biotechnology Information for analysis. Multiple amino acid sequence alignments were performed with the MegAlign module of a demonstration version of the Lasergene sequence analysis software system for the Power Macintosh computer (DNA STAR, Inc., Madison, Wis.).
Siderophore assays. Chrome azurol S (CAS) agar plates for scoring of B. bronchiseptica siderophore production were prepared as described by Armstrong and Clements (3). The CAS universal siderophore detection assay (40) was used to monitor siderophore production by Bordetella strains grown in liquid culture by measuring the decrease in A 630 of the CAS dye reaction mixture as reported previously (3) . Siderophore activities are expressed relative to uninoculated complete culture medium. Siderophore assays were performed in triplicate in multiple experimental trials. Purified alcaligin used in control siderophore activity assays and spectroscopic analysis was isolated from B. bronchiseptica cultures by the simplified large-scale method described by Brickman and coworkers (10) . Determination of hydroxamates was by the method of Csaky (15) with hydroxylamine hydrochloride as standard. Visible and UV spectroscopy used a Beckman DU-65 spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.).
Inhibition of alcaligin siderophore production by a competitive inhibitor of Odc activity. Wild-type B. bronchiseptica B013N was cultured overnight in ironreplete SS, and then bacterial cells from 200 l of culture were harvested by centrifugation, washed twice with 1-ml volumes of iron-depleted SS, and used to seed 20 ml of iron-depleted SS. The resulting suspension was divided among sterile culture tubes, and a 0.5 M stock solution of aqueous DAB, a competitive inhibitor of Odc activity (13) , was added to the first tube to provide 2 mM final concentration. DAB was diluted serially 1:5 through the series of culture tubes, yielding starting cultures containing eric53@example.org, 0.400, 0.080, 0.016, 0.003, and 0.001 mM final concentrations. A control culture had no DAB added. Cultures were incubated with shaking at 37ЊC for 18 h and then assayed for siderophore production by the CAS method.
Odc enzyme assays. Odc activities of Bordetella strains were assayed by a modification of the method described by Tabor and coworkers (44, 45) for mass screening of polyamine biosynthetic mutants. Odc activities were determined for Bordetella strains grown under iron-replete or iron-depleted SS culture conditions to examine whether expression of Odc activity was iron regulated. Bordetella strains grown for approximately 20 h in iron-replete SS were subcultured 1:200 into iron-replete or iron-depleted SS and grown with shaking at 37ЊC for an additional 16 to 18 h, corresponding to mid-to late logarithmic growth phase. A volume of each culture equivalent to 2 optical density units (600-nm wavelength) of bacterial cells (usually 0.5 to 1 ml) was pelleted gently in a microcentrifuge, washed twice with 1-ml volumes of a cold buffer consisting of 20 mM Tris-HCl (pH 8.0)-5 mM dithiothreitol-1 mM EDTA (TDE buffer), and resuspended in 100 l of cold TDE. Twofold serial dilutions of cell suspensions were prepared in cold TDE buffer, and then to each dilution an equal volume of cold TDE-2% toluene emulsion was added. Fifty-microliter volumes of the resulting cell suspensions in TDE-1% toluene, equivalent to 2 ϫ 10 14 C]ornithine (0.3 Ci/ml) was added to each well, and then the plates were covered with filter paper wetted with a freshly prepared saturated solution of barium hydroxide to trap evolved 14 CO 2 as insoluble barium carbonate. The 96-well tray lids were replaced, and the plates were wrapped in plastic wrap, clamped securely to provide tight contact of the filter paper over the wells, and incubated at 37ЊC. After 2 to 3 h, the filter paper was removed, rinsed briefly in acetone, air dried, and subjected to autoradiography for 2 to 5 days. Autoradiographs were optically scanned, and digitized images were integrated with the National Institutes of Health Image version 1.57 image processing and analysis program for the Macintosh computer. The areas of the dried filters corresponding to each well were excised, and captured [ 14 C]barium carbonate was quantified with EcoLume scintillant (ICN Biomedicals, Inc.) in a Beckman LS5000TD liquid scintillation counter (Beckman Instruments, Inc.). Nonenzymatic background levels of 14 CO 2 evolution in Odc assays were determined with B013N cell suspensions that were boiled for 10 min prior to dilution with TDE-2% toluene. Siderophore levels of the cultures used in these experiments were monitored by CAS assay.
Construction of a B. pertussis odc mutant by allelic exchange. The 5Ј-coding region of the B. pertussis odc gene was deleted from a cloned 8.5-kb XhoI DNA fragment by substitution of an internal 3.8-kb EcoRI B. pertussis DNA fragment with a 2.1-kb Tn5-derived kanamycin resistance cassette in the allelic exchange plasmid vector pRTP1 (42) . The resulting deletion-replacement plasmid construct was conjugally transferred to a spontaneous streptomycin-resistant derivative of B. pertussis UT25, UT25Sm1 (2), and the mutation was transferred to the chromosome by homologous recombination as described by Stibitz and coworkers (42) . Reciprocal exchange was verified by Southern hybridization analysis. The resulting ⌬oaf76:e4bb:d472:9fe5:10c0:37a3:766a:5b99Kan B. pertussis mutant was designated P12.
Nucleotide sequence accession number. The GenBank accession number assigned to the partial B. pertussis UT25 odc nucleotide sequence is U34895.
RESULTS
Transposon insertions defining group III siderophore-deficient mutants of B. bronchiseptica interrupt a genetic region predicted to encode an amino acid decarboxylase. Molecular cloning of mini-Tn5/lacZ1 transposon insertion markers associated with group III B. bronchiseptica siderophore-deficient mutants BRM3 and BRM5, resulting in recombinant plasmids pBRM3 and pBRM5, was accomplished in a previous study (3) . B. bronchiseptica genomic sequences flanking the inser- VOL. 178, 1996 BORDETELLA ALCALIGIN SIDEROPHORE BIOSYNTHESIS GENE 55 tions were used in DNA hybridization analysis to demonstrate the existence of homologous sequences in the related species B. pertussis. Restriction mapping estimated that the chromosomal sites of the BRM3 and BRM5 insertions were separated by approximately 200 to 300 bp. BRM3 and BRM5 displayed no significant growth defect other than the inability to cope with iron starvation.
To isolate the B. pertussis homolog of the mutated group III siderophore gene(s), an approximately 700-bp NotI-SalI fragment of pBRM3 was used to probe a B. pertussis UT25 cosmidbased genomic library by in situ DNA hybridization. Four distinct recombinant cosmids that hybridized strongly with the B. bronchiseptica DNA probe at high stringency were identified. Restriction mapping and Southern hybridization analysis of the four cosmids, pCP3.1, pCP3.2, pCP3.3, and pCP3.4, determined that they shared B. pertussis DNA sequences homologous to the B. bronchiseptica DNA region interrupted by the insertions defining the group III siderophore-deficient mutants (data not shown). All four B. pertussis cosmids restored wild-type levels of iron-regulated siderophore production to mutants BRM3 and BRM5 when introduced by conjugal transfer, as determined by the CAS siderophore assay and absorption spectroscopy of iron-depleted culture supernatants. Subcloning and complementation analysis established that the cosmids shared an 8.5-kb XhoI B. pertussis UT25 genomic DNA fragment that encoded the complementing activity (Fig. 1) . Cosmid pCP3.3 was selected for further examination. Complementation analysis of pCP3.3 plasmid subclones and deletion derivatives mapped the genetic region encoding the complementing activity to an approximately 2.8-kb B. pertussis DNA fragment extending from a B. pertussis NotI restriction site to a terminus generated by exonuclease III treatment; the genetic limits of this fragment are depicted in Fig. 1 . The minimal complementing plasmid pRK/143.1 carries the 2.8-kb fragment in the broad-host-range plasmid vector pRK415.
Nucleotide sequencing analysis of pBRM3 and pBRM5 with synthetic oligonucleotide primers complementary to the miniTn5/lacZ1 transposon ''I'' and ''O'' ends confirmed that the BRM3 and BRM5 chromosomal insertions were separated by 222 bp. Nucleotide sequences flanking the cloned transposon insertions were identical to corresponding sequences determined later for the complementing B. pertussis DNA. Nucleotide sequencing of pCP3.3 plasmid subclones and deletion derivatives and subsequent BLAST database searches of the translated B. pertussis DNA sequences identified high-scoring segment pairs with a family of pyridoxal phosphate-dependent amino acid decarboxylase protein sequences. These decarboxylases included LDC, the lysine decarboxylase of Hafnia alvei (17) ; CadA, the inducible lysine decarboxylase of E. coli (32) ; Adi, the biodegradative arginine decarboxylase of E. coli (43) ; SpeC, the constitutive biosynthetic Odc of E. coli (4); and SpeF, the inducible Odc of E. coli (25) . The putative B. pertussis decarboxylase homolog was encoded by a DNA region corresponding to the B. bronchiseptica genomic region disrupted by the transposon insertions defining BRM3 and BRM5 (Fig. 1) .
Over the highest-scoring 63-amino-acid segment of similarity with the decarboxylases, multiple sequence alignments found 54% identity of the deduced partial Bordetella amino acid sequence with LDC, 52% identity with CadA, 46% with Adi, and 38% with both SpeC and SpeF. A multiple amino acid sequence alignment of this region is shown in Fig. 2 . Percent identity between the deduced B. pertussis polypeptide and each of the same known decarboxylases over a 20-amino-acid region spanning the highly conserved pyridoxal phosphate cofactorbinding site ranged from 50 to 80.
Expression of a putative Bordetella amino acid decarboxylase protein in E. coli. The 2.8-kb B. pertussis DNA fragment including the region encoding the putative pyridoxal phosphatedependent amino acid decarboxylase homolog was subcloned into plasmid vector pGEM4Z to produce pT7/odc, allowing bacteriophage T7 promoter-directed expression of Bordetella proteins in E. coli K38 (pGP1-2). Plasmid pT7/odc-encoded proteins were exclusively 35 S radiolabeled and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A B. pertussis DNA-encoded product of approximately 86 kDa in apparent molecular mass was consistently observed in autoradiographs of SDS-polyacrylamide gels (Fig. 3) , as was the T7-transcribed vector ␤-lactamase product(s) also seen in pGEM4Z vector control samples. The observed 86-kDa molecular-mass product is within the calculated limits of the coding capacity of the 2.8-kb DNA fragment and approximates the 82-to 84-kDa mass of the homologous decarboxylases identified in database searches.
Restoration of alcaligin siderophore production to mutant BRM3 by culture medium supplementation with amino acid decarboxylase products. In an attempt to circumvent the siderophore biosynthesis defect associated with the loss of a putative amino acid decarboxylase activity in group III mutant BRM3, iron-depleted SS culture medium was supplemented with cadaverine, agmatine, or putrescine, which are the prod- 
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ucts of lysine decarboxylase, arginine decarboxylase, or Odc activities, respectively. Iron-regulated alcaligin production by BRM3 was restored to wild-type levels by medium supplementation with putrescine (1,4-diaminobutane) and only partially restored by supplementation with agmatine (1-amino-4-guanidobutane) (Fig. 4) . Cadaverine supplementation had no effect on siderophore production by BRM3, nor did supplementation with ornithine, arginine, or spermidine in other experiments. All supplements had no effect on alcaligin production by wild-type B013N, except putrescine, which resulted in a slight enhancement of siderophore production. In E. coli, putrescine can be produced by two alternative biochemical pathways (34) : the first and most efficient route is direct decarboxylation of ornithine, and the second is a twostep process involving decarboxylation of arginine to form agmatine, followed by the conversion of agmatine to putrescine by the action of agmatine ureohydrolase (Fig. 5) . Full restoration of siderophore production to BRM3 by putrescine and partial restoration by agmatine were consistent with the predicted involvement of an amino acid decarboxylase activity, possibly either an Odc or an arginine decarboxylase, in the production of putrescine, the proposed precursor of the Bordetella siderophore alcaligin on the basis of structural analysis.
Inhibition of Bordetella alcaligin production by an inhibitor of Odc. Supplementation of iron-depleted SS culture medium with the Odc inhibitor DAB resulted in strong reduction in siderophore levels achieved by wild-type B. bronchiseptica B013N (Fig. 6) . Alcaligin production was highly sensitive to DAB inhibition; the lowest inhibitor concentration tested, 0.001 mM, decreased siderophore production by approximately 20%. At 0.080 mM DAB, inhibition was near maximal at approximately 50% inhibition of siderophore production compared with untreated cultures. DAB concentrations used 
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BORDETELLA ALCALIGIN SIDEROPHORE BIOSYNTHESIS GENE 57
were not growth inhibitory over the culture period examined, and control assays established that DAB did not interfere with the CAS siderophore assay.
Restoration of siderophore production to mutant BRM3 by the speC constitutive Odc-encoding gene of E. coli. Concordant with the decarboxylase product feeding results, the siderophore biosynthesis defect of BRM3 was functionally complemented in trans by plasmid pRK/3.2P, encoding the heterologous Odc activity SpeC of E. coli (8) ( Table 1 ). Alcaligin levels produced by BRM3 (pRK/3.2P) approximated those of wildtype B. bronchiseptica B013N and BRM3 carrying the group III-complementing B. pertussis plasmid pRK/143.1.
Restoration of Odc activity to BRM3 by plasmids complementing its siderophore biosynthesis defect. In Odc assays, group III siderophore-deficient mutant BRM3 was found to lack significant levels of Odc activity compared with the wildtype parent strain B013N (Table 1) . Counts determined were in close agreement with integrated values resulting from computer analysis of digitized autoradiographic images. Activities for mutant BRM3 and BRM3 carrying the plasmid vector pRK415 were essentially the same as nonenzymatic background activity of boiled B013N suspensions. Levels of Odc activity comparable to those expressed by wild-type B013N were restored to BRM3 by the B. pertussis plasmid, pRK/143.1, that complemented its siderophore biosynthesis defect, indicating that the amino acid decarboxylase activity required for alcaligin biosynthesis is an Odc. Bordetella Odc enzymatic activities appeared to be largely independent of iron concentration in the culture medium, suggesting that the previously reported Fur-mediated iron regulation of Bordetella siderophore biosynthesis (9) is exerted over other alcaligin biosynthetic activities.
Construction and analysis of a group III-analogous mutant of B. pertussis. Siderophore-deficient B. pertussis mutant P12 was constructed by allelic exchange by using a deletion-replacement pRTP1 derivative to delete approximately 1 kb of N-terminal coding sequences of the B. pertussis Odc-encoding gene required for alcaligin biosynthesis. Deletion of the 3.8-kb B. pertussis genomic DNA fragment depicted in Fig. 1 and replacement by the 2.1-kb Tn5-derived kanamycin resistance cassette were confirmed by Southern hybridization analysis of genomic DNA isolated from a dozen independent kanamycinresistant derivatives. As with B. bronchiseptica mutant BRM3, B. pertussis P12 was incapable of alcaligin siderophore production unless supplied with the wild-type DNA sequences in trans or unless the iron-depleted growth medium was supplemented with putrescine (Fig. 7) . These data establish that the Odc, for which we propose the genetic designation odc, is required for biosynthesis of the siderophore alcaligin (Fig. 8) in B. pertussis as well as in B. bronchiseptica.
DISCUSSION
We report the identification of the odc gene encoding an enzymatic activity essential for alcaligin siderophore biosynthesis in Bordetella spp. This Odc supplies the alcaligin precursor 1,4-diaminobutane, commonly known as putrescine. The involvement of an amino acid decarboxylase activity in the biosynthesis of alcaligin was predicted on the basis of database searches of translated DNA sequences corresponding to the genomic region interrupted by transposon insertions defining previously reported B. bronchiseptica alcaligin-deficient mu- tants BRM3 and BRM5. Evidence supporting the involvement of an Odc activity was gained in mutant feeding experiments in which culture medium supplementation with decarboxylated ornithine (putrescine) fully restored alcaligin biosynthetic capacity to BRM3. Accordingly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by expression of a heterologous Odc activity, SpeC of E. coli. Ultimately, BRM3 was shown to be defective in Odc activity compared with its wild-type parent B013N in crude enzyme assays, and wild-type Odc activity levels were restored to BRM3 by plasmids shown to complement its alcaligin biosynthesis defect. An Odc mutant of B. pertussis constructed by allelic exchange was likewise unable to produce alcaligin. The identification of putrescine as a precursor of alcaligin in Bordetella spp. is in agreement with earlier predictions based on the known structure of the siderophore.
The siderophore alcaligin was first purified from the taxonomically related species A. denitrificans subsp. xylosoxydans, isolated from freshwater lake sediment (35, 36) . Structural analysis found alcaligin to be a symmetric 20-membered ring, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone. The dihydroxamate molecule can thus be considered a cyclic dimer of two structural units consisting of 1-amino-4-(N-hydroxylamino)-2(S)-butanol and succinic acid. Consistent with this twofold symmetric structure, high-resolution proton and carbon nuclear magnetic resonance spectroscopy recently determined that the molecule displays a twofold symmetric conformation in aqueous solution, although evidence suggests that multiple C 2 -symmetric conformations of alcaligin coexist in aqueous and methanolic solution (10) . Alcaligin shares structural features with the ferrioxamine family of siderophores (30) , which are produced by actinomycetes as well as certain enterobacterial species such as H. alvei (55) . All ferrioxamines incorporate hydroxylated polyamines and succinic acid in their molecular structures. It is possible that LDC of H. alvei identified in database searches as a homolog of Bordetella Odc may be required for production of the cadaverine portion of Hafnia ferrioxamines. Alcaligin most resembles the only other known cyclic dihydroxamate siderophore, bisucaberin, produced by the marine bacterium Alteromonas haloplanktis (23, 48) . However, bisucaberin includes two residues of N-hydroxycadaverine instead of the hydroxyl-substituted N-hydroxyputrescine residues of alcaligin.
Certain catechol-type siderophores, such as agrobactin (37) , vibriobactin (20) , and parabactin (47) also contain polyamines, and in Paracococcus denitrificans, along with parabactin siderophore production, spermine production increases by an order of magnitude in response to iron starvation, although spermidine and not spermine donates its triamine backbone to the siderophore (5) .
Remarkable parallels exist between the Bordetella odc gene and the recently described hdc gene of Vibrio anguillarum required for biosynthesis of the siderophore anguibactin (49), a trait necessary for virulence. As with Bordetella odc, the V. anguillarum hdc gene was identified after transposon mutagenesis on the basis of a siderophore-deficient phenotype. Nucleotide sequence analysis of the mutated region of plasmid pJM1 identified deduced amino acid sequence similarities with known histidine decarboxylases. Anguibactin siderophore production by V. anguillarum hdc mutants was restored by culture medium supplementation with histamine, the product of histidine decarboxylation, confirming the predicted role of histamine as an essential precursor of that siderophore. Further, neither odc nor hdc is repressible by iron, although alcaligin and anguibactin siderophore production have been shown to be negatively regulated by the action of a Fur repressor (9, 50) , indicating that iron concentration controls other siderophore biosynthetic activities.
On the basis of the elucidation of the alcaligin biosynthesis role of odc in Bordetella spp. and the known structure of alcaligin, we hypothesize the subsequent action of an oxygenase catalyzing the hydroxylation of putrescine. This putative Bordetella oxygenase would supply an activity analogous to the IucD L-lysine N 6 -hydroxylase of E. coli (22) , involved in the biosynthesis of the hydroxamate siderophore aerobactin, and to the L-ornithine N 5 -oxygenases PvdA of Pseudomonas aeruginosa (51) and Sid1 of Ustilago maydis (31) , required for pyoverdin and ferrichrome siderophore biosynthesis, respectively. Preliminary nucleotide sequencing of the B. pertussis DNA corresponding to the B. bronchiseptica genomic region interrupted by transposons defining the group I alcaligin siderophore-deficient mutants BRM1 and BRM9 (GenBank accession number U34894) identified an open reading frame predicted to encode such a Bordetella homolog of the IucD, PvdA, and Sid1 oxygenases (24) . Confirmation of the role of this putative Bordetella oxygenase in the biosynthesis of alcaligin awaits further biochemical analysis.

{'paper_id': '7466674', 'title': 'The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin.', 's2_url': 'https://api.semanticscholar.org/CorpusID:7466674', 'source': 'S2ORC'}