Siderophore-Mediated Iron Uptake in Alcaligenes eutrophus CH34 ...

JOURNAL OF BACTERIOLOGY, Sept. 1996, p. 5499–5507 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 178, No. 18

Siderophore-Mediated Iron Uptake in Alcaligenes eutrophus CH34 and Identification of aleB Encoding the Ferric Iron-Alcaligin E Receptor ANJA GILIS,1 MUHAMMAD AYUB KHAN,1 PIERRE CORNELIS,2 JEAN-MARIE MEYER,3 MAX MERGEAY,1 AND DANIEL VAN DER LELIE1* Milieutechnologie, Vlaamse Instelling voor Technologisch Onderzoek (VITO), B-2400 Mol,1 and Vlaams Instituut voor Biotechnologie, Vrije Universiteit Brussel, B-1640 Sint-Genesius-Rode,2 Belgium, and Laboratoire de Microbiologie et de Ge´ne´tique, Universite´ Louis-Pasteur, Unite´ de Recherche Associe´e au Centre National de la Recherche Scientifique 1481, F-67000 Strasbourg, France3 Received 25 April 1996/Accepted 9 July 1996

Siderophore production in response to iron limitation was observed in Alcaligenes eutrophus CH34, and the corresponding siderophore was named alcaligin E. Alcaligin E was characterized as a phenolate-type siderophore containing neither catecholate nor hydroxamate groups. Alcaligin E promoted the growth of siderophore-deficient A. eutrophus mutants under iron-restricted conditions and promoted 59Fe uptake by ironlimited cells. However, the growth of the Sid2 mutant AE1152, which was obtained from CH34 by Tn5-Tc mutagenesis, was completely inhibited by the addition of alcaligin E. AE1152 also showed strongly reduced 59Fe uptake in the presence of alcaligin E. This indicates that a gene, designated aleB, which is involved in transport of ferric iron-alcaligin E across the membrane is inactivated. The aleB gene was cloned, and its putative amino acid sequence showed strong similarity to those of ferric iron-siderophore receptor proteins. Both wild-type strain CH34 and aleB mutant AE1152 were able to use the same heterologous siderophores, indicating that AleB is involved only in ferric iron-alcaligin E uptake. Interestingly, no utilization of pyochelin, which is also a phenolate-type siderophore, was observed for A. eutrophus CH34. Genetic studies of different Sid2 mutants, obtained after transposon mutagenesis, showed that the genes involved in alcaligin E and ferric iron-alcaligin E receptor biosynthesis are clustered in a 20-kb region on the A. eutrophus CH34 chromosome in the proximity of the cys-232 locus. 37). Consequently, the efflux of the heavy metals results in a pH increase outside the cytoplasmic membrane. Metals are sequestered from the external medium through the bioprecipitation of metal carbonates formed in the saturated zone around the cell (14). This latter phenomenon can be exploited in bioreactors designed to remove metals from effluents. The fact that A. eutrophus CH34 is resistant to a broad spectrum of heavy metals prompted us to investigate whether this strain produces siderophores and whether these siderophores are involved in heavy metal resistance, in order to obtain a better insight into the interactions between siderophore-mediated iron uptake and heavy metal resistance. This work reports the identification and chemical classification of alcaligin E, a novel siderophore produced by A. eutrophus CH34, and some of the genetic determinants involved in its biosynthesis and uptake. The aleB gene, encoding the ferric iron-alcaligin E receptor, is identified, and its role in the utilization of alcaligin E and heterologous siderophores by A. eutrophus CH34 is investigated. (This work is part of the Ph.D. theses of A. Gilis and M. A. Khan.)

Iron, the fourth most abundant element in the earth’s crust, forms insoluble ferric hydroxide complexes under aerobic conditions and at neutral pH, thus severely restricting the bioavailability of this metal. In virtually all microorganisms, iron plays an irreplaceable role as cofactor for a variety of functional proteins and enzymes. Therefore, microorganisms have evolved specialized high-affinity transport systems in order to obtain sufficient amounts of this essential element. Most bacteria have the ability to produce and excrete siderophores, small compounds exhibiting very high affinity for ferric iron (33). A cognate-specific transport system mediates the uptake of the ferric iron-siderophore complex into the cell (16, 34). Many microorganisms are also able to utilize iron complexed to siderophores produced by other bacterial or fungal species. In general, the biosynthesis of the siderophore and associated transport machinery is initiated under conditions of iron limitation. Metal-tolerant Alcaligenes eutrophus strains are a group of strongly related strains that are well adapted to environments containing high levels of heavy metals. A. eutrophus CH34 is the main representative and the most studied strain of this group. It harbors two megaplasmids, pMOL28 and pMOL30, which carry multiple resistance determinants to different heavy metals (7, 26). Among the best characterized is the czc operon of pMOL30, which determines resistance to Co, Cd, and Zn by a chemiosmotic cation/proton-antiporter efflux system (14, 35–

MATERIALS AND METHODS Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are described in Table 1. A. eutrophus and Escherichia coli were grown at 30 and 378C, respectively. Resistance to heavy metal salts was tested in Tris-minimal medium (26). Fe(III) was omitted from this medium in order to screen for siderophore production in iron-limiting conditions. The chrome azurol S (CAS) shuttle solution (46) was used for routine testing of siderophore production in liquid media. To detect siderophore production on solid medium, the CAS solution was added to autoclaved Tris-minimal medium. Antibiotic-resistant clones were selected on media supplemented, per milliliter, with 20 mg of tetracycline, 100 mg of ampicillin, or 50 (for E. coli) or 1,000 (for A. eutrophus)

* Corresponding author. Mailing address: Milieutechnologie, Vlaamse Instelling voor Technologisch Onderzoek (VITO), Boeretang 200, B-2400 Mol, Belgium. Phone: 32-14-335166. Fax: 32-14-320372. Electronic mail address: [email protected]. 5499

5500

J. BACTERIOL.

GILIS ET AL. TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid

Strains A. eutrophus CH34 AE104 AE232 AE1093 AE1152 AE1593 AE1594 AE1595 E. coli HB101 CM404 CM448 CM666 CM987 CM1570 Plasmids pRK2013 pLAFR3 pSUP10141 pUC18 pUT/mini-Tn5-Km1 pMOL871 pMOL872 pMOL873 pMOL874 pMOL875 pMOL1032

Relevant characteristic(s)

Source or reference

Wild type Plasmid-free derivative of CH34 cys-232 leu-27 met-81 trpE50 ale-1093::Tn5-Tc aleB1152::Tn5-Tc ale-1593::mini-Tn5-Km1 ale-1594::mini-Tn5-Km1 ale-1595::mini-Tn5-Km1

26 26

Plasmid free pRK2013 pSUP10141 pLAFR3 pUT/mini-Tn5-Km1 pUC18

4

Kmr Tra1, unable to replicate in A. eutrophus Tcr cosmid Tn5-Tc (Tcr Kmr) Cmr, unable to replicate in A. eutrophus Apr Lac1 Apr Kmr Tcr aleB1, hybrid pLAFR3 derivative Tcr aleB1, hybrid pLAFR3 derivative Tcr aleB1, hybrid pLAFR3 derivative Tcr aleB1, hybrid pLAFR3 derivative Tcr aleB1, hybrid pLAFR3 derivative Tcr ale-15951, hybrid pLAFR3 derivative

15

43 This This This This This

study study study study study

51 47 39 12 This study This study This study This study This study This study

mg of kanamycin. Succinate medium was used for the production of siderophores for extraction procedures (28). Characterization and partial purification of alcaligin E. Thin-layer chromatography was performed with silica gel 60F aluminum plates (Merck Laboratories, Darmstadt, Germany) and 1-butanol–glacial acetic acid–H2O (vol/vol/vol) as a solvent system. After development of the chromatography plates, they were sprayed with CAS solution or 0.1 M FeCl3 z 6H2O to detect the presence of siderophores. In order to determine the type of siderophore, different chemical tests were performed. The Csaky test (10) was used to determine hydroxamate groups, and hydroxylamine hydrochloride was used as a positive control. Phenolate groups were detected by the ferric chloride ferricyanide reagent (17) with phenol as a positive control. The Arnow test (2) was performed to screen for catecholate groups, and catechol was used as a positive control. All tests were performed on 10-fold-concentrated (by freeze drying) culture supernatant of cells grown under iron-limiting conditions. Fe(III) was added to control cultures in order to repress the siderophore production. Alcaligin E was partially purified as follows: alcaligin E was extracted from NaCl-saturated succinate culture supernatants (2 liters reduced to 200 ml under vacuum) with 70 ml of chloroform-phenol (1/1 [vol/wt]) under vigorous shaking and solubilized back to water by the addition of 2 volumes of diethyl ether and 1 volume of distilled water to the chloroform-phenol solution, with shaking. The aqueous solution was washed three times with diethyl ether and the residue, dried under vacuum, was kept at 2208C until used. The size of the partially purified alcaligin E was estimated by gel filtration on a Sephadex G15 column, with vitamin B12 (molecular weight, 1,357) and pyochelin (molecular weight, 325) as size standards. Conjugation experiments for transposon mutagenesis using Tn5-Tc and miniTn5-Km1. Triparental matings for transposon mutagenesis were done with CH34

as the receptor strain, CM404 (with pRK2013) (15) as the helper strain for mobilization, and CM448 (with pSUP10141/Tn5-Tc) (47) or CM987 (with pUT/ mini-Tn5-Km1) (12) as the donor strain. For the detection of siderophorenegative mutants, transconjugants were selected on Tris-minimal medium without Fe(III) to which the CAS solution was added. Molecular cloning techniques and electroporation. Restriction enzymes, phosphatase, and T4 DNA ligase were purchased from GIBCO BRL (Merelbeke, Belgium) and used as recommended by the supplier. Molecular cloning techniques, plasmid DNA isolation from E. coli, and colony hybridizations were performed as described by Sambrook et al. (44). Plasmid DNA was isolated from A. eutrophus as described by Taghavi et al. (52). Total DNA was isolated from A. eutrophus as described for Bacillus subtilis according to the method of Bron and Venema (5). Electrocompetent cells of E. coli were prepared and transformed with the Bio-Rad (Richmond, Calif.) gene pulser according to the manufacturer’s instructions. Electrocompetent cells of A. eutrophus were prepared and transformed as described by Taghavi et al. (52). Restriction enzyme-digested DNA was transferred to Hybond N1 membranes (Amersham International plc, Buckinghamshire, England) by the method of Southern (50). Hybridizations were performed by using the fluorescein gene image labeling and detection systems (Amersham International plc). Hybridization signals were visualized by exposure of Southern and colony blots to Hyperfilm-MP (Amersham International plc) with an intensifying screen. Construction of an A. eutrophus CH34 cosmid library. Total DNA of A. eutrophus CH34 was partially digested with Sau3AI and subjected to sucrose gradient ultracentrifugation (5 to 25% linear gradient) to isolate DNA fragments with sizes of 20 to 30 kb. The gradient was subjected to ultracentrifugation in an SW41 rotor for 16 h at 30,000 rpm. After ultracentrifugation, 26 fractions of 0.4 ml each were drawn in Eppendorf tubes, and 20 ml of each fraction was subjected to electrophoresis to determine the size of the fractionated DNA, which was then recovered by ethanol precipitation. pLAFR3 cosmid arms were prepared according to the method of Staskawicz et al. (51). Both cosmid arms were mixed with partially Sau3AI-digested chromosomal DNA, ligated, and packaged in vitro by using the Gigapack Gold packaging mix (Stratagene, La Jolla, Calif.). E. coli HB101 cells were transfected with this mixture, and the transfectants were selected for tetracycline resistance. DNA sequencing and analysis. For DNA sequencing, both subcloning and primer walking strategies were used. Sequencing reactions were carried out by using the AutoRead Sequencing Kit (Pharmacia, Uppsala, Sweden). The reactions were run on the Automated Laser Fluorescent A.L.F. DNA sequencer (Pharmacia) according to the manufacturer’s instructions. The sequences were analyzed with the PC gene program (version 6.85; Intelligenetics, Mountainview, Calif.) and the Genetics Computer Group (University of Wisconsin) (version 8.0) program. Outer membrane preparations and SDS-PAGE. Outer membrane proteins were prepared as described by Mizuno and Kageyama (30). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Lugtenberg et al. (24), with 15% (wt/vol) acrylamide in the running gel. Plate bioassays. Cross-feeding experiments were performed with Luria-Bertani (LB) agar medium supplemented with 100 mg of ethylenediamine di(ohydroxyphenylacetic acid) (EDDHA) per ml as described by Hohnadel and Meyer (19). Plates were inoculated randomly with the help of sterile glass beads at a concentration of 106 cells per plate. Sterile paper disks (6 mm in diameter) were impregnated with 10 ml of filter-sterilized 1 mM siderophore solutions (3 mM for siderophores forming a complex with iron, i.e., cepabactin at a ratio of 3:1) and deposited, after drying because of siderophores solubilized in methanol (e.g., enterobactin, cepabactin, and pyochelin), at the surface of the inoculated agar. The siderophores tested are listed in Table 2. Their origins as well as details on their purification procedures have been described elsewhere (19, 27). Ornibactins from Burkholderia vietnamensis were purified according to the method of Meyer et al. (29). The ability of the siderophores to promote bacterial growth by competing for iron with EDDHA was checked after 24, 48, and 72 h of incubation at 308C. 59 Fe uptake. A. eutrophus iron-starved cells were harvested from 24-h cultures in succinate medium (28), whereas iron-fed cells were obtained from 100 mM FeCl3-supplemented succinate medium. The capacity of cells to incorporate siderophore-chelated 59Fe was determined according to a previously described procedure (8), with partially purified alcaligin E dissolved in the minimal amount of distilled water (see above) or the pure siderophores described in Table 2. In 59 Fe uptake studies with partially purified alcaligin E, the supernatant of a Sid2 mutant of CH34, obtained under iron-limiting conditions, was used as a negative control. Control experiments without bacteria verified that the labeled iron was fully solubilized under the conditions used. Nucleotide sequence accession number. The nucleotide sequence of the aleB region has been deposited in the EMBL nucleotide sequence database under accession no. X97499.

RESULTS Siderophore production by A. eutrophus CH34. (i) Identification and chemical characterization of alcaligin E. By the

SIDEROPHORE-MEDIATED IRON UPTAKE IN A. EUTROPHUS

VOL. 178, 1996

TABLE 2. Heterologous siderophore-mediated iron uptake in A. eutrophus CH34 and AE1152 Siderophore

Plate bioassay resulta

Desferriferrioxamine B Desferriferrioxamine D2 Desferriferrioxamine E Ornibactin Desferriferrichrysin Desferriferricrocin Desferriferrichrome A Pyoverdinesc Enterobactin Pyochelin Cepabactin Coprogen Citric acid

1 1 1 1 1 1 6 2 2 2 2 2 2

Fe uptakeb

59

1 ND 1 1 1 1 ND 2 ND 2 2 ND ND

a Suppression (1) or no suppression (2) of the inhibitory effect of EDDHA on the growth of A. eutrophus CH34. b Iron incorporation (1) or no incorporation (2) into iron-starved A. eutrophus CH34 cells. ND, not determined. c Pyoverdines from P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, P. aeruginosa Pa6, P. putida ATCC 12633, Pseudomonas chlororaphis ATCC 9446, P. fluorescens CCM 2798, P. fluorescens ATCC 13525, and P. fluorescens ATCC 17400 were tested. Details on the purification procedures of these siderophores were published elsewhere (27).

CAS test, A. eutrophus CH34 was found to produce siderophores under iron-limiting growth conditions. The presence of 1 mM Fe(III) in the culture medium markedly decreased siderophore production, while the addition of 2 mM Fe(III) completely repressed siderophore production. The plasmidfree derivative of A. eutrophus CH34 (strain AE104) also produced siderophores under iron-limiting growth conditions, indicating that the genetic determinants for siderophore biosynthesis are chromosomally encoded. After separation of the siderophore-containing CH34 supernatant by thin-layer chromatography, only one spot that reacted with the CAS solution and FeCl3 z 6H2O was found. When the same experiment was performed with a Sid2 derivative of CH34, obtained after transposon mutagenesis, no spot that reacted with the CAS solution or FeCl3 z 6H2O was found. Therefore, we concluded that only one siderophore is produced by A. eutrophus CH34. This siderophore was named alcaligin E, since the name alcaligin had been previously used to describe the dihydroxamate siderophore produced by Alcaligenes xylosoxidans subsp. xylosoxidans (38). Subsequently, the siderophore type of alcaligin E was determined by different chemical tests. The results for the Csaky and Arnow tests, which specifically detect hydroxamate and catecholate groups, respectively, were negative. However, the ferric chloride-ferricyanide reagent (for detection of phenolate groups) turned blue (positive reaction) after the addition of a 10-fold-concentrated wild-type culture supernatant grown under iron-limiting conditions. As a control, a Sid2 derivative obtained after transposon mutagenesis was tested. With the culture supernatant of this strain, no reaction was obtained. This means that alcaligin E is a phenolate-type siderophore that contains neither a catecholate nor a hydroxamate group. To date, the only siderophore known to contain a similar type of iron-binding group is pyochelin, produced by Pseudomonas aeruginosa (9), Pseudomonas fluorescens (11), and Burkholderia cepacia (49). However, several data made us conclude that alcaligin E is different from pyochelin and is consequently a novel siderophore. The molecular weight of alcaligin E was estimated by gel filtration to be about 1,470. Moreover, by cross-feeding experiments and 59Fe uptake studies (see below), it was shown

5501

that pyochelin did not promote iron uptake by A. eutrophus CH34. Attempts to extract alcaligin E from pH 3-acidified culture supernatants by chloroform or ethyl acetate (9) (a method of choice for the purification of pyochelin) were unsuccessful. Amberlite-XAD chromatography did not allow the purification of alcaligin E either. Finally, the CAS-reacting compound was extractable in its native form by the chloroform-phenyl-diethyl ether procedure (28). Because of apparently very weak bacterial production, the amounts of material recovered were too small, however, for quantification or for a further purification of the siderophore. (ii) Isolation and characterization of alcaligin E biosynthetic mutants. Tn5-Tc as well as mini-Tn5-Km1 mutagenesis was performed in order to obtain mutants in which the biosynthesis of alcaligin E was impaired. Five different siderophorenegative mutants (Sid2 phenotype) were isolated: AE1093 (ale-1093::Tn5-Tc), AE1152 (aleB1152::Tn5-Tc), AE1593 (ale1593::mini-Tn5-Km1), AE1594 (ale-1594::mini-Tn5-Km1), and AE1595 (ale-1595::mini-Tn5-Km1). To test whether transposon insertion was the basis of the Sid2 phenotype, EcoRI- or BglII-digested chromosomal DNA of each mutant was introduced by electroporation into CH34 and AE104. These restriction enzymes, which do not cut the transposon (EcoRI for Tn5-Tc and BglII for mini-Tn5-Km1), were chosen in order to generate DNA fragments containing the transposon flanked by chromosomal DNA. After electroporation, transformants were selected for tetracycline or kanamycin resistance and analyzed for siderophore production under iron-limiting conditions. The majority of the transformants obtained in this way were found to be Sid2, except those obtained by transformation with AE1093 digested DNA. The DNA of the latter transformants showed a hybridization pattern different from that of AE1093 DNA, indicating that the inheritance of the resistance marker in these transformants did not result from gene replacement (52). This means that, perhaps with the exception of AE1093, the insertion of the transposon is the basis of the Sid2 phenotype for each of the Sid2 mutants. It was decided to exclude AE1093 from further studies. In mutant AE1152, the uptake of alcaligin E was affected: the addition of culture supernatant containing alcaligin E to the iron-depleted growth medium strongly inhibited the growth of AE1152, whereas the growth of the other siderophore mutants was stimulated (data not shown). This result suggests that because of the Tn5-Tc insertion, a gene involved in the transport of ferric iron-alcaligin E across the membrane is inactivated. SDS-PAGE analysis of outer membrane proteins (Fig. 1) enabled the identification of three iron-regulated outer membrane proteins (IROMPs) with estimated sizes of 85.3, 77.8, and 74.5 kDa in CH34. For strain AE1152, which contains the aleB1152::Tn5-Tc insertion, the 85.3-kDa IROMP was absent, but, instead, a 69.9-kDa IROMP was found. The 69.9-kDa IROMP is likely to be a truncated form of the wild-type 85.3kDa IROMP as a consequence of the Tn5-Tc insertion. Subcloning of DNA flanking the transposon insertion site and screening of a CH34 cosmid bank with an aleB and an ale-1595 probe. In order to gain information concerning the genetic basis of alcaligin E biosynthesis and the ferric ironalcaligin E uptake gene, which seemed to be mutated in AE1152, the DNA flanking the transposon insertion of the ale mutants was subcloned in pUC18. The left and right flanking DNAs of the aleB1152::Tn5-Tc insertion site were cloned independently. SalI restriction of total AE1152 DNA was done in order to clone the left flanking DNA along with IS50L and the Kmr gene of Tn5-Tc. BamHI

5502

GILIS ET AL.

FIG. 1. SDS-PAGE analysis of outer membrane proteins of CH34 and AE1152 grown in iron-deficient minimal medium with or without the addition of FeCl3. Lanes: 1, CH34 without FeCl3; 2, CH34 with 30 mM FeCl3; 3, AE1152 without FeCl3; 4, AE1152 with 30 mM FeCl3. The positions and sizes (in kilodaltons) of the molecular mass standards and the wild-type (arrowhead in lane 1, 85.3 kDa) and mutant (truncated) (arrowhead in lane 3, 69.9 kDa) AleB are indicated.

digestion was used to clone the right flanking DNA along with IS50R and the Tcr gene of Tn5-Tc. E. coli HB101 was transformed with the ligation mixture, and transformants were selected for Kmr (left flanking DNA) or Tcr (right flanking DNA). For ale-1593::mini-Tn5-Km1, ale-1594::mini-Tn5-Km1 and ale-1595::mini-Tn5-Km1, the left and right flanking DNAs of the transposon insertion were cloned on one BglII fragment (an enzyme that does not cut the transposon). Transformants were selected for Kmr. A genomic library of A. eutrophus CH34 was constructed in pLAFR3. In order to identify recombinant cosmids that carry ale genes, colony hybridizations were performed with two different probes: an aleB probe (presumably representing part of a ferric iron-alcaligin E transport gene) and an ale-1595 probe (presumably representing part of an alcaligin E biosynthetic gene). As the aleB probe, a 0.85-kb BglII fragment derived from the left flanking DNA of aleB1152::Tn5-Tc was used (Fig. 2B). In total, 4,500 colonies were checked, and five different overlapping clones, all containing the 0.85-kb BglII fragment, were isolated (pMOL871 to pMOL875) (Fig. 2). The ale-1595 probe consisted of the cloned ale-1595::mini-Tn5-Km1 BglII fragment. A total of 1,000 colonies were checked, and one clone was found to be positive (pMOL1032) (Fig. 2A). Complementation of ale mutants with aleB1 or ale-15951 cosmids. Complementation studies of the Sid2 mutants were performed with the aleB1 and ale-15951 cosmid clones by introducing them by triparental matings from E. coli into the different mutants. Because of interference of the tetracycline resistance gene present in the Tn5-Tc mutants, these studies could be performed only with the mini-Tn5-Km1 mutants. Kmr and Tcr transconjugants were selected and subsequently checked for their Sid phenotype. Plasmid extractions were carried out with each type of transconjugant to confirm the presence of the cosmid. The results of the complementation studies are summarized in Table 3. The aleB1 cosmids pMOL872 and pMOL874 were able to complement the Sid2 phenotype of AE1595 (ale-1595::mini-Tn5-Km1). This means that aleB1152::Tn5-Tc and ale-1595::mini-Tn5-Km1 are located at neighboring sites on the chromosome (within a region of 16 kb). The ale-15951 cosmid pMOL1032 could complement the Sid2 phenotype of the three mini-Tn5-Km1 mutants, AE1593, AE1594, and AE1595. The facts that a single cosmid clone can complement the Sid2 phenotype of three sid-

J. BACTERIOL.

erophore mutants and that two aleB1 cosmid clones complement the Sid2 phenotype of AE1595 are indications that the corresponding genes involved in alcaligin E biosynthesis and ferric iron-alcaligin E transport are clustered. The restriction map of the chromosomal ale region was determined by hybridization studies and restriction analysis, and the positions of aleB1152, ale-1593, ale-1594, and ale-1595 were located on this map (Fig. 2A). Sequencing and analysis of aleB. As already mentioned, the left and right flanking DNA sequences of the aleB1152::Tn5-Tc insertion were cloned independently along with IS50L and IS50R, respectively. Sequencing was started with a 20-kb primer designed on the O end of IS50 and continued to both ends by primer walking, with subclones of pMOL871 in pUC18. In total, 4,290 bp was sequenced. Within this sequence, an open reading frame, extending from base 299 to base 2839, potentially encodes 847 amino acids. Several translation initiation (GTG) codons were found at positions 320, 425, 431, and 467. Only the first two were preceded by sequences resembling ribosome binding sites. Comparison of the putative AleB sequence with other sequences in the GenBank, SwissProt, and EMBL databases revealed that the IutA, PbuA, FpvA, PupA, and PupB proteins of E. coli, Pseudomonas sp. strain M114, P. aeruginosa ATCC 15692, and P. putida WCS358, respectively, are the most similar to AleB. IutA (23) is the receptor for ferric aerobactin of E. coli, PbuA (32) is the ferric pseudobactin receptor of Pseudomonas sp. strain M114, FpvA (41) is the P. aeruginosa ATCC 15692 receptor for ferric pyoverdine, whereas PupA (3) and PupB (21) are receptors for ferric iron-pseudobactin of P. putida. The GTG at position 320 is the most probable translation start site, as the resultant product would possess a typical N-terminal signal sequence (55), consistent with the outer membrane location of ferric iron-siderophore receptors to which AleB shows similarity. The predicted AleB precursor consists of 840 amino acids, and the most probable potential cleaving site conforming to the 23,21 rule (54) is located between positions 37 and 38. This predicted signal sequence is followed by a polypeptide of 803 amino acids, which would result in a mature protein with a calculated molecular mass of 87.98 kDa. This is in good agreement with the estimated size of AleB from SDS-PAGE analysis (85.3 kDa). The termination codon TGA is followed by an inverted repeat and a string of T residues characteristic of rho-independent transcription termination (57). An alignment of the deduced amino acid sequences of different ferric siderophore receptors is shown in Fig. 3. In the deduced amino acid sequence of AleB, regions corresponding to domains II, III, and IV of TonB-dependent receptors (25) were identifiable at the same relative positions as those in the other receptors (Fig. 3). This result suggests the presence of a TonB-like protein involved in the iron uptake system of A. eutrophus CH34. However, the most-N-terminal domain I is not conserved in AleB. Upstream of the aleB coding region, a sequence (TGCTG ACCCTGC, at positions 146 to 157) that shows similarity to previously defined 216 to 225 sequences of iron-regulated genes was found (40). No sequence resembling the iron box consensus sequence (13) was found. Downstream of the putative alcaligin E receptor gene is a noncoding region with an inverted repeat (DG of 220.8) followed by a ribosomal binding site and the start of an open reading frame (at position 2835) whose putative protein shows similarity to CysK and CysM of E. coli K-12 and Salmonella typhimurium (6, 48). Chromosomal mapping of aleB locus. Sequencing analysis revealed an open reading frame downstream of aleB with sim-

VOL. 178, 1996

SIDEROPHORE-MEDIATED IRON UPTAKE IN A. EUTROPHUS

5503

FIG. 2. (A) Map of part of the A. eutrophus CH34 chromosome composed of aleB1 and ale-15951 cosmids, showing the positions of the ale-1593 (arrow 1), ale-1594 (arrow 2), ale-1595 (arrow 3), and aleB (arrow 4) mutations. Open arrows indicate insertion of mini-Tn5-Km1; the closed arrow indicates insertion of Tn5-Tc. The positions of the inserts of the different cosmids are shown relative to the chromosome part. (B) Map of aleB1 cosmid pMOL871. The open arrow indicates the aleB gene. The closed arrow indicates the open reading frame downstream of the aleB gene that shows homology to CysM and CysK of E. coli K-12 and S. typhimurium. The 0.85-kb BglII fragment flanking the insertion site of mutant AE1152, which was used as a probe to screen the genomic bank of strain CH34 in pLAFR3, is indicated. Ba, BamHI; E, EcoRI; bold line, extremities of the pLAFR3 part of the cosmids.

5504

GILIS ET AL.

J. BACTERIOL.

TABLE 3. Complementation analysis of siderophore biosynthetic mutants with recombinant aleB1 and ale-15951 pLAFR3 cosmids Complementation of wild-type siderophore production by the cosmidsa Strain

AE1593 AE1594 AE1595

Mutation genotype

ale-1593::mini-Tn5-Km1 ale-1594::mini-Tn5-Km1 ale-1595::mini-Tn5-Km1

pMOL871

pMOL872

pMOL873

pMOL874

pMOL875

pMOL1032

2 2 2

2 2 1

2 2 2

2 2 1

2 2 2

1 1 1

a pMOL871 to pMOL875 contain the aleB locus, while pMOL1032 contains the ale-1595 locus. The cosmids were introduced in the Sid2 strains by triparental matings. Transconjugants were further tested for their siderophore production. 1, complementation from Sid2 to Sid1 phenotype; 2, no complementation.

ilarity to proteins involved in cysteine biosynthesis. In order to confirm this, a triparental mating was performed to transfer the aleB1 cosmid clone pMOL871 into AE232. Transconjugants were selected on minimal medium containing tetracycline, cysteine, leucine, methionine, and tryptophan. Replica plating showed that the transconjugants became Cys1, indicating that pMOL871 can complement cys-232 and that the considered ale genes are located near this locus. Alcaligin E-mediated iron uptake. The uptake of iron mediated by alcaligin E was monitored by following the incorporation of 59Fe liganded to semipurified alcaligin E into ironstarved A. eutrophus CH34 and AE1152 cells (Fig. 4). The iron uptake seemed to be very efficient in iron-starved wild-type cells, showing a linear response in the 15-min experiment. Iron uptake in iron-starved aleB mutant cells was three to four times less efficient than that in wild-type cells. This finding fits with the observation that the growth of this mutant is decreased in the presence of alcaligin E-containing culture supernatant derived from wild-type cells, with the IROMP character of AleB, and with the similarity that was found for AleB with different ferric iron-siderophore receptors. Therefore, we conclude that aleB encodes the ferric iron-alcaligin E receptor. Specificity of AleB receptor in heterologous siderophoremediated iron uptake. In order to test the specificity of the AleB receptor, uptake experiments with heterologous siderophores were performed. Plate bioassays revealed that the growth inhibition of strains CH34 and AE1152 by EDDHA could be suppressed by some of the heterologous siderophores tested (Table 2). Desferriferrioxamines B, D2, and E, ornibactin, desferriferrichrysin, and desferriferricrocin promoted strong bacterial growth within 24 h of incubation around the paper disks impregnated with these siderophores. A slight and delayed (48-h) growth occurred with desferriferrichrome A, whereas no growth was visible after 3 days of incubation for the other siderophores tested (Table 2). The ability of desferriferrioxamines B and E, ornibactin, desferriferrichrysin, and desferriferricrocin to efficiently mediate iron uptake in A. eutrophus CH34, as was suggested by the plate bioassays, was assessed by monitoring the incorporation of the 59Fe ligand on the siderophores. All five siderophores allowed, with variable efficiency, iron incorporation into ironstarved cells, whereas pyoverdines, pyochelin, and cepabactin, which were inefficient in the plate bioassays, failed to promote iron uptake (Table 2; Fig. 4). As a control, no incorporation was observed in experiments involving A. eutrophus CH34 cells grown in an iron-enriched medium with desferriferrioxamines B and E or ornibactin as the iron ligands (data not shown). The heterologous siderophores that promoted iron uptake in A. eutrophus CH34 wild-type cells promoted iron uptake in aleB mutant cells to the same extent (Fig. 4). This finding shows that AleB is not involved in the uptake of these heterologous siderophores.

DISCUSSION A. eutrophus CH34, a soil bacterium that contains several heavy metal resistance systems (for an overview, see reference 7) was found to produce siderophores under iron-limiting conditions. The name alcaligin E was chosen for this siderophore, since the name alcaligin had previously been used to describe the dihydroxamate siderophore isolated from A. xylosoxidans subsp. xylosoxidans (38) and recently identified in the taxonomically related bacterial species Bordetella pertussis and Bordetella bronchiseptica (31). However, A. eutrophus is not related to these species and recently was taxonomically placed in the new genus Ralstonia (56). Chemical tests revealed that alcaligin E can be classified as a phenolate type of siderophore. Since a negative result was obtained for the Arnow test, only one hydroxyl group is present on the benzene ring and not two, as is the case for the catecholate type of siderophores. The negative Csaky test result indicates that no hydroxamate groups are present. To date, the only siderophore known to have the same type of iron-binding group is pyochelin. Our data show that pyochelin and alcaligin E are different siderophores. Transposon mutagenesis with Tn5-Tc and mini-Tn5-Km1 led to the isolation of four different alcaligin E-deficient mutants (Sid2 phenotype): AE1152 (aleB1152::Tn5-Tc), AE1593 (ale-1593::mini-Tn5-Km1), AE1594 (ale-1594::mini-Tn5-Km1), and AE1595 (ale-1595::mini-Tn5-Km1). When semipurified alcaligin E was added to the iron-depleted growth medium of AE1593, AE1594, and AE1595, the growth of these mutants was stimulated. These results show the siderophore character of alcaligin E. However, under the same conditions, the growth of mutant AE1152 was strongly inhibited, suggesting that in AE1152 the uptake of alcaligin E is affected because of inactivation of a gene involved in ferric iron-alcaligin E transport across the membrane. The iron-regulated character, the outer membrane location, and sequencing analysis of aleB1152:: Tn5-Tc flanking DNA yielded more evidence that aleB encodes the ferric iron-alcaligin E receptor. At the amino acid level, the sequence of AleB showed strong homology with known ferric iron-siderophore receptors. The highest score was obtained with IutA, the ferric iron-aerobactin receptor of E. coli: 25.3% identity and 8.1% conserved changes. With FptA, the ferric iron-pyochelin receptor from P. aeruginosa, the similarity was less but still substantial: 14.9% identity and 6.6% conserved changes. It has been observed previously that there is not necessarily a relationship between receptor structure and ligand specificity among siderophores and their receptors, as structurally different siderophores can be recognized by significantly homologous ferric iron-siderophore receptors (1, 20). Therefore, it may not be surprising that AleB, the receptor of a phenolate-type siderophore, has stronger similarity to IutA (a hydroxamate-type siderophore receptor) than to FptA (a phenolate-type siderophore receptor). Within the

VOL. 178, 1996

SIDEROPHORE-MEDIATED IRON UPTAKE IN A. EUTROPHUS

5505

FIG. 3. Multiple alignments of the amino acid sequences of the putative ferric iron-alcaligin E receptor protein (AleB) and IutA (25.3% identical to AleB and 8.1% conserved changes), PbuA (16.8% identical to AleB and 8.8% conserved changes), PupA (14.4% identical to AleB and 8.1% conserved changes), PupB (15.7% identical to AleB and 8.2% conserved changes), and FptA (14.9% identical to AleB and 6.6% conserved changes) receptor proteins. Boldface letters indicate the first amino acid of the mature receptor protein. Conserved regions in TonB-dependent receptor proteins (25) are underlined. Residues that are identical or similar in the majority of the displayed sequences are shown against a shaded background. Similar amino acids are I, L, and V; F, Y, W, and H; and D, R, K, E, and H.

5506

GILIS ET AL.

J. BACTERIOL.

FIG. 4. 59Fe uptake (in picomoles per milligram of cell [dry weight]) mediated by alcaligin E and different heterologous siderophores in iron-starved A. eutrophus AE1152 (A) and CH34 (B) cells. {, alcaligin E; h, desferriferrioxamine B; 1, desferriferrioxamine E; ■, ornibactins; 3, desferriferricrocin; F, desferriferrichrysin.

sequence of AleB, three regions with strong similarity to conserved domains in TonB-dependent receptor proteins of E. coli (25, 42) are present. This finding suggests the presence of a TonB-like protein involved in the iron uptake system of A. eutrophus CH34. However, AleB does not contain the consensus most-N-terminal domain I, called the TonB box, which has been shown to represent a site of physical interaction of various receptors with the TonB protein (18, 45, 53). The same observation was made for PupB (21) from P. putida WCS385, FptA (1) from P. aeruginosa, and FpvA (41) from P. aeruginosa. It was hypothesized that the alignment of these receptors in region I represents degenerate TonB boxes or that in transport via these receptors TonB interacts with regions II, III, and IV (1). The inactivation of the ferric iron-alcaligin E receptor gene resulted in the absence of expression of alcaligin E biosynthesis, indicating positive regulation of the biosynthetic ale genes mediated by the ferric iron-alcaligin E receptor gene. In P. putida, the ferric iron-pseudobactin receptor (PupB) triggers, in the presence of pseudobactin, a signal transduction pathway resulting in the activation of the pupB gene (22). A similar regulation might exist for the ale genes. Cosmid clones carrying ale genes were isolated from a genomic library of A. eutrophus CH34 in pLAFR3. Complementation studies of the Sid2 mutants with these cosmid clones and hybridization analysis revealed the sites of chromosomal insertion of the Sid2 mutant strains. The transposon insertion in the four mutants seemed to be located at neighboring sites on the chromosome. This is an indication that the genes involved in alcaligin E biosynthesis and ferric iron-alcaligin E transport are organized in a gene cluster. Evidence that these genes are located near the cys-232 locus on the chromosomal map of A. eutrophus CH34 was found (43), in accordance with the observation of a cysM- or cysK-like gene downstream of aleB. Iron uptake in aleB mutant cells was reduced three to four times relative to that in wild-type cells. Since the growth of this mutant is strongly inhibited in the presence of alcaligin E, it seems likely that alcaligin E is bound only to the cell membrane (to the truncated receptor) and not transported into the cell. The truncated receptor would then be missing elements required for efficient transport of ferric alcaligin E through the membrane, such as TonB box II that is missing in this putative protein. Heterologous siderophore-mediated iron uptake was assessed by plate bioassays and by 59Fe uptake experiments. The results of both assays were in complete agreement and

showed that desferriferrioxamines B and E, ornibactin, desferriferrichrysin, and desferriferricrocin efficiently mediated iron uptake in A. eutrophus CH34, whereas pyoverdines, pyochelin, and cepabactin did not promote iron uptake. The siderophores that promoted iron uptake by A. eutrophus CH34 have both bacterial and fungal origins and belong to several different structural families. The observation that pyochelin is unable to promote iron uptake in A. eutrophus CH34 clearly demonstrates that pyochelin and alcaligin E are different siderophores. This means that pyochelin is no longer structurally unique in being a phenolate type of siderophore that contains neither hydroxamate- nor catecholate-chelating groups. The heterologous siderophores that promoted iron uptake in A. eutrophus CH34 promoted to the same extent iron uptake in the aleB mutant, indicating that AleB is not involved in the uptake of these siderophores. Consequently, other ferric ironsiderophore receptors that recognize heterologous siderophores from related and unrelated strains as well as from fungal origin must be present in A. eutrophus CH34. This phenomenon is believed to be important in competition for iron in soil and rhizosphere bacteria. Attempts to purify alcaligin E are in progress. The purified siderophore will be used to determine binding constants with iron and different heavy metals. There is preliminary evidence that alcaligin E accounts for the solubilization of precipitated heavy metals. This suggests a correlation between siderophore production and heavy metal resistance. Therefore, the study of alcaligin E could be important in the optimalization of bioremediation processes based on heavy metal resistance. Further work should also address the regulation of alcaligin E genes by iron and different heavy metals and the role of A. eutrophus siderophores with respect to heavy metal resistance. ACKNOWLEDGMENTS This investigation was supported by the VLAB-ETC03 grant from the Flemish Regional Government. We thank G. Nuyts, G. Seyer and W. Verrijdt for valuable technical assistance. REFERENCES 1. Ankenbauer, R. G., and H. N. Quan. 1994. FptA, the Fe(III)-pyochelin receptor of Pseudomonas aeruginosa: a phenolate siderophore receptor homologous to hydroxamate siderophore receptors. J. Bacteriol. 176:307–319. 2. Arnow, L. E. 1937. Colorimetric determination of the components of 3,4dihydroxyphenylalanine-tyrosine mixtures. J. Biol. Chem. 118:531–537. 3. Bitter, W., J. D. Marugg, L. A. de Weger, J. Tomassen, and P. J. Weisbeek.

VOL. 178, 1996

4. 5. 6. 7.

8. 9. 10. 11. 12.

13. 14. 15.

16. 17. 18.

19. 20.

21.

22. 23.

24. 25. 26. 27. 28.

SIDEROPHORE-MEDIATED IRON UPTAKE IN A. EUTROPHUS

1991. The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein. Mol. Microbiol. 5:647–655. Boyer, H. W., and D. Roulland-Dussoix. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459–472. Bron, S., and G. Venema. 1972. Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction of a transformable eight-fold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat. Res. 15:1–10. Byrne, C. R., R. S. Monroe, K. A. Ward, and N. M. Kredich. 1988. DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK region to ptsH. J. Bacteriol. 170:3150–3157. Collard, J. M., P. Corbisier, L. Diels, Q. Dong, C. Jeanthon, M. Mergeay, S. Taghavi, D. van der Lelie, A. Wilmotte, and S. Wuertz. 1994. Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: mechanisms and applications. FEMS Microbiol. Rev. 14:405–414. Cornelis, P., D. Hohnadel, and J. M. Meyer. 1989. Evidence for different pyoverdine-mediated iron uptake systems among Pseudomonas aeruginosa strains. Infect. Immun. 57:3491–3497. Cox, C. D., K. L. Rinehart, M. L. Moore, and J. C. Cook. 1981. Pyochelin: novel structure of an iron-chelating growth promoter from Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 78:302–308. Csaky, T. Z. 1948. On the estimation of bound hydroxylamine in biological materials. Acta Chem. Scand. 2:450–454. Cuppels, D. A., R. D. Stipanovic, A. Stoessl, and J. B. Stothers. 1987. The constitution and properties of a pyochelin-zinc complex. Can. J. Chem. 65:2126–2130. de Lorenzo, V., M. Herrero, V. Jakubzik, and K. N. Timmis. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 172:6568–6572. de Lorenzo, V., S. Wee, M. Herrero, and J. B. Neilands. 1987. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (Fur) repressor. J. Bacteriol. 169:2624–2630. Diels, L., Q. Dong, D. van der Lelie, W. Baeyens, and M. Mergeay. 1995. The czc operon of Alcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals. J. Ind. Microbiol. 14:142–153. Figursky, D. H., R. F. Pohlman, D. H. Bechhofer, A. S. Prince, and C. A. Kelton. 1982. The broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli. Proc. Natl. Acad. Sci. USA 79:1935– 1939. Guerinot, M. L. 1994. Microbial iron transport. Annu. Rev. Microbiol. 48: 743–772. Hathway, D. E. 1969. Plant phenols and tannins, p. 308–345. In Chromatographic and electrophoretic techniques. Interscience, New York. Heller, K., R. J. Kadner, and K. Gu ¨nter. 1988. Suppression of the btuB451 mutation by mutations in the tonB gene suggests a direct interaction between TonB and TonB-dependent receptor proteins in the outer membrane of Escherichia coli. Gene 64:147–153. Hohnadel, D., and J. M. Meyer. 1988. Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains. J. Bacteriol. 170:4865– 4873. Koebnik, R., K. Hantke, and V. Braun. 1993. The TonB-dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict co-evolution of receptor structure and substrate specificity. Mol. Microbiol. 7:383–393. Koster, M., J. van de Vossenberg, J. Leong, and P. J. Weisbeek. 1993. Identification and characterization of the pupB gene encoding an inducible ferric-pseudobactin receptor of Pseudomonas putida WCS358. Mol. Microbiol. 8:591–601. Koster, M., K. van Klompenburg, W. Bitter, J. Leong, and P. J. Weisbeek. 1994. Role for the outer membrane ferric siderophore receptor PupB in signal transduction across the bacterial cell envelope. EMBO J. 13:2805–2813. Krone, W. J. A., F. Stegehuis, G. Koningstein, C. van Doorn, B. Roosendaal, F. K. de Graaf, and B. Oudega. 1985. Characterisation of the pCOLV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli: isolation and purification of the protein and analysis of its nucleotide sequence and primary structure. FEMS Microbiol. Lett. 26:153–161. Lugtenberg, B., J. Meijers, R. Peters, P. van der Hoek, and L. van Alphen. 1975. Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K12 into four bands. FEBS Lett. 58:254–258. Lundigran, M. D., and R. J. Kadner. 1986. Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. J. Biol. Chem. 261:10797–10801. Mergeay, M., D. Nies, G. Schlegel, J. Gerits, P. Charles, and F. Van Gijsegem. 1985. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol. 162:328–334. Meyer, J. M. 1992. Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation. J. Gen. Microbiol. 138:951–958. Meyer, J. M., and M. A. Abdallah. 1978. The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification and physicochemical properties.

5507

J. Gen. Microbiol. 107:319–328. 29. Meyer, J. M., V. Tran Van, A. Stintzi, O. Berge, and G. Winkelman. 1995. Ornibactin production and transport properties in strains of Burkholderia vietnamensis and Burkholderia cepacia (formerly Pseudomonas cepacia). Biometals 8:309–317. 30. Mizuno, T., and M. Kageyama. 1978. Separation and characterization of the outer membrane of Pseudomonas aeruginosa. J. Biochem. 84:179–191. 31. Moore, C. H., L.-A. Foster, D. G. Gerbig, D. W. Dyer, and B. W. Gibson. 1995. Identification of alcaligin as the siderophore produced by Bordetella pertussis and Bordetella bronchiseptica. J. Bacteriol. 177:1116–1118. 32. Morris, J., D. F. Donnelly, E. O’Neill, F. McConnell, and F. O’Gara. 1994. Nucleotide sequence analysis and potential environmental distribution of a ferric pseudobactin receptor gene of Pseudomonas sp. strain M114. Mol. Gen. Genet. 242:9–16. 33. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev. Biochem. 50:1–24. 34. Neilands, J. B. 1982. Microbial envelope proteins related to iron. Annu. Rev. Microbiol. 36:285–309. 35. Nies, D. H. 1995. The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli. J. Bacteriol. 174:8102–8110. 36. Nies, D. H., A. Nies, L. Chu, and S. Silver. 1989. Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus. Proc. Natl. Acad. Sci. USA 86:7351–7355. 37. Nies, D. H., and S. Silver. 1995. Ion efflux systems involved in bacterial resistances. J. Ind. Microbiol. 14:186–199. 38. Nishio, T., N. Tanaka, J. Hiratake, Y. Katsube, Y. Ishida, and J. Oda. 1988. Isolation and structure of the novel dihydroxamate siderophore alcaligin. J. Am. Chem. Soc. 110:8733–8734. 39. Norrander, J., T. Kempe, and J. Messing. 1983. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106. 40. O’Sullivan, D. J., and F. O’Gara. 1991. Regulation of iron assimilation: nucleotide sequence analysis of an iron-regulated promoter from a fluorescent pseudomonad. Mol. Gen. Genet. 228:1–8. 41. Poole, K., S. Neshat, K. Krebes, and D. E. Heinrichs. 1993. Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa. J. Bacteriol. 175:4597–4604. 42. Postle, K. 1990. TonB and the Gram-negative dilemma. Mol. Microbiol. 4:2019–2025. 43. Sadouk, A., and M. Mergeay. 1993. Chromosome mapping in Alcaligenes eutrophus CH34. Mol. Gen. Genet. 240:181–187. 44. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 45. Scho ¨ffler, H., and V. Braun. 1989. Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane. Mol. Gen. Genet. 217:378–383. 46. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47–56. 47. Simon, R., U. Priefer, and A. Pu ¨hler. 1983. A broad-host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784–790. 48. Sirko, A., M. Hryniewics, D. Hulanicka, and A. Bo ¨ck. 1990. Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster. J. Bacteriol. 172:3351–3357. 49. Sokol, P. A. 1986. Production and utilization of pyochelin by clinical isolates of Pseudomonas cepacia. J. Clin. Microbiol. 23:560–562. 50. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503–517. 51. Staskawicz, B., N. Dahlbeck, N. Keen, and C. Napoli. 1987. Molecular characterization of cloned avirulence gene from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacteriol. 169:5789–5794. 52. Taghavi, S., D. van der Lelie, and M. Mergeay. 1994. Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments. Appl. Environ. Microbiol. 60:3585–3591. 53. Tuckman, M., and M. S. Osburne. 1992. In vivo inhibition of TonB-dependent processes by a TonB box consensus pentapeptide. J. Bacteriol. 174:320– 323. 54. von Heijne, G. 1984. How signal sequences maintain cleavage specificity? J. Mol. Biol. 173:243–251. 55. von Heijne, G. 1985. Signal sequences: the limits of variation. J. Mol. Biol. 184:99–105. 56. Yabuuchi, E., Y. Kosako, I. Yano, H. Hotta, and Y. Nishiuchi. 1995. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb. nov. and Ralstonia eutropha (Davis, 1962) comb. nov. Microbiol. Immunol. 39:897–904. 57. Yager, T. D., and P. H. von Hippel. 1987. Transcription elongation and termination in Escherichia coli, p. 1241–1257. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C.