INTRODUCTION

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The genera Salmonella and Escherichia diverged from a common ancestor some 100 million to 160 million years ago (26). The lineage of the genus Salmonella subsequently split into two species, S. enterica and S. bongori (22, 28). During formation of these species a large amount of DNA was acquired by plasmid- or phage-mediated horizontal gene transfer (3). As a result of horizontal transfer, more than 10% of the S. enterica serotype Typhimurium genome consists of genetic material that is not present in Escherichia coli (25, 30). To fully comprehend the biological characteristics that distinguish S. enterica from closely related species such as E. coli and S. bongori, it is necessary to study the functions of proteins that are encoded by genetic material that was acquired by way of horizontal transfer.

Genetic material that is absent from closely related bacteria but is present in all phylogenetic lineages of S. enterica was likely received during horizontal transfer events that contributed to the formation of this species. Two genetic regions on the S. enterica chromosome, Salmonella pathogenicity island 2 and the iroBC operon, indeed show this phylogenetic distribution (4, 19, 24). In a study on the distribution of iroB among 197 bacterial isolates, this gene was found to be present in all S. enterica serotypes tested but absent from S. bongori serotypes and from 15 other bacterial species tested (4). The phylogenetic distribution thus suggests that the gene products encoded in the iroA locus confer properties that set S. enterica apart from other bacterial species. What are the characteristics that were obtained by S. enterica during acquisition of the iroA locus?

The iroA locus was first described in S. enterica serotype Typhimurium based on its iron-regulated expression (12). An insertion that created a transcriptional fusion between lacZ and the iroA locus was mapped close to the tct locus (13), an area of the S. enterica serotype Typhimurium chromosome that is not present in E. coli (31). The first genes of the iroA locus, designated iroBC, were identified in S. enterica serotype Typhi during a genetic screen for genes that are regulated by the iron response regulator Fur (5). Regulation by Fur results in expression of iroBC under iron-limited growth conditions. In contrast, during growth under iron sufficiency expression is prevented by binding of the Fur-Fe²⁺ repressor complex to a Fur DNA binding site in the iroB promoter region. In addition to iroBC, the Fur repressor protein controls expression of some 28 genes in S. enterica, including those that function in iron acquisition and some genes involved in defense against oxidative stress (38-40). However, the iroBC gene products show homology to proteins that have so far not been associated with iron uptake or defense against oxidative stress in other bacteria. IroB shows homology with bacterial glycosyltransferases, and IroC is a member of the ATP binding cassette (ABC) family of transport proteins (5). Interestingly, IroC has little homology to bacterial ABC transport proteins involved in the import of siderophores but rather shows strong homology to ABC export proteins of the eukaryotic multidrug resistance family. To obtain further clues about the function of genes encoded in the iroA locus, we analyzed iroN, an open reading frame located downstream of the iroBC operon.

	MATERIALS AND METHODS

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Bacterial strains, media, growth conditions, and outer membrane preparations. A collection of Salmonella serotypes representing S. bongori and six subspecies of S. enterica has been described by Reeves et al. (28). All other bacterial strains used in this study are listed in Table 1. All bacteria were routinely cultured aerobically at 37°C in Luria-Bertani (LB) broth or on LB plates. Antibiotics, when required, were included in the culture medium or plates at the following concentrations: kanamycin, 100 mg/liter; chloramphenicol, 30 mg/liter; and carbenicillin, 100 mg/liter. To create iron-limiting or iron-sufficient growth conditions, 0.2 mM 2,2'-dipyridyl or 0.04 mM FeSO₄, respectively, was added. Desferal (desferrioxamine B) was purchased from Ciba Geigy (Basel, Switzerland). N-(2,3-Dihydroxybenzoyl)-L-serine (DBS) and benzaldehyde-2,3-dihydroxybenzhydrazone were synthesized and kindly provided by L. Heinisch, Hans-Knölle Institut, Jena, Germany. The myxochelin derivatives 2-N,6-N-bis(2,3-dihydroxybenzoyl) lysine (9), 2-N,6-N-bis(2,3-dihydroxybenzoyl) lysine amide (37), myxochelin A (21), myxochelin B, and myxochelin C (36) were synthesized in the L or D configuration and kindly provided by W. Trowitzsch-Kienast and H. D. Ambrosi, Technische Fachhochschule, Berlin, Germany. Cross-feeding with bacterial supernatants and utilization of siderophores was detected by an agar diffusion assay (29). The strain to be tested was poured in 3 ml of 2% Noble agar onto a nutrient broth-dipyridyl (NBD) agar plate. Filter paper disks impregnated with ferrioxamine B (5 µl of a 1-mg/ml solution of Desferal in 0.1 M FeCl₃), DBS (5 µl of a 1-mg/ml solution), benzaldehyde-2,3-dihydroxybenzhydrazone (5 µl of a 1-mg/ml solution), or myxochelin derivatives (5 µl of a 1-mg/ml solution) were laid onto the top agar, and after incubation overnight at 37°C, growth stimulation around the filter disk was recorded. Growth promotion in broth culture was determined in NBD supplemented with a 500× siderophore stock solution (1 mg/ml). Bacterial outer membranes were isolated as previously described (17). Outer membrane proteins were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were visualized by Coomassie blue staining.

Recombinant DNA techniques. Plasmid DNA was isolated by using ion-exchange columns from Qiagen. Standard methods were used for restriction endonuclease analyses, ligation, and transformation of plasmid DNA (2). Sequencing was performed with an ALF automated sequencer (Pharmacia).

Suicide vector constructs and isolation of mutants. For mutational analysis of iroN, a 0.4-kb XbaI-KpnI fragment of pTY961 containing an internal part of the iroN open reading frame was introduced into the suicide vector pGP704 (20) to give rise to pTY966. Plasmid pTY966 was conjugated into S. enterica serotype Typhimurium IR715 and S. enterica serotype Typhi AJB22. Exconjugants were designated AJB52 and AJB54, respectively. Strains AJB64 and AIR49 were generated by P22 transduction of aroA::Tn10 from S. enterica serotype Typhimurium CL1509 into strains AJB52 and AJB20, respectively. E. coli S17-1 pir was used for propagation of all suicide vector constructs and as a donor for introduction of these constructs into IR715 or AJB22 by conjugation. Chromosomal DNA of mutants was routinely tested by Southern hybridization with suitable DNA probes to confirm mutational inactivation of the gene of interest.

Southern hybridization. The inserts of plasmids pTY961 and pTY911 (5) were used to generate DNA probes specific for iroN and iroCDE, respectively. Chromosomal DNA was isolated as recently described (2). Chromosomal DNA of strains shown in Fig. 4 was restricted with EcoRI, and the fragments were separated on a 0.5% agarose gel. Southern transfer of DNA onto a nylon membrane was performed as previously described (2). Hybridization was performed at 65°C in solutions without formamide. Two 15-min washes were performed under nonstringent conditions at room temperature in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS. Hybrids were detected by using a labeling and detection kit (nonradioactive) from Boehringer Mannheim.

Computer analysis. The nucleotide sequences were compared to SWISS-PROT, PIR, and GenPept at the National Center for Biotechnology Information by using the program blastX and to GenBank and EMBL by using the program blastN (1). Multiple alignments were performed with the program CLUSTAL, which is part of the program package PCGENE.

Nucleotide sequence accession number. The sequence reported (Fig. 1) has been deposited at GenBank under accession no. U97227.

	RESULTS

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Sequence analysis of a DNA region located downstream of iroBC. The nucleotide sequence of a 4,837-bp DNA region of S. enterica serotype Typhi AJB70 that is located downstream of iroBC was determined. Directly downstream of iroC were two open reading frames transcribed in the same orientation. These open reading frames were designated iroDE (Fig. 1). The close proximity of the open reading frames and the lack of transcriptional terminators suggest that the iroBCDE cluster forms an operon. The deduced amino acid sequence of iroD showed 28% sequence identity with Fes, the E. coli enterochelin esterase (27). The amino-terminal 169 amino acids of IroE displayed 38% identity with the deduced amino acid sequence of an open reading frame located downstream of pfeA, the enterochelin receptor gene of Pseudomonas aeruginosa (data not shown) (10). Downstream of iroDE was a third open reading frame transcribed in the opposite orientation. This open reading frame, termed iroN, encoded a polypeptide of 727 amino acids with a calculated molecular mass of 79.5 kDa. Cleavage of a predicted N-terminal signal sequence of 25 amino acids would yield a mature protein with a calculated molecular mass of 76.8 kDa. The region between iroE and iroN contained a putative transcriptional terminator (stem bp 2261 to 2270; loop bp 2271 to 2273; stem bp 2274 to 2283 of the GenBank sequence). A putative Fur-DNA binding site (4576-GATAATTATTATCATTAGC-4558) that matches the E. coli consensus sequence (34) in 16 of 19 bases was located 86 bp upstream of the iroN start codon. The G+C content of the entire 10,837-bp DNA region containing the iroBCDE and iroN genes was 55%, which is slightly higher than the 52% average G+C content characteristic of S. enterica.

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Restriction map of a DNA region located at about four centisomes on the S. enterica serotype Typhi chromosome. Positions and sizes of inserts carried in cosmids (pTY908 and pTY2117) or plasmids depicted were determined previously (5). Arrows above the map indicate positions and orientations of open reading frames identified by sequence analysis. E, EcoRI.

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Percentage sequence identity determined by pairwise alignment of amino acid sequences from IroN, PfeA, BfeA, FepA, and Cir with the program CLUSTAL.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Multiple sequence alignment of IroN, PfeA, FepA, and BfeA with the program CLUSTAL. Dashes represent gaps introduced by the program to improve the alignment; identical amino acids are indicated by asterisks; dots indicate amino acids with similar properties.

Phylogenetic distribution of iroN. To obtain information on the distribution of iroN, we used 20 strains representing all phylogenetic lineages of the genus Salmonella, including S. bongori and S. enterica subspecies I, II, IIIa, IIIb, IV, and VI. The phylogenetic relationship among these strains has previously been established by multilocus enzyme electrophoresis (28). Analysis of the distribution of iroN among these strains can therefore identify the branch of the phylogenetic tree in which this gene was acquired. To compare the distribution of iroN with that of other genes of the iroA locus DNA, probes specific for iroCDE (pTY911), and iroN (pTY961) were used for Southern hybridization (Fig. 4).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Phylogenetic distribution of genes of the iroA locus. The phylogenetic tree on the left was established by Reeves and coworkers (28). (A) Restriction map of the region from S. enterica serotype Typhi AJB70 (E, EcoRI). Positions of genes (arrows) identified in the iroA locus and of DNA fragments used as probes (black bars) are indicated. (B) Results of hybridization with these DNA probes. +, hybridization signal; , no hybridization signal.

Identification of the iroN gene product. Homologies to siderophore receptors from other bacteria suggested that the iroN gene product is localized in the outer membrane. To detect IroN in outer membrane preparations, we constructed mutants of S. enterica serotype Typhimurium (AJB52 and AJB64) and S. enterica serotype Typhi (AJB54) in which the iroN open reading frame was disrupted by integration of suicide vector pTY966 via homologous recombination. S. enterica serotype Typhi and S. enterica serotype Typhimurium have been shown to contain three major iron-regulated outer membrane proteins which are 69, 78, and 83 kDa in size, respectively (7, 11). The 69- and 83-kDa proteins likely represent the S. enterica FhuA and FepA receptor proteins, respectively. Inactivation of iroN in AJB52, AJB54 (data not shown), and AJB64 (Fig. 5) resulted in loss of the 78-kDa outer membrane protein. These data therefore identify the 78-kDa outer membrane protein as the iroN gene product. The size predicted for the mature IroN protein (76.8) is in good agreement with the apparent molecular weight determined by SDS-PAGE. Furthermore, a 78-kDa protein could be detected in outer membrane preparations of strain H5058 upon introduction of a cosmid (pTY908) carrying the iroN gene of S. enterica serotype Typhi (Fig. 5), indicating that IroN also localizes to the outer membrane when expressed in E. coli. Outer membrane preparations of E. coli strains that were lacking the iroN gene [H5058 or H5058(pTY2117)] did not contain this 78-kDa protein. Sequence analysis of the insert of cosmid pTY908 revealed no open reading frames other than iroN that could encode this 78-kDa outer membrane protein (3a).

View larger version (54K): [in this window] [in a new window]   FIG. 5.   Outer membrane profiles of bacterial strains which carry the iroN gene (lanes 3, 5, and 6) or in which iroN is lacking (lanes 7 and 8) or inactivated (lane 2). The position of IroN is indicated by an arrow. Positions and sizes of bands from standard proteins are indicated (lanes 1 and 4). (A) SDS-PAGE of outer membrane preparations of S. enterica serotype Typhimurium AJB64 (lane 2) and IR715 (lane 3) grown under iron limitation. (B) Outer membrane profiles of E. coli H5058 (lanes 7 and 8) and H5058(pTY908) (lanes 5 and 6) grown in LB supplemented with 0.2 mM 2,2'-dipyridyl (lanes 6 and 8) or 0.04 mM FeSO4 (lanes 5 and 7).

IroN serves as a receptor for catecholate siderophores. The effect of mutations in iroN on siderophore utilization was tested in S. enterica serotype Typhimurium strains carrying a mutation in aroA. S. enterica aroA mutants are unable to produce the siderophore enterochelin and therefore exhibit strongly reduced growth under iron deficiency (NBD plates). Levels of growth stimulation of strains CL1509 (aroA) and AJB64 (aroA iroN) and AIR49 (aroA iroBC) by different siderophores were compared on NBD agar plates (Table 2). In E. coli, the catecholate-type siderophore enterochelin, composed of a circular trimer of DBS, is transported across the outer membrane via the receptor protein FepA. During its transport into the cytosol, enterochelin is hydrolyzed by Fes esterase to N,N',N"-tri-(2,3,-dihydroxybenzoyl)-dipeptide (DBS₃), dimers (DBS₂), and monomers of DBS. These breakdown products of enterochelin can be used as siderophores, and each is translocated across the outer membrane by any of three different E. coli outer membrane receptor proteins: FepA, Cir, and Fiu (16). Since IroN showed the highest degree of homology to enterochelin receptors of E. coli, B. pertussis, and P. aeruginosa, we investigated the ability of an S. enterica serotype Typhimurium iroN mutant to utilize catecholate-type siderophores, including enterochelin, DBS, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine amide, myxochelin A, myxochelin B, and myxochelin C (Fig. 6). As a control, we studied uptake of ferrioxamine B, a hydroxamate siderophore. All siderophores stimulated growth of all S. enterica strains, although the growth stimulation by benzaldehyde-2,3-dihydroxybenzhydrazone and DBS was less in the iroN aroA mutant (AJB64) than in the aroA mutant (CL1509) and the iroBC mutant (AIR49) (Table 2). When utilization of myxochelin A was tested in broth culture, addition of the siderophore promoted growth of an S. enterica serotype Typhimurium iroBC mutant (AIR49) better than of an iroN mutant (AJB64) (Fig. 7). These data indicated that IroN contributes to but is not the sole receptor involved in myxochelin A uptake in S. enterica.

View larger version (24K): [in this window] [in a new window]   FIG. 6.   Structures of catecholate siderophores used in this study.

View larger version (13K): [in this window] [in a new window]   FIG. 7.   Growth curves of S. enterica serotype Typhimurium (A) and E. coli (B) strains in NBD broth culture without supplements or supplemented with myxochelin A (MyxA). Growth was measured as optical density at 620 nm (OD620).

	DISCUSSION

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Several lines of evidence suggest that the genes iroBCDE and iroN, part of the S. enterica iroA locus, form a functional unit. Expression of iroBCDE and that of iroN are both iron regulated, indicating that these genes may be functionally linked. The iron response regulator Fur is likely involved in iron-responsive expression of genes in the iroA locus, as typical Fur DNA binding sites are present in the iroB and iroN promoter regions (5). Both the iroBCDE operon and the iroN gene are present in S. enterica but are absent from closely related bacteria (4) (Fig. 4). This phylogenetic distribution can best be explained by acquisition of the entire iroA locus during a single horizontal transfer event in a lineage ancestral to the species S. enterica. A function of the iroA locus in iron acquisition is suggested by homologies of iroDE and iroN to genes associated with siderophore utilization. The iroN gene encodes an outer membrane siderophore receptor with high homology to FepA, BfeA, and PfeA, the enterochelin receptors of E. coli, B. pertussis, and P. aeruginosa, respectively (Fig. 2 and 3) (6, 10, 23). The iroD gene product shows homology to Fes, an E. coli enzyme involved in enterochelin utilization (27). Finally, iroE has homology to an open reading frame located downstream of pfeA, the enterochelin receptor gene of P. aeruginosa (10). Although these data suggest that the iroA locus functions in iron acquisition, there is at present no evidence implicating the iroBC genes in siderophore biosynthesis or uptake.

Acquisition of the iroN gene by S. enterica introduced two characteristics which now set this species apart from related bacteria. First, localization of IroN in the outer membrane exposes this protein to the host immune system. Fur-regulated siderophore receptors of S. enterica serotype Typhimurium are highly expressed during infection, as shown by in vivo studies (18). Furthermore, antibodies against iron-regulated outer membrane proteins have been shown to be present in sera from patients who recovered from typhoid fever, an infection caused by S. enterica serotype Typhi (11). Thus, acquisition of iroN introduced a new antigen that is characteristic of S. enterica and likely presents a target for the host immune system. Second, acquisition of iroN provided S. enterica with a new protein involved in iron uptake. Some of the substrates that are utilized by the S. enterica IroN receptor protein are excreted by soil bacteria. For instance, the siderophores 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine and 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine amide are produced by Azotobacter vinelandii (9), and myxochelin A is a product of the myxobacterium Angiococcus disciformans (21). It could therefore be speculated that IroN facilitates growth of S. enterica in soil, a step frequently encountered during the fecal oral transmission of this ubiquitous pathogen. Although it is possible that some soil bacteria produce siderophores which are transported exclusively by IroN in S. enterica, this was not the case for the catecholates used in this study (Table 2). However, the presence of IroN resulted in an increased growth rate of S. enterica in broth culture containing myxochelin A (Fig. 7), suggesting that this receptor may confer a selective advantage in the environment under certain growth conditions.

In E. coli, IroN mediated uptake of a variety of catecholate siderophores, including DBS, enterochelin, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine amide, and myxochelin A. However, transport of these siderophores was not abolished in a S. enterica iroN mutant. A possible explanation for the utilization of catecholate siderophores by an S. enterica iroN mutant is that the substrate specificity of IroN overlaps with that of other siderophore receptor proteins present in serotype Typhimurium, such as orthologs of FepA and Cir (18, 38). Overlapping substrate specificities of catecholate receptors were first identified in E. coli, where DBS is transported by the outer membrane receptors FepA, Cir, and Fiu (16). Furthermore, all catecholates transported by E. coli fepA cir fiu mutants expressing IroN [H5058(pTY908) and H5058(pTY994)] were also utilized by the isogenic E. coli parent (AB2847), indicating that these siderophores are substrates of the E. coli FepA, Cir, and/or Fiu receptor proteins (Table 2). In analogy, uptake of catecholate siderophores in an S. enterica iroN mutant may thus be mediated by the orthologs of the FepA and/or Cir receptor proteins present in this organism (18, 38).