INTRODUCTION

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The genus Yersinia contains at least 11 species, 3 of which are enteropathogenic for humans. Yersinia pestis is the agent of bubonic plague, while Y. pseudotuberculosis and Y. enterocolitica are pathogenic for humans. Y. enterocolitica causes a broad range of diseases ranging from acute bowel disease to extraintestinal manifestations such as reactive arthritis and uveitis. Human-pathogenic Yersinia species can be divided into highly pathogenic Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica biotype 1B (so-called American serotypes), which are mouse lethal at low doses, and low-pathogenic Y. enterocolitica biotypes 2 to 5 (so-called European serotypes), which are not mouse lethal at low doses (10).

A prerequisite for any expression of pathogenicity by Yersinia is the presence of a 70-kb pYV virulence plasmid that is found in high- and low-pathogenic strains (4, 21, 27). Differences in mouse virulence seem to be chromosomally determined. Highly pathogenic strains possess a chromosomal cluster of iron-regulated genes designated the high-pathogenicity island (HPI). This island is absent in low-pathogenic or nonpathogenic strains and was found to be unstable in Yersinia strains. Its loss leads to a marked reduction in mouse virulence (36).

Two proteins encoded by iron-repressible genes have been detected only in highly pathogenic Yersinia strains, being putatively located on the HPI: HMWP1 (high-molecular-weight protein 1; 260 kDa, encoded by irp1) and HMWP2 (190 kDa, encoded by irp2) (9, 15). Inactivation of irp2 in Y. pseudotuberculosis results in a considerable reduction of mouse virulence (8). HMWPs are suspected to be important for siderophore yersiniabactin production and therefore involved in the expression of a CAS (chrom azurol S ferric ion indicator dye)-positive phenotype in highly pathogenic Yersinia strains (31). The receptor of yersiniabactin, FyuA (ferric yersiniabactin uptake), is a receptor with dual function: it is a receptor of the siderophore and a receptor of Y. pestis bacteriocin pesticin. Thus, highly pathogenic strains are pesticin sensitive (Pst^s) because of such a dual nature of FyuA (44). Yersiniabactin and FyuA were shown to be produced only by mouse-lethal strains (32).

In Y. pestis, the fyuA gene, the irp2 gene, and the hms locus (encoding hemin storage) are located on a 102-kb fragment designated the pgm (pigmentation) locus. This fragment is flanked by two copies of the insertion sequence (IS) element IS100 (23, 41), which might be the reason for frequent deletions of the pgm locus. ybtA, a gene encoding a protein belonging to the AraC family of transcriptional regulators, was recently detected upstream the irp2 gene in Y. pestis. YbtA is believed to be a transcriptional activator of the yersiniabactin receptor and of the siderophore biosynthetic genes (22). Bearden et al. (3) have identified an approximately 22-kb region of the pgm locus of Y. pestis which encodes several iron-regulated proteins. Some of them (YbtT and YbtE) were shown to be involved in the biosynthesis of a putative siderophore of Y. pestis.

The HPI of Y. enterocolitica contains the fyuA and irp2 genes but does not harbor genes for the hemin storage (24). This locus is much more stable than the pgm locus of Y. pestis. No flanking IS100 elements, but at least two IS elements, IS1328 and IS1400, were identified downstream fyuA in Y. enterocolitica O8 (7, 43). Irp2 and fyuA are separated by approximately 12 kb. This fragment may contain additional irp genes involved in siderophore synthesis, including irp1 (encoding HMWP1). In this study, we have determined the nucleotide sequence of the irp1 to irp5 genes of Y. enterocolitica O8, shown their involvement in yersiniabactin biosynthesis, and demonstrated the siderophore-directed regulation of yersiniabactin synthesis and receptor genes.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in the study are listed in Table 1. Strains were grown in Luria-Bertani (LB) broth or on LB agar plates (Difco Laboratories, Detroit, Mich.) at 28°C (Yersinia) or 37°C (Escherichia coli). Iron-deficient medium (NBD) was made by adding 200 µM --dipyridyl (Sigma, St. Louis, Mo.) to NB medium (nutrient broth [Difco] with 5 g of NaCl per liter as described previously [44]).

DNA manipulation. Bacterial chromosomal DNA was isolated by the method of Davis et al. (14). A gene bank was prepared from Y. enterocolitica WA-314 serotype O8. The chromosomal DNA was partially digested with Sau3A and ligated into the BamHI site of vector pLAFR2 (25).

DNA sequencing and sequence comparison. The subcloned fragments EcoRI-2 and -3 from cosmid 17A11 (Fig. 1) were treated with exonuclease III (Nested Deletion kit; Pharmacia Biotech). Vector primers for templates generated by exonuclease digestion were used.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Genetic organization of the Y. enterocolitica O8 WA-314 irp2-fyuA gene cluster. The genes are depicted as boxes. Arrows above indicate the direction of transcription. E, EcoRI; S, SalI.

PCR conditions. PCR amplifications were performed in an automated thermal cycler (TRIO Thermoblock; Biometra or GeneAmp PCR System 2400; Perkin-Elmer) as described by Saiki et al. (48) with TaqI polymerase and different pairs of oligonucleotides (Roth; Karlsruhe, Mannheim, Germany). The initial denaturation step (94°C, 7 min) was followed by 35 cycles of denaturing, annealing, and extension with one final extension step. Annealing and extension temperatures were set according to the primers used. PCR amplification products were separated in 1.6% agarose gels followed by purification with a QIAquick PCR purification kit or gel purified by using QIAquick gel extraction kit 250 (Qiagen GmbH, Hilden, Germany).

Construction of irp1 and ipr2 mutants. The EcoRI-2 fragment from cosmid 17A11 (Fig. 1) was ligated into the EcoRI site of vector SuperCos1. Fragment EcoRI-2 harbors a SalI site in the open reading frame (ORF) of irp1. A kanamycin cassette containing a SalI fragment from plasmid pUC-4K was inserted into it. Fragment EcoRI-2 of irp1 with the kanamycin cassette was ligated into the pKAS 32 suicide vector (designated pKAS-1SKan). pKAS 32 contains the rpsL gene, which encodes the S12 protein of the ribosomes (55). Insertion of the suicide vector into the chromosome results in a Sm^s phenotype of a formerly Sm^r strain. Kan^r (kanamycin resistance) Sm^r arose after an allelic exchange (double crossover) had taken place and the vector was lost. The construct was transformed into E. coli S17-1  pir⁺ tra⁺ (39, 54) followed by mobilization into WA-CS. Mutants were selected on agar plates containing kanamycin (40 µg/ml), streptomycin (100 µg/ml), and nalidixic acid (100 µg/ml), and the presence of the kanamycin cassette in irp1 was confirmed by Southern hybridization. To exclude a polar effect on the CAS phenotype, we created a second irp1 mutant by using a kanamycin cassette without transcriptional terminator. The EcoRI/SalI fragment of irp1 harboring an EcoRV cutting site (182 bases downstream of the EcoRI site) was inserted into the pKS vector, and the kanamycin cassette from pSB 315 cut by HindII was ligated into the EcoRV restriction site. The construct was excised with KpnI/SacI and inserted into the pKAS 32 suicide vector (resulting in pKAS-E1Kan) followed by mobilization and selection as described above.

FyuA-GFP reporter gene studies. We translationally fused 267 amino acids (aa) of FyuA (including the upstream regulatory sequences and the putative YbtA binding site) and the product of the reporter gene gfp (encoding green fluorescent protein [GFP]) mut3 by using standard PCR cloning procedures and primers with designed restriction sites (HindIII-BamHI [FyuA] and BamHI-SalI [GFP mut3]). The resultant plasmid, pCJG3.3N, was transferred into WA-C and WA-CS irp1::Kan^r by electroporation.

Growth experiments: feeding assay with yersiniabactin containing culture supernatant, desferrioxamine, purified yersiniabactin, and pyochelin. Y. enterocolitica mutant H1852 fur fyuA (siderophore hyperproducer) was cultivated aerobically in iron-deficient NBD medium for 12 h at 28°C (29). After centrifugation, the supernatant containing siderophore was sterilized by filtration and used for feeding experiments. Desferrioxamine (Desferal) was obtained from Ciby Geigy. Purified yersiniabactin and pyochelin preparations used for final confirming experiments were kindly provided by R. Reissbrodt (Wernigerode, Germany) and H. Budzikiewicz (Cologne, Germany).

Pesticin assay. Pesticin-producing strain Y. pestis EV76 was grown overnight at 26°C, and pesticin production was induced by mitomycin C (0.3 µg ml¹) for an additional 16 h. Cells were collected by centrifugation, and the supernatant was used as a crude pesticin preparation after sterilization with 0.1% chloroform (35). Sensitivity to pesticin was monitored by serial dilution of the supernatant (1:2) on mid-log-phase bacterial cultures (10⁶ microorganisms) in 0.6% LB agar used as an overlay (double-layer technique) with 1.2% LB agar as a support. Plates were incubated at 37°C for 18 h.

Screening for iron-chelating compounds. Strains to be tested were plated on CAS agar (52) and incubated for 2 days at 26°C. A clearly visible red-orange halo around bacterial colonies was indicative of siderophore production (i.e., colonies were CAS positive).

SDS-PAGE and Western blotting. The bacteria were cultured under iron-limiting conditions in NBD broth, centrifuged, washed, and solubilized by boiling in Laemmli buffer (total cell lysate) (38). Equal amounts of all strains (50 µg of protein) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 7.5% polyacrylamide gel at a constant current of 40 mA. The gel was stained with Roti-Blue (Roth) or electroblotted to nitrocellulose membranes (BA85; Schleicher & Schüll, Inc., Dasserl, Germany) as described previously (33). HMWP-specific antibodies were kindly provided by E. Carniel (Institut Pasteur, Paris, France). These antibodies had been obtained by using a purified HMWP fraction to immunize BALB/c mice. They specifically recognized the two HMWPs. Antibodies directed against one HMWP also recognize the other one (6).

Nucleotide sequence accession number. The sequence determined was deposited at the EMBL/GenBank database under accession no. Y12527.

	RESULTS

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Cloning of irp1 and nucleotide sequence determination. A Y. enterocolitica WA-C gene bank was screened for the presence of cosmids harboring the irp2-fyuA fragment by Southern hybridization. One of the cosmids, 17A11 with an insert of approximately 23 kb, hybridized with the irp2 terminal probe, fyuA, and the IS1328 element, indicating that this cosmid carries the DNA fragment covering the region between irp2 and fyuA. 17A11 was digested with EcoRI, resulting in six fragments of 23 (pLAFR2 vector portion), 8.2, 6.2, 3.3, 2.6, and 2.1 kb. The 3.3-kb EcoRI fragment hybridized with the fyuA probe, the 2.6-kb EcoRI fragment hybridized with the irp2 probe, and the 2.1-kb EcoRI fragment hybridized with the IS1328 probe. Double digestion with EcoRI and SalI revealed a physical restriction map of the 23-kb insert shown in Fig. 1. The 6.2-kb EcoRI fragment (designated fragment EcoRI-3) hybridized with the irp2 probe (bp 4581 to 5007 of irp2) downstream the EcoRI site. The 8.2-kb EcoRI fragment (designated fragment EcoRI-2) did not hybridize with any of the probes. It has been postulated that irp1 constitutes one operon with irp2 (8). We assumed that fragments EcoRI-2 and -3 comprise most likely the irp1 gene encoding HMWP1 with the size of 240 kDa. Therefore, both fragments were subcloned into the pKS vector and sequenced.

DNA and protein sequence homology. To identify similarities of irp1 to irp5 and their deduced polypeptides to known sequences, a search in the EMBL gene data library was performed with the FastA program. The irp1 DNA sequence has highest identity to a cosmid from Mycobacterium tuberculosis (57.5% identity in a 1,049-bp overlap [bases 490 to 1530]; unpublished, accession no. Z83857), 53.1% identity over 971 bp (bp 187 to 1131) to the polyketide synthase gene of Bacillus subtilis W168 (53), 52.9% identity in a 2,763-bp overlap (bp 1 to 2678) to the eryA gene of Saccharopolyspora erythraea (17), 51.4% identity over 1,713 bp (bp 195 to 1866) to the polyketide synthase gene of Streptomyces antibioticus (58), 64.2% identity in 162 bp (bp 175 to 335) to the polyketide immunosuppressant gene of Streptomyces hygroscopicus (1, 40, 51), and 52.9% identity in a 1,481-bp overlap (bp 5716 to 7132) to irp2 of Y. enterocolitica (28). Interestingly, similarities to all of these related sequences are located within the first 3,000 nucleotides, whereas the following 6.3 kb show similarity only to irp2. A potential -ketoacyl synthase active site could be identified in HMWP1 between aa 184 and 210. -Ketoacyl-ACP (acyl carrier protein) synthase is the enzyme that catalyzes the condensation of malonyl-ACP with the growing fatty acid chain and is also found as a component in polyketide antibiotic synthases.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Common amino acids motifs found in HMWP1 and HMWP2.

Presence of irp1 in various Yersinia species and E. coli. The presence of irp1 in various Yersinia and E. coli isolates was tested by Southern hybridization. The chromosomal DNAs of various strains (Table 2) were digested with EcoRI, and Southern hybridization was performed with an irp1 probe (corresponding to bp 7901 to 8139). As expected, irp2/fyuA-negative strains were also devoid of irp1. In irp2/fyuA-positive strains (Y. enterocolitica WA-CS and 8081, Y. pestis KIM and KUMA, Y. pseudotuberculosis PB1 and 346, and E. coli Phi), a band that hybridized with the irp1 probe was detected (Fig. 3).

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Southern hybridization of chromosomal DNAs from Yersinia and E. coli strains with the irp1 probe. The chromosomal DNA was digested with EcoRI, and the resulting fragments were separated on a 1% agarose gel prior to Southern blotting. Hybridization was performed with a DIG-labeled PCR probe generated with primers i8513 and i8730. Lane 1, Y. pestis KUMA; lane 2, Y. pestis KIM; lane 3, Y. pestis KIM pgm; lane 4, Y. pseudotuberculosis 346; lane 5, Y. pseudotuberculosis 201; lane 6, Y. pseudotuberculosis PB1; lane 7, Y. enterocolitica 8081; lane 8, Y. enterocolitica WA-CS; lane 9, Y. enterocolitica Y5.27; lane 10, Y. enterocolitica Y-96-C; lane 11, Y. enterocolitica Y-108-C; lane 12, E. coli Phi; lane 13; E. coli DH5.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Comparison of the region from bp 1041 to 1191 of irp1 in Y. enterocolitica 8081, Y. enterocolitica WA-CS (I), Y. pestis KUMA, Y. pestis KIM, Y. pseudotuberculosis 346, Y. pseudotuberculosis PB1, and E. coli Phi (II). Nonmatching bases are boldfaced and underlined.

irp1- and irp2-encoded proteins are involved in yersiniabactin synthesis. The possible relationship between the CAS phenotype and HMWP production was analyzed by mutagenesis of gene irp1. A kanamycin cassette was inserted into irp1 gene of WA-CS, and allelic exchange was performed. The resulting mutant, WA-CS irp1::Kan^r, was tested on CAS agar and found to be CAS negative, indicating loss of yersiniabactin production (Fig. 5). The polar effect of the kanamycin cassette on the irp operon was ruled out by interruption of irp1 with a kanamycin cassette lacking a transcriptional terminator. This mutant was also unable to form a halo on CAS agar. The same result was achieved by inserting the total suicide vector pGPCAT carrying a fragment of irp2 (designated pGPIRP2) into the irp2 gene by homologous recombination (data not shown). These results demonstrate that mutants disrupted in either irp1 or irp2 lost the ability to synthesize the siderophore yersiniabactin.

View larger version (113K): [in this window] [in a new window]   FIG. 5.   CAS agar plate showing siderophore-producing Y. enterocolitica WA-CS (A; with halo) and mutant WA-CS irp1::Kanr (B).

Yersiniabactin-dependent expression of the irp operon. To evaluate the effect of the irp1 inactivation on the expression of HMWP1, a total-cell lysate of WA-CS and WA-CS irp1::Kan^r grown under iron-poor conditions (NBD medium) was subjected to SDS-PAGE. HMWP1 could not be detected in the mutant strain, while HMWP2 was still visible as a very faint band after Roti-Blue staining (Fig. 6). The parent strain WA-CS expressed both HMWPs in comparable amounts. An additional weak protein band could be detected between HMWP1 and HMWP2. This band is thought to be a modified form of HMWP2 (5a) and reacts with the HMWP antibodies (Fig. 7). Production of HMWP1 and -2 was also decreased in an fyuA mutant impaired in its ability of ferric yersiniabactin uptake (Fig. 6). HMWP1 and HMWP2 were detected with anti-HMWP1 and anti-HMWP2 antisera in the wild-type strain but not in the mutant WA-CS irp1::Kan^r (Fig. 7).

View larger version (59K): [in this window] [in a new window]   FIG. 6.   Expression of HMWP1 and -2 in WA-CS irp1::Kanr and WA fyuA mutants. SDS-PAGE (7.5% gel) of total-cell proteins from iron-starved strains WA-CS (lane 1), WA-CS irp1::Kanr (lane 2), and WA fyuA (lane 3).

View larger version (33K): [in this window] [in a new window]   FIG. 7.   SDS-PAGE (7.5% gel) (A) and corresponding immunoblot (B) with cell lysates of WA-CS (lane 1), WA-CS irp1::Kanr (lane 2), and WA-CS irp1::Kanr with an addition of purified yersiniabactin (lane 3). The strains were grown under iron starvation in NBD medium. Western blotting was performed with HMWP polyclonal antibodies kindly provided from Elisabeth Carniel.

View larger version (54K): [in this window] [in a new window]   FIG. 8.   Effects of pesticin and yersiniabactin on expression of the HMWPs. SDS-PAGE (7.5% gel) of total-cell proteins of iron-starved strains WA-CS and WA-CS irp1::Kanr with addition of pesticin and culture supernatant containing yersiniabactin. Lane 1, WA-CS in NBD medium; lane 2, WA-CS in NBD medium with pesticin (1:1,024); lane 3, WA-CS in NBD medium with yersiniabactin supernatant (1:50); lane 4, WA-CS irp1::Kanr in NBD medium; lane 5, WA-CS irp1::Kanr in NBD medium with pesticin; lane 6, WA-CS irp1::Kanr in NBD medium with yersiniabactin supernatant.

View larger version (17K): [in this window] [in a new window]   FIG. 9.   Fluorescence of FyuA-GFP in Y. enterocolitica WA-CS (- -) and WA-CS irp1::Kanr () in NB medium (A), NBD medium (B), NBD medium plus purified yersiniabactin (C), and NBD medium plus desferrioxamine B (D). (E) Positive (WA-CS[pGFP mut 3]; -- --) and negative (WA-CS; --  --) controls. Au, arbitrary units.

	DISCUSSION

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Since the rediscovery of siderophore production by highly pathogenic Yersinia species in 1987, it has been generally appreciated that this high-affinity ferric iron uptake system significantly contributes to virulence of yersiniae (31). Recently, the chemical structure of yersiniabactin has been determined (18). There was an indication that the putative genes for biosynthesis of yersiniabactin reside within the HPI on the chromosome (28). The objectives of this study were to demonstrate that the irp1 and irp2 genes, encoding HMWP1 and HMWP2, are involved in yersiniabactin biosynthesis and to characterize the irp operon. Two genes, irp2, encoding iron-repressible HMWP2, which is proposed to be involved in nonribosomal protein synthesis, and fyuA, encoding yersiniabactin/pesticin FyuA receptor, represent the yersiniabactin biosynthetic cluster. A small araC-like ybtA gene seems to be a positive regulator for irp gene expression (22) and precedes the irp operon.

In this study, we analyzed the whole irp operon and identified four additional ORFs on a 13-kb DNA fragment between the irp2 and fyuA genes. The largest one, located immediately downstream irp2, comprises 9.5 kb with the capacity to code for a 384-kDa polypeptide. The theoretical molecular mass of the polypeptide encoded by that ORF is higher than expected from the results of the SDS-PAGE (240 kDa). This can be due to the complex secondary structure of this extremely large protein as well as to its possible atypical migration in denaturing conditions. The insertional inactivation of irp1 by allelic exchange leads to loss of HMWP1 and HMWP2 in SDS-PAGE (Fig. 6).

Inactivation of irp1 or irp2 resulted in the loss of siderophore production as monitored on CAS agar (Fig. 5). Thus, irp1 and irp2 are involved in yersiniabactin biosynthesis.

HMWP1 shares a unique motif with the polyketide synthases. The -ketoacyl-ACP synthase active site is highly conserved among the three multifunctional polypeptides of the rapamycin-producing polyketide synthases of S. hygroscopicus as well as in the polyketide synthase of S. antibioticus (1). Polyketide synthases are involved in the synthesis of a large and highly diverse group of heterocyclic compounds including antibiotics, antitumor compounds, and heterocyclic immunosuppressants. Polyketide metabolites are produced by successive condensation of simple acid units as propionate and acetate. Prolonging of the acid chain is catalyzed by the polyketide synthases (17). The similarity implies that HMWP1 could be involved in the synthesis of a siderophore or of an antibiotic.

The last 6.3 kb of irp1 have no significant similarity to any known sequence besides a 52.9% identity over 1,481 bp (bp 5716 to 7132) to irp2. HMWP2 is known to be homologous to AngR (involved in the anguibactin biosynthesis of V. anguillarum), and it was predicted to direct nonribosomal synthesis of small molecules involved in the nonribosomal synthesis of antibiotics or siderophores (28).

Two other defined ORFs, irp4 and irp5, have pronounced identity to a thioesterase-like protein from anguibactin biosynthetic gene cluster of V. anguillarum (20) and EntE (2,3-dihydroxybenzoic acid-activating enzyme) from E. coli (57), respectively. irp4 and irp5 are nearly identical to the ybtT and ybtE genes described as a part of a siderophore biosynthetic region in Y. pestis (3). Insertional inactivation of the ybtE gene yielded mutants unable to grow in iron-deficient medium at 37°C. A ybtE mutant could be cross-fed by a culture supernatant from a wild-type strain. It is reasonable to propose that yersiniabactin or a siderophore with a similar structure can represent this siderophore in Y. pestis. Taking into account structural similarities of yersiniabactin and anguibactin siderophores (Fig. 10), one can assume that irp2 to irp5 represent a yersiniabactin biosynthetic gene cluster with the following organization: irp2-irp1-irp3-irp4-irp5. An additional gene located between irp2 and irp1 as proposed for Y. pestis (3) could not be identified.

View larger version (17K): [in this window] [in a new window]   FIG. 10.   Chemical structures of yersiniabactin, pyochelin, and anguibactin.

Insertional inactivation of the irp1 and irp2 yersiniabactin biosynthetic genes results not only in elimination of the corresponding polypeptide bands but also in downregulation of the other proteins involved in yersiniabactin synthesis and binding; namely, irp1 inactivation was followed by considerable reduction of HMWP2 and FyuA proteins (Fig. 6 and 9). Nonsense mutation in the fyuA gene resulted in downregulation of the irp1 and irp2 genes. It was expected that inactivation of the irp2 gene, being the first gene in the polycistronic operon, would lead to a reduction of the irp1 gene product since irp1 is devoid of promoter/operator sequences. The unexpected reduction of HMWP2 and FyuA production as a result of irp1 inactivation suggests that the yersiniabactin biosynthetic operon is subjected to autoregulation by its product, the siderophore. Indeed, the addition of yersiniabactin to the siderophore-negative mutant WA-CS irp1::Kan^r upregulates the production of HMWP2 (Fig. 7 and 8) and its receptor, as demonstrated by the translational fyuA-gfp fusion (Fig. 9). Consistent with these data, Y. pestis supernatant was also found to influence the expression of the ybt (yersiniabactin biosynthetic genes in Y. pestis)-encoded proteins (3).

Exogenous siderophore desferrioxamine B taken up by yersiniae through the FoxA receptor (2) did not induce fyuA. Thus, the yersiniabactin molecule specifically induces fyuA expression, while sublethal doses of neither pesticin, desferrioxamin B, nor the structurally related molecule pyochelin could serve as a signal for induction of the irp operon.

Siderophore-dependent expression of the cognate receptors was demonstrated for the iron dicitrate system in E. coli (61) and for pyoverdine, pyochelin, and enterobactin receptors in Pseudomonas aeruginosa (16, 34, 59). Moreover, phenolate siderophore pyochelin shows high structural similarity with yersiniabactin (Fig. 10). Several other features are common between yersiniabactin and pyochelin receptors. The presence of pyochelin, which exhibits a low affinity for iron in vitro, has been correlated with increased virulence and in vivo growth (13). The pyochelin receptor gene fptA is positively regulated by a pchR product, an AraC-type transcriptional regulator (34). On the basis of the similarity of ybtA to the araC-type regulators, it was proposed that the YbtA activator requires ferric yersiniabactin to interact with the palindrome sequences upstream psn (designation of the yersiniabactin receptor in Y. pestis with 99% similarity to fyuA) and irp2 for the maximum induction of these genes (22). Thus, the absence of yersiniabactin results in low expression of irp2 and fyuA genes in the irp1 mutant due to the inability of YbtA to form a complex with yersiniabactin, to bind to the palindrome sequences, and therefore to activate the yersiniabactin operon. Due to the lack of sequencing data of the pyochelin biosynthetic cluster, it is not possible to demonstrate the relationship of these two siderophore systems on the molecular level. Nevertheless, these three siderophore systems, yersiniabactin, pyochelin, and anguibactin, have a high degree of similarity in structure and function (Fig. 10).

Although it exhibited no siderophore production, the mutant WA-CS irp1::Kan^r was still able to grow in iron-deficient medium. This indicates that at least one efficient iron uptake system is present in Y. enterocolitica in addition to the yersiniabactin system. The TonB-independent yfu system discovered recently in Y. enterocolitica (49) may be a candidate for such a system.

irp2 and fyuA genes present on the HPI were shown to be highly conserved (45). The same is true to the irp1 gene start and end portions, which were shown to be identical or highly similar in all three highly pathogenic species, Y. enterocolitica, Y. pseudotuberculosis O1, and Y. pestis. The irp4 and irp5 genes were also found to be nearly identical to the corresponding genes identified in Y. pestis KIM. Thus, ybtA-fyuA genes comprise a highly conserved gene cluster (HPI) present in highly pathogenic yersiniae. A high G+C content and a codon usage different from that in Yersinia housekeeping genes were found in all genes constituting the HPI. Therefore, a horizontal transfer may be responsible for the dissemination of the yersiniabactin biosynthetic operon in Yersinia. Moreover, recent studies have demonstrated that conserved HPI is widely distributed among representatives of certain pathotypes of E. coli (50). Such a wide dissemination of HPI and its impact on the virulence of Yersinia implies an important, possible multifunctional role of yersiniabactin system in vivo and the availability of an efficient mechanism for its genetic transfer.