INTRODUCTION

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The genus Yersinia consists of 11 species. Strains of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica are pathogenic for mammals. Pathogenicity determinants have been localized on plasmids and on the chromosome of yersiniae. Pathogenic Yersinia can be divided into a high-pathogenicity group (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica biogroup [BG] 1B) and a low-pathogenicity group (Y. enterocolitica BG 2 to 4), on the basis of the lethal infectious dose in the mouse model (9). Lethality for mice (50% lethal dose < 1,000 microorganisms) depends on the presence of the yersiniabactin (ybt) locus, which carries genes for biosynthesis, transport, and regulation of the siderophore yersiniabactin (22-24, 28, 31). In Y. pestis, the unstable 102-kb chromosomal fragment is associated with pigmentation (pgm), i.e., the ability of bacterial cells to form pigmented colonies on hemin or Congo red agar plates at 26°C (31). The pgm locus is composed of yersiniabactin (ybt) and hemin storage (hms) loci (15, 25, 27, 29). In contrast, Y. pseudotuberculosis contains nonclustered ybt and hms loci and only the ybt locus is present in Y. enterocolitica (5, 8). The in vitro instability of the pgm locus in Y. pestis has been observed as a complete or partial (hms or ybt locus) deletion of the 102-kb fragment due to the presence of two IS100 flanking sequences (12, 14, 15). The ybt locus comprises 36 to 43 kb (8). Sequencing of genes involved in yersiniabactin synthesis and uptake revealed a G+C content higher than that of the host genome (21, 36). The ybt locus is flanked by an asn tRNA gene at one extremity and carries a gene for a putative integrase (4, 8). These are features typical of pathogenicity islands (20). Therefore, the ybt locus is termed a high-pathogenicity island (HPI) to emphasize its involvement in the mouse-lethal phenotype (8). Interestingly, the ybt cluster has been detected in certain pathotypes of Escherichia coli, suggesting that it originates from a horizontal transfer (40).

Five synthesis genes, irp1 to irp5 (ybtU, ybtT, and ybtE are the orthologs of irp3, irp4, and irp5 in Y. pestis) of the ybt locus are clustered in one large 19-kb operon (2, 28). The genes for the yersiniabactin receptor FyuA (also named Psn in Y. pestis) and an AraC-type yersiniabactin regulator YbtA flank the biosynthetic genes (16, 17, 33). An RS3 repeated sequence and two IS elements were identified downstream of fyuA in Y. enterocolitica (8, 34).

Two distinct variants of the HPI were identified in Y. pseudotuberculosis/Y. pestis (Yps HPI) and in Y. enterocolitica (Yen HPI) (36). The part of the island that contains yersiniabactin synthesis, receptor, and regulator genes is highly conserved in HPIs of both evolutionary lineages (28, 36), while the right end (downstream of fyuA) differs markedly between the Yps HPI and the Yen HPI (5, 8). The Yps HPI is able to occupy any of the three asparagine tRNA genes in Y. pseudotuberculosis, suggesting that the HPI has retained its mobility functions (4). The HPI is flanked by 24-bp (21) or 17-bp (4) direct repeats that are duplications of the 3' end of asn tRNA.

In this work, we determined the complete molecular genetic structure of the HPI in Y. enterocolitica WA-314 and compared it with that of the Y. pseudotuberculosis HPI to gain insight into the divergent evolution of the HPI and Yersinia in general.

	MATERIALS AND METHODS

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Bacterial strains, media, and growth conditions. The bacterial strains used in the study are listed in Table 1. The strains were grown in Luria-Bertani (LB) broth or on LB agar plates (Difco Laboratories, Detroit, Mich.) at 28°C (Yersinia) or 37°C (E. coli). Iron-chelating compounds were screened on a chrome azurol S ferric ion indicator dye (CAS) agar (41). A red-orange halo around bacterial colonies indicated siderophore production (i.e., colonies were CAS agar positive). Y. pestis spontaneous mutants, unable to accumulate the Congo red dye (pgm), were selected on LB medium containing 15 µg of Congo red per ml.

DNA manipulations. Bacterial DNA was isolated by the method of Davis et al. (11). A Y. enterocolitica gene bank was prepared from WA-314 serotype O:8, and cosmid 12H2 was used to determine the 5' end of the HPI (35). The D11 cosmid of the Y. pseudotuberculosis PB1 serotype O1A gene bank was used to determine the 3' end of the HPI (36). PCR amplifications were performed in an automated thermal cycler (GeneAmp PCR system 2400; Perkin-Elmer) as described by Saiki et al. (39) with TaqI polymerase and different pairs of oligonucleotides (Roth, Karlsruhe, Germany, and Metabion, Munich, Germany). Self-designed PCR primers used to define the ends of HPI and to amplify specific HPI sequences are listed in Table 2. Southern blot hybridizations were performed with digoxigenin (DIG)-labeled PCR probes, using different primer pairs plus DIG-11-dUTP, as specified by Boeringer Mannheim Biochemica.

DNA sequencing and sequence comparison. Cosmid 12H2 (35) was used to determine sequences located between ybtA and the left end of the island associated with asn tRNA. Four ClaI fragments of the 12H2 cosmid subcloned in pBluescript KSII cloning vector (Stratagene), and the original 12H2 cosmid was used to determine the sequence of the 5' boundary of the HPI by primer walking.

Nucleotide sequence accession numbers. The sequences determined in this study were deposited at the EMBL/GenBank database under accession no. AJ132668, AJ132945 and AJ236887.

	RESULTS

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To complete the structure of the Yen HPI in Y. enterocolitica WA-C, serotype O:8, we sequenced both ends of the island (Fig. 1). The borders of the island can be defined by 17-bp direct repeats flanking the HPI. The Yen HPI is 43,393 bp. The left (5') end, which is associated with the asn tRNA gene, is nearly identical to that of the Yps HPI, while the right (3') end shows significant differences between the Y. enterocolitica O:8 HPI and the Yps HPI (5, 8). Therefore, the DNA sequence of the 3' end of the HPI in Y. pseudotuberculosis PB1, O1A (Yps HPI), was also determined and compared to the 3' end of the Yen HPI. The comparison of Yen and Yps evolutionary lineages of the HPI reveals that the HPI has two distinct parts (Fig. 2). The first part spans the region between asn tRNA and the fyuA stop codon and represents the functional "core" of the island. It is nearly identical (98 to 99% identity) in both evolutionary lineages and has a G+C content significantly higher than that of the Yersinia genome (57.5 mol% versus 46 to 48 mol% for the Yersinia chromosome) (3). A considerably lower G+C content is found downstream of the fyuA gene. This AT-rich variable part differs completely between the HPIs of the two evolutionary lineages.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Complete structure of the HPI in Y. enterocolitica WA-C. The Left (5') (A) and right (3') (B) ends of the island are shown. Arrows show the positions of the ORFs and the direction of transcription. Positions of restriction sites are depicted by vertical lines; numbers above the lines show the distance from the beginning of the sequenced DNA fragment in base pairs.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Complete structure and the G+C content of the HPIs in Y. pseudotuberculosis PB1 and Y. enterocolitica WA-C. Arrows below the graph show position of genes and direction of transcription. Vertical arrows show the borders of the HPI, the left DR17 within asn tRNA defines the left border, and the right DR17 defines the right border.

Left (5') end of the HPI in Y. enterocolitica. The nucleotide sequence of the 8.1-kb fragment at the 5' end of the Yen HPI contains five ORFs flanked by ybtA and asn tRNA (Fig. 1A). Four ORFs, irp6 to irp9, have the opposite transcriptional polarity to ybtA. These four genes display 98 to 99% identities to the ybtP, ybtQ, ybtX, and ybtS genes, respectively, recently described in Y. pestis (18, 19). YbtP and YbtQ are thought to be involved in the uptake of the ferric yersiniabactin and YbtS might be involved in biosynthesis of the yersiniabactin in Y. pestis.

Integrase. The next ORF has an opposite transcriptional polarity to irp6 to irp9 (Fig. 1A). It has high similarity to the genes encoding putative integrase from Y. pseudotuberculosis (4) and to the P4 prophage integrase (6). In contrast to the putative integrase genes in Y. pseudotuberculosis and Y. pestis (4, 21), a TAA stop codon interrupts this ORF, henceforth designated intB, 415 bp downstream of the ATG start codon. The 420-amino-acid (aa) polypeptide that can be predicted from the intB pseudogene sequence shows 98% identity to the putative integrase of Y. pseudotuberculosis and 49.7% identity to the integrase of the prophage P4.

ERIC sequence. A 127-bp enterobacterial repetitive intergenic consensus (ERIC) sequence (26), also known as the intergenic repeated unit (IRU) (42), was recognized in the promoter of the ybtA yersiniabactin regulator (Fig. 3). D7 and D340 primers, designed to amplify the ybtA promoter, yielded a 229-bp product in Y. pseudotuberculosis PB1, Y. pestis KIM, and HPI-positive E. coli strains (K49, K235, D-1041-86, C-4441, and 12860). In contrast, the same primers amplified a larger (354-bp) product in all Y. enterocolitica 1B isolates (O:8, O:13, O:20, and O:21). Sequencing of the Y. enterocolitica 1B amplicons demonstrated that the 125-bp DNA insertion represents an ERIC sequence.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Insertion of a 125-bp ERIC sequence into the ybtA promoter of the Yen HPI. The upper panel shows the structure of the ybtA promoter in Y. pestis and Y. enterocolitica. The lower panel depicts aligned nucleotide sequences of both promoters. Identical bases are boxed in grey. Black arrows show two repeated sequences (RS1 and RS2) of the ybtA promoter. The asterisk in the RS2* sequence depicts an interruption in the repeated sequence. Open arrows show the position and direction of the inverted repeats (IR) of the ERIC. Yen WA-C, Y. enterocolitica WA-C; Yp KIM, Y. pestis KIM; FBS, Fur protein-binding site; RS, repeated sequences, SD, potential ribosome-binding site.

Right (3') end of the HPI. The size of the 3' end of the HPI differs between the evolutionary groups. We determined a 14-kb sequence downstream of the fyuA gene in Y. enterocolitica WA-C and compared it to the corresponding sequence of Y. pseudotuberculosis PB1.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Fragment of the Y. pseudotuberculosis PB1 chromosome with the 3' end of the Yps HPI. The large arrow on the left indicates the position of fyuA. Arrows show positions of the ORFs and their direction of transcription. Black bars within the Y. pseudotuberculosis sequence represent regions of identity with the Y. enterocolitica DNA. Triangles indicate positions of the IS elements within the Yen HPI. PstI, EcoRI, BamHI, and KpnI depict the positions of recognition sites of the corresponding enzymes. Small black arrows under the graph indicate PCR primers used. The DIG-11-dUTP-labelled PCR products obtained with the same primer pairs were used as hybridization probes. + and , presence or absence, respectively, of hybridization products. +*, larger PCR amplicon in Y. enterocolitica WA-C; Y.ent, Y. enterocolitica; Y.pstbc, Y. pseudotuberculosis.

Junctions of the high-pathogenicity island. The 5' intB junction of the HPI in Y. enterocolitica 8081, serotype O:8, is associated with the 3' end of the asnT tRNA (8). asnT tRNA is also used by the HPI in E. coli (40). Integration of pathogenicity islands into highly conserved tRNA copies is a common feature of these islands (20). Four identical copies of the asn tRNA genes (asnT, asnU, asnV, and asnW) have been identified on the chromosome of E. coli K-12 (E. coli MG1655 Genome Project; accession no. AE000289, AE000290, and AE000291) and three have been identified in Yersinia (4). The sequence upstream of the asnT tRNA gene in Y. enterocolitica WA-C O:8 has 84% identity to the sequences upstream of the HPI in Y. pseudotuberculosis O:1 (accession no. AJ009592 [4]). This agrees with the proposal that the Yen HPI in Y. enterocolitica WA-C and the Yps HPI in Y. pseudotuberculosis PB1 occupy the same asn tRNA copy.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Left (5') and right (3') junctions of the Yen HPI. Arrows in the graph show position and orientation of the genes. Black arrows under the graph show PCR primers used. The + and  indicate the presence or absence of a PCR product amplified with W250 plus W598 and asn468 plus c15-205 primer pairs. Numbers correspond to the size of the PCR products in base pairs. Y.ent, Y. enterocolitica.

The HPI att site is unoccupied in Y. enterocolitica biogroup 1A. Primers W250 (located upstream of the asnT tRNA) and Ye262 (anneals downstream of DR₁₇) flank the HPI (Fig. 5). As expected, they failed to amplify the whole HPI in Y. enterocolitica WA-C and 8081. However, these primers successfully amplified a 515-bp fragment in avirulent Y. enterocolitica NF-O (O:5, BG 1A). The sequence of this amplicon contains a 16-bp DNA stretch identical to DR₁₇ and overlaps with the sequences flanking the junctions of the HPI in Y. enterocolitica WA-C (Fig. 6).

View larger version (31K): [in this window] [in a new window]   FIG. 6.   Unoccupied attachment site of the HPI in Y. enterocolitica NF-O. The NF-O sequence was amplified with primers W250 and Ye262 (Fig. 5) and aligned with the 5' and 3' boundaries of the Yen HPI. Grey boxes show identical nucleotide sequences and a 16-bp part of the DR17. Numbers on the right show relative positions of nucleotides in base pairs. 5' end, 5' boundary of the Yen HPI, asnT RNA; 3' end, 3' boundary of the Yen HPI; NF-O, a PCR product amplified by the W250 and Ye262 primers in Y. enterocolitica NF-O.

Deletions in Y. enterocolitica HPI are imprecise. The yersiniabactin receptor FyuA has a dual function, since it also serves as a receptor for the Y. pestis bacteriocin pesticin (31, 33). Thus, pesticin sensitivity can be used as a selective marker for the presence of FyuA. Previously we selected several Y. enterocolitica pesticin-resistant spontaneous mutants that failed to produce the yersiniabactin on CAS agar (35). This is indicative that yersiniabactin biosynthetic genes were coinactivated with the yersiniabactin receptor gene. The WA fyuA3 and WA fyuA4 mutants were resistant to the pesticin due to deletions in fyuA. We have analyzed both mutants for the presence of the HPI genes (Table 5). intB through irp4 sequences were detected in the WA fyuA3 mutant but were absent from the WA fyuA4 mutant. Both mutants lost IS1328 and IS1329 insertion sequences and either of the junctions (Fig. 5). However, WA fyuA4 contains the IS1400 element as well as the right junction of the HPI. WA fyuA3 and WA fyuA4 were negative with the W250 and Ye262 primers that amplified the island-free attachment site in Y. enterocolitica NF-O. In summary, WA fyuA4 has lost the HPI sequences upstream of IS1400, whereas WA fyuA3 has lost the right extremity of the island downstream of the irp4 gene. Thus, spontaneous elimination of the Yen HPI, which results in the inactive yersiniabactin iron acquisition system, occurs through imprecise deletions in contrast to the precise excision described for the Yps HPI (4).

	DISCUSSION

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The HPI of Yersinia spp. is responsible for lethality for mice and for biosynthesis and uptake of the siderophore yersiniabactin. Sequence comparison depicts two evolutionary lineages of the HPI, Y. enterocolitica (Yen HPI) and Y. pestis/Y. pseudotuberculosis (Yps HPI), at the nucleotide level (21, 36) as well as in the genetic organization of the fyuA end of the island (8). We have determined the complete size of the Yen HPI to be 43,393 bp, which is 7.3 kb larger than that of the corresponding HPI in Y. pseudotuberculosis. Such a difference infers the presence of additional genes on the Yen HPI, which might support functions different from yersiniabactin-mediated iron acquisition.

Yersiniabactin-mediated iron acquisition is thought to be the main function of the HPI (19), and genes involved in yersiniabactin biosynthesis, transport, and regulation (irp1 to irp9, ybtA, and fyuA) are clustered on the "core" of the HPI. The P4-like integrase gene intB resides on the 5' end of the HPI next to the asn tRNA bacterial attachment site and belongs to the "core" of the island as well. The genes of the "core" show extremely high conservation in both evolutionary lineages and are characterized by a higher G+C (57.5 mol%) content than average in yersiniae (48 mol%) (2, 4, 19, 36). In contrast, the second component of the HPI is an AT-rich region that is completely different in the Yen HPI and Yps HPI. Thus, the genes of the HPI variable region are not expected to be involved in yersiniabactin production.

We could identify 13 ORFs within the fyuA end of the Yen HPI (Table 3). Six of them encode putative transposases of the four insertion sequences IS1328, IS1329, IS1400, and IS1222. The fyuA end of the Yps HPI in Y. pseudotuberculosis contains eight ORFs (Table 4). Two of them encode a putative transposase of IS100, while two other ORFs encode products with similarities to putative DNA-binding proteins. These proteins might be the remnants of self-transmissible elements, which were lost during stabilization of the HPI. The ORF2 and ORF5 products might be involved in DNA recognition, but they are not sufficient for the horizontal transfer of the island. Moreover, these proteins are not present in the Y. enterocolitica or E. coli HPI.

Yersinia might have acquired the HPI horizontally from a common progenitor with a high G+C content. The HPI has evolved divergently in the two evolutionary lineages, although the DNA similarity between the yersiniabactin genes of the two groups is about 98%. Y. enterocolitica 1B strains are the only ones that produce a halo on a CAS indicator agar, indicating yersiniabactin production. Actually, the yersiniabactin is the sole endogenous siderophore of Y. enterocolitica. Y. pestis, although containing a complete set of yersiniabactin genes, appears to be CAS negative. The fine-tuning of yersiniabactin genes to specific requirements of the Y. enterocolitica cell might be achieved in a stepwise mode by a single integration of the ERIC element into the promoter of the ybtA yersiniabactin regulator in Y. enterocolitica. The ERIC sequence modifies the structure of the ybtA operator and thus might be responsible for the different expression of the yersiniabactin biosynthetic genes in yersiniae (36a).

The HPI is associated with the asn tRNA genes in Y. enterocolitica (8), Y. pseudotuberculosis (4), Y. pestis (21), and E. coli (40). The Yps HPI can be inserted into any of the three asn tRNA copies of Y. pseudotuberculosis (4) and can use different asn tRNA genes in Y. pestis and Y. pseudotuberculosis (21). The comparison of the HPI integration sites in two Y. pseudotuberculosis strains, described in recent publications (4, 21), demonstrates that the Yps HPI integrates into two different asn tRNA copies in these strains. In contrast to the Yps HPI, the Yen HPI is stably integrated into the same asnT RNA gene in all serotypes of Y. enterocolitica BG 1B strains. Therefore, the HPI seems to be "immobile" in Y. enterocolitica, perhaps due to the inactivated putative integrase. Y. enterocolitica is thus not expected to be the original donor of the HPI that is widely distributed among the members of the Enterobacteriaceae (40). The presence of the Y. pestis-type HPI in the Enterobacteriaceae supports this prediction.

The Yen HPI, like the Yps HPI, is flanked by a 17-bp perfect duplication of the 3' end of the asn tRNA gene. Such duplications indicate a site-specific recombination event that results in integration of prophages and plasmids (7). The excision of the integrated units is predominantly precise and leads to the reconstruction of the original attachment site. A precise excision seems to be responsible for the HPI disintegration in Y. pseudotuberculosis O:1A (4). In contrast, deletions of the Yen HPI sequences in two pesticin-resistant mutants resulted in different endpoints and extensions. Moreover, the direct repeats of the island do not play a role in these deletion events. Three complete IS elements, which are present in the Yen HPI, might be responsible for the above deletions. Consequently, in contrast to Y. pseudotuberculosis, the precise excision of the Yen HPI is a rare or perhaps even impossible event in Y. enterocolitica.

We have identified an "island-free" bacterial att site for the HPI in apathogenic Y. enterocolitica NF-O (serotype O:5, BG 1A) (Fig. 6). The same site is "occupied" in HPI-negative yersiniae of BG 2, 3, and 4. This indicates that the asn tRNA genes might be used as integration sites for a foreign DNA in human pathogenic yersiniae.

The widespread presence of the HPI in E. coli of different pathotypes (40) implies an efficient mechanism of its transfer. Temperate phages or transmissible plasmids are candidates as HPI vehicles. The presence of a variable AT-rich "additive" to the highly conserved "core" points to a passive HPI transfer by a head-full phage transduction. It is also possible that the mobility genes were already lost by the island as in the locus of enterocyte effacing (LEE) island in enteropathogenic E. coli strains compared to enterohemorrhagic E. coli O157:H7 (30). Alternatively, the LEE island in O157:H7 may acquire the mobility genes. ORF2 and ORF5 with possible DNA-binding ability might be remnants of a mobility fraction of the ancestral HPI.

Different pathotypes of E. coli carry the yersiniabactin "core" of the HPI (40). In addition to the yersiniabactin iron acquisition system, E. coli has the enterochelin system, with higher affinity for iron (10). Reportedly, isolated irp2-positive Y. pseudotuberculosis O:3 strains do not express siderophore activity on the CAS agar and lack the yersiniabactin receptor (4, 36). Therefore, one can envisage alternative functions of the HPI-encoded genes besides production of the yersiniabactin and iron uptake. Modulation of cellular host defense (1, 13) may be a complementary function of the yersiniabactin. Determination of alternative functions and mechanisms of HPI transfer will provide interesting insights into the evolution of genomic islands.