INTRODUCTION

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Plasmid-encoded gene products are required for full expression of virulence in many enteropathogenic bacteria, including those of the genera Shigella (53, 54) and Yersina (17, 20), as well as Salmonella (12, 33, 35, 47, 56). Nontyphoidal Salmonella serovars are important agents of gastroenteritis and can cause systemic infection, such as bacteremia (septicemia), in animals and humans. Many of these serotypes typically carry large plasmids which are essential to the production of systemic infection in animal models (21, 23). Although the virulence plasmids of these Salmonella strains are variable in size, ranging from 50 to 94 kb, their distribution is dependent on the serotype. For example, S. enterica serovar Choleraesuis, S. enterica serovar Enteritidis, S. enterica serovar Dublin, S. enterica serovars Gallinarum and Pullorum, and S. enterica serovar Typhimurium harbor the 50-, 60-, 80-, 90-, and 94-kb virulence plasmids, respectively.

Strains of serovar Typhimurium cured of the virulence plasmid are strongly attenuated in their subsequent spreading infection to the mesenteric lymph nodes, spleen, and liver (23), while the presence of the virulence plasmid of Salmonella does not appear to be required for bacterial adherence to and invasion of cultured eukaryotic cells or for colonization of the cecum or invasion of Peyer's patches in the mouse (24, 42). All of these virulence plasmids contain a highly conserved 8-kb region, which contains the spv (Salmonella plasmid virulence) locus that can confer complete virulence on a strain of serovar Typhimurium cured of the plasmid (25).

The spv region consists of spvR, a gene that encodes a transcriptional factor of the LysR family, and the spvABCD operon of structural genes (1, 2, 22, 25, 37). The spv operon is required for the systemic phase of disease in specific hosts, i.e., serovar Choleraesuis in pigs (15), serovar Dublin in cattle (39, 61), serovars Gallinarum and Pullorum in fowl (5, 6), and serovars Typhimurium and Enteritidis in mice (24, 33, 47). The importance of these genes for the establishment of a systemic infection by serovar Typhimurium has also been shown by in vivo expression technology, which has demonstrated that the genes are induced during infection of the animal (28), and by signature-tagged mutagenesis, which has identified them as essential virulence genes (29). Recently, it has been reported that SpvB is an ADP-ribosylating enzyme of an unknown host protein (49). However, the molecular functions of other Spv proteins have not yet been determined.

Other virulence-associated loci on the virulence plasmid of serovar Typhimurium include the pef (plasmid-encoded fimbria) operon, which has been implicated in bacterial adherence to intestinal epithelial cells and is required for fluid accumulation in infant mice (8, 19), and the rck (resistance to complement killing) gene, which encodes an outer membrane protein whose expression renders the bacteria host serum resistant (26, 27). In addition, a recent in vivo expression study of serovar Typhimurium using the gfp reporter gene has identified mig-5, a macrophage-inducible gene that codes for carbonic anhydrase. Insertional mutation in mig-5 resulted in a decrease in bacterial colonization in the mouse spleen, demonstrating that the gene product of mig-5 is a virulence factor (60). However, the presence of these genes in every serotype has not been proved and their role in pathogenesis remains unclear.

To establish virulence determinants of the large virulence plasmids of nontyphoidal Salmonella, we tried to determine the genetic organization of the 50-kb virulence plasmid, designated pKDSC50, of serovar Choleraesuis strain RF-1 (35). A detailed restriction map of this plasmid has already been made available (36). In addition, the spv region on pKDSC50 has been subjected to a detailed genetic analysis and sequenced (44-46). In this report, we present the entire DNA sequence of the 50-kb virulence plasmid, pKDSC50, from serovar Choleraesuis strain RF-1. The complete DNA sequence of the plasmid could provide important insight into the evolution and origin of the virulence plasmids of Salmonella serovars.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains used in this study are listed in Table 1. The 50-kb virulence plasmid pKDSC50 was isolated from serovar Choleraesuis strain 2N-3, an isogenic derivative of RF-1 cured of a 6.7-kb cryptic plasmid (35). The large virulence plasmids were prepared from Salmonella strains grown overnight at 37°C in Luria-Bertani medium and obtained by the method of Kado and Liu (34).

Subcloning for sequencing and DNA sequence. Library construction for DNA sequencing was based on the previously established restriction map of pKDSC50 (36). DNA fragments generated with the restriction endonucleases EcoRI and SalI were cloned into Escherichia coli DH5 (Gibco BRL, Grand Island, N.Y.) using the sequencing vector pBluescript II SK(+) (Stratagene, Heidelberg, Germany). Series of nested deletions were generated from each clone. Purified pBluescript II SK(+) templates were sequenced using cycle-sequencing reactions with fluorescein isothiocyanate-labeled forward and reverse primers (Amersham-Pharmacia Biotech, Piscataway, N.J.). Gaps including regions between EcoRI, SalI, and EcoRI and SalI fragments in the pKDSC50 molecule were amplified by PCR using the original plasmid template for pKDSC50. The PCR products were cloned into a pBluescript II SK(+) vector. To resolve the ambiguity in the sequence of pKDSC50, 15 sequence data were obtained by the primer-walking method using fluorescein isothiocyanate-labeled synthetic oligonucleotides designated from contig ends according to the method described above. All sequence samples were run on a DSQ-2000L sequencer (Shimadzu, Kyoto, Japan).

DNA sequence analysis and annotation. A 6.9-kb DNA sequence of pKDSC50, which contains IS630 (accession no. D10689) and spvRABC regions (accession no. E03417), was previously published by our group (44-46). This sequence was combined with sequences determined in this study to obtain a simple circular sequence of pKDSC50. Open reading frames (ORFs) were initially identified using GENETYX-Mac software (version 10.1) and a BLAST database of putative genes. For subsequent analysis, each ORF was compared to the current nonredundant protein database of the National Center for Biotechnology Information by using BLAST software through the Internet. Only ORFs encoding peptides of more than 50 amino acids were analyzed.

PCR and DNA-DNA hybridization. The primers for PCR amplification of genes between repB and repC on the large virulence plasmids of Salmonella serovars are listed in Table 2. Dot blot hybridization was carried out using the standard protocol. In the Southern hybridization test, approximately 100 ng of Sau3AI-digested plasmid DNA of each Salmonella strain was blotted onto a GeneScreen Plus membrane (NEN Life Science Products, Boston, Mass.) using a Bio-dot microfiltration apparatus (BioRad Laboratories, Hercules, Calif.). The blots were prehybridized in hybridization buffer containing 0.5 M Na₂HPO₄ (pH 7.2), 1 mM EDTA, and 7% sodium dodecyl sulfate at 60°C for 1 h and then hybridized overnight at 60°C with probe DNA that was made by PCR with gene-specific primers (Table 2) using pLT2, the large virulence plasmid of serovar Typhimurium strain LT2, as the template and fluorescein labeled using a random prime labeling and detection system (Amersham-Pharmacia Biotech). Hybridization reactions were detected with horseradish peroxidase-conjugated rabbit antifluorescein antibody and Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products) and used to expose X-ray film.

Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession no. AB040415 and AB041905.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

General overview. Large virulence plasmid pKDSC50 from serovar Choleraesuis strain RF-1 was isolated, purified, and finally cloned into the E. coli sequencing vector. Subsequently, the total nucleotide sequence of pKDSC50 was determined. The entire DNA sequence of pKDSC50 consists of 49,503 bp forming a circular plasmid. The nucleotide sequence from bp 501 to bp 6417, which contains IS630 and the spvRABC, was previously published (44-46). pKDSC50 contains 48 identified ORFs, which were searched against a nonredundant protein database summarized in Table 3. Of the 48 putative ORFs, 28 (58%) were known to be encoded by other virulence plasmids of Salmonella; 9 (19%) were homologous to genes of other conjugative plasmids, including F and R plasmids; and only 1 (2%) was an insertion sequence. Four (8%) ORFs which were predicted to encode truncated proteins were unrelated to Salmonella. The remaining six (13%) ORFs had no regions of significant homology to protein sequences in the current database. In addition to these ORFs, four noncoding elements were found on pKDSC50 (Fig. 1 and Table 3).

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Map of the whole pKDSC50 plasmid. The circle shows ORFs with their orientations denoted by their positions; ORFs outside the ring have a clockwise orientation: and those inside the ring have a counterclockwise orientation. ORFs encoding virulence-associated factors are indicated by red boxes; ORFs encoding proteins related to replication and plasmid maintenance functions are indicated by blue boxes; ORFs encoding proteins homologous to the tra operon are indicated by green boxes; and the IS element is indicated by a yellow box. For the nomenclature of the ORFs, see Table 3.

Virulence-associated genes. A BLAST search revealed several homologues to known virulence-associated genes which localized to the large plasmid derived from other Salmonella serovars. Among these were Spv proteins, Pef proteins, and other proteins essential for virulence, such as Mig-5, a carbonic anhydrase that is required for systemic infection in the mouse (60), and another possible virulence-associated regulatory protein, TlpA, which functions as a thermometer by regulating its own transcription according to temperature (32).

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Distribution of the pef and rck genes among selective Salmonella serovars. (A) The genetic organization of the pef operon on pLT2, the 94-kb virulence plasmid of serovar Typhimurium (19) is shown. Gene-specific PCR products obtained by using a set of primers listed in Table 2 are indicated as thin lines with each DNA size amplified. (B) The pef and rck genes were detected by PCR and DNA-DNA hybridization (Blot). In Southern dot hybridizations, fluorescein-labeled pefB, pefA, pefC, pefD, orf5, and rck gene-specific PCR products from pLT2 of serovar Typhimurium were used as probes. Note that PCR and Southern blot analyses yielded identical results, except in the case of the pefC gene of the virulence plasmid from serovar Choleraesuis strain AH1, which was PCR negative and Southern blot positive.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Comparison of genetic organization of the pef region from Salmonella serovars Choleraesuis, Typhimurium, and Enteritidis. On pKDSC50, a 6,880-bp DNA sequence downstream of pefD on pLT2 is completely deleted. On pS72, the virulence plasmid of serovar Enteritidis, orf5 was truncated and orf6 was replaced with a 1,296-bp DNA fragment unrelated to the pef operon (see text for details). The identities of amino acid sequences with the pef-, srg-, and rck-encoded proteins are given as percentages.

Replication and plasmid maintenance. Three potential plasmid replication regions were found, one resembling the RepFIIA replicon, another resembling the RepFIB replicon, and the third containing genes involved in stable maintenance in the host. The first replication region showed very high homology to the repB (RepFIIA) region of serovar Enteritidis plasmid pFM82139 (accession no. U64796) (52) in the translated ORFs (99% identical to RepA, 100% identical to Tap and CopB). Tap is necessary for the translation of repA through translational coupling (9), and CopB is involved in plasmid copy number control. The same sequence showed high similarity to the RepFIIA replicon of plasmids pB171, the enteropathogenic E. coli (EPEC) adherence factor plasmid of EPEC strain B171 (O111:NM) (accession no. AB024946) (59); pO157 of EHEC strain O157:H7 (AB011549 and AF074613) (10, 41); pYVe227 of Yersinia enterocolitica (AF102990); pCD1 of Y. pestis (AF074612) (31); and R100 (NR1) (NC002134). The second replication region exhibited 95% identity (96% similarity) to plasmid pFM82139 of serovar Enteritidis; 97% identity (100% similarity) to the RepFIB replicon of pB171, the EPEC adherence factor plasmid of EPEC; 91% identity (98% similarity) to pO157 of EHEC; and 62% identity (93% similarity) to pMT1 of Y. pestis (AF074611) (40) (Fig. 4). Outside of the encoding region of the RepFIB replicon, repeats B through J, which contained the consensus sequence 5'-ANATAAGCTGTAGNNNGNAAA-3', were also found on plasmid pKDSC50. Both the RepFIIA and RepFIB replication regions of serovars Typhimurium and Enteritidis, named repB and repC, respectively, have been proven to be functional replicons (51, 58). The third replication region contains genes necessary for stable maintenance of the plasmid. pKDSC50 contains the par region of the serovar Typhimurium virulence plasmid consisting of four loci, incR, parA, parB, and parS, which are required for incompatibility and partition (11), between bp 36888 and bp 40587. Another region composed of rsd, the trans-acting resolvase gene, and crs, the cis-acting resolution site, which encodes a multimer resolution function involved in plasmid stability (38), was found. In addition to these loci, the samAB operon was located downstream of the parAB operon (D90202), which has been shown to be involved in the mediation of UV mutagenesis (48). ORF15, which was located in the rsd-crs region, showed high similarity (99%) to the ccdB genes of the F plasmid (P05703). The ccd operon, which is responsible for postsegregational killing of segregant cells, consisted of two genes, ccdA and ccdB, on the F plasmid, but the corresponding region on the pKDSC50 plasmid did not contain a ccdA homologue, indicating that the ccd system of pKDSC50 is unlikely to be functional.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Primary structure of the RepFIIA and RepFIB regions. Comparison of the repB (RepFIIA) (A) and repC (RepFIB) (B) regions of pKDSC50 and the corresponding regions of other plasmids from entropathogenic bacteria. These regions included pFM82139 of serovar Enteritidis (accession no. U64796), the pB171 plasmid of EPEC (AB024946), and the pO157 plasmid of EHEC O157:H7 (AB011549), as well as the pMT1 (AF074611) and pCD1 (AF074612) plasmids of Y. pestis, the pYVe227 plasmid of Y. enterocolitica (AF102990), and the R100 (NR1) plasmid (NC_002134). ORFs are indicated by arrows under the DNA of plasmid pKDSC50. Nucleotide positions are indicated on the line. Homologous amino acid sequences of these plasmids are shown by thick lines with the percentages of similarity and identity under the arrow, indicating homologous regions in the nucleotide position in each sequence.

Transfer region. Although some virulence plasmids of serovar Typhimurium are mobilized by conjugation under inducing conditions (3), almost all of the Salmonella virulence plasmids, including pKDSC50, are known to be nonconjugative (50). Genes from bp 25817 to 34819 in pKDSC50 were found to be homologous to the tra region genes from the F and R100 (NR1) plasmids, which included finO, traX, traI, trbH, traD, traT, and traG. Only two genes, finO (75% identical and 97% similar to the R100 plasmid) and traT (91% identical and 98% similar to the R100 plasmid), were complete; all of the other genes were truncated. In addition, the region downstream of the traG gene, which contains the tra genes required for DNA transfer, was completely deleted. This suggests that pKDSC50 is defective in DNA transfer by conjugation.

IS element. pKDSC50 contains a single copy of IS630. However, the nucleotide sequence from bp 45662 to bp 48829 was highly homologous to the sequence downstream of the ntn operon (AF043544), which is involved in the catabolism of 4-nitrotoluene and toluene in Pseudomonas sp. strain TW3. ORF45 (RlgA) has 75% similarity to ORF4, which is, in turn, similar to Xanthomonas campestris transposase gene tnpR. Recently, it has been reported that RlgA is a member of the resolvase family of site-specific recombinases and that mutation in rlgA on the virulence plasmid from serovar Typhimurium did not affect the stability of the plasmid (43). ORF46 and ORF47 correspond to one-half of the N-terminal part and one-third of the C-terminal part of ORF3, respectively; ORF3 is similar to the Deinococcus radiodurans putative transposase. ORF48 showed strong homology (97% similarity) to ORF2, which is similar to a Pseudomonas syringae protein of unknown function. These observations raise the possibility that a DNA element of plasmid pKDSC50 was acquired from Pseudomonas through horizontal transfer of a mobile element.

Base distribution. In addition to the presence of sequences related to different mobile genetic elements, the mosaic nature of pKDSC50 was further indicated by the results of a base composition analysis of the plasmid. Although the average G+C content of the plasmid was 52.1%, which is close to the value of the Salmonella chromosome, a more detailed analysis revealed that the region of the plasmid containing the spv operon had a significantly different G+C content (45.7%) than the surrounding region of the DNA (Fig. 5). Moreover, the G+C content of the predicted coding regions ranged from 39 to 70%, suggesting that development of the pKDSC50 plasmid may occur through the acquisition of DNA fragments from various microorganisms whose DNAs have a lower or higher G+C content.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   G+C content of pKDSC50 with a window of 100 bp across. The line indicates 50% G+C content, and selected ORFs are shown as hatched boxes drawn to the correct scale. The graph was generated using the GENETYX-Mac program (Software Development Co., Ltd., Tokyo, Japan).

Conclusions. The complete sequence of pKDSC50, the 50-kb virulence plasmid of serovar Choleraesuis, reveals a plasmid size unique to each Salmonella serovar. Comparison of the nucleotide sequences determined for the 93,939 bp of pLT2, the virulence plasmid of serovar Typhimurium strain LT2, obtained from the Salmonella genome database (Genome Sequencing Center, Washington University), and the 49,503 bp of pKDSC50 showed that 47 out of the 48 ORFs of pKDSC50 are highly homologous to the corresponding ORFs of pLT2 and that both plasmids contain the genes in the same order. Furthermore, two large deletions downstream of the pef region and most of the tra region were present on pKDSC50 (Fig. 6). This suggests that the virulence plasmid of serovar Choleraesuis is a variant of the virulence plasmid of serovar Typhimurium and was generated by deletion events that occurred during their divergence from a common predecessor. In addition, all genes of the 60-kb virulence plasmid of serovar Enteritidis thus far detected, and their genetic orders, are identical to these plasmids (13, 52), suggesting that the virulence plasmids from serovars Typhimurium, Choleraesuis, and Enteritidis share a common ancestry.

View larger version (17K): [in this window] [in a new window]   FIG. 6.   Linear genetic maps of the virulence plasmids pLT2 of Salmonella serovar Typhimurium and pKDSC50 of serovar Choleraesuis. The nucleotide sequence of pLT2 was obtained from the Salmonella genome database (http://genome.wustl.edu/gcs/). Areas of pKDSC50 that are not present in plasmid pLT2 are shown as open bars. The nucleotide positions of the deletions in the pKDSC50 sequence and the corresponding positions in pLT2 are indicated. The positions of genes are shown above the plasmids.