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Journal of Bacteriology logoLink to Journal of Bacteriology
. 2003 Sep;185(17):5055–5065. doi: 10.1128/JB.185.17.5055-5065.2003

Composition, Acquisition, and Distribution of the Vi Exopolysaccharide-Encoding Salmonella enterica Pathogenicity Island SPI-7

Derek Pickard 1,*, John Wain 2, Stephen Baker 1, Alexandra Line 3, Sonia Chohan 3, Maria Fookes 3, Andrew Barron 3, Peadar Ó Gaora 1, José A Chabalgoity 4, Niren Thanky 1, Christoph Scholes 1, Nicholas Thomson 3, Michael Quail 3, Julian Parkhill 3, Gordon Dougan 1
PMCID: PMC180996  PMID: 12923078

Abstract

Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNApheU sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNApheU site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNApheU site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNApheU-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.


Salmonella enterica subspecies I serovar Typhi is a host-adapted, human-restricted pathogen that causes typhoid fever (38). In addition to some isolates of S. enterica serovar Paratyphi C (11), S. enterica serovar Dublin, and Citrobacter freundii (35), most clinical isolates of S. enterica serovar Typhi express the Vi exopolysaccharide (23, 41). In contrast to the human host-restricted taxon S. enterica serovar Typhi, S. enterica serovar Paratyphi C is pathogenic for both humans and other animal species (30). S. enterica serovar Dublin Vi-positive isolates have been found mainly associated with cattle, while C. freundii is rarely associated with pathogenicity and the role of the Vi antigen in this species is equivocal. Vi expression is associated with a cluster of 10 genes located at position 4409519 on the S. enterica serovar Typhi chromosome, known as the viaB operon (29, 37), that comprises both Vi antigen biosynthetic genes (tviB to tviE) and export genes (vexA to vexE). Vi expression is under control of the rcsB-rcsC (2, 24) and ompR-envZ (39) two-component regulator systems that lie outside the pathogenicity island. The regulators in turn interact with the first gene of the viaB gene cluster, tviA (48), and regions upstream of the tviA promoter. The DNA sequences of viaB in S. enterica serovar Typhi strains CT18 (37) and Ty2 (21), as well as a Vi-positive isolate of C. freundii (accession number AF316551), have been determined. The viaB operon in S. enterica serovar Typhi resides on a 134-kb pathogenicity island that is located between the partially duplicated copies of the pheU tRNA gene, which has been designated Salmonella pathogenicity island 7 (SPI-7) (37).

There are multiple genes encoded within SPI-7 that are not directly associated with expression of the Vi capsule, but none of them are present in the genome of the completely sequenced strain S. enterica serovar Typhimurium strain LT2 (20, 31). However, little else is known about the conservation of SPI-7 in Vi-positive serovars or indeed about possible related or unrelated DNA insertions at the tRNApheU site in Vi-negative serovars. It is known that tRNA genes are common sites for insertion of horizontally acquired DNA in S. enterica, but little is known about how variable such tRNA-associated DNA insertions are in other S. enterica serovars. The abilities of different S. enterica serovars or isolates to cause distinct disease syndromes are likely to be reflected in their genetic makeups, and so variability, especially in horizontally acquired regions, may have relevance for pathogenicity and host specificity. We present here a detailed comparison of the SPI-7 regions from representative isolates of several Vi-positive S. enterica strains belonging to different serovars. We also describe the distribution of DNA insertions in the tRNApheU region of the S. enterica chromosome from selected isolates from Salmonella Reference Collection B (SARB) (8) and a bioinformatic analysis which indicated that specific genomic regions from a variety of soil and plant bacteria, including a recently sequenced gene island in Pseudomonas aeruginosa, share a number of gene clusters with SPI-7.

MATERIALS AND METHODS

Bacterial strains and culture.

The S. enterica strains used in this study are listed in Table 1. The majority of the S. enterica strains were from SARB (8). Bacteria were routinely cultured in Luria-Bertani broth or on Luria-Bertani agar. S. enterica serotypes were determined by using typing sera from Murex (Dartford, United Kingdom), and biochemical traits were analyzed by using Analytical Profile Index (API 20E) strips obtained from BioMerieux (Marcy-l'Etoile, France).

TABLE 1.

Bacterial isolates

Tazon Strain Source
C. freundii ID 7821 (Vi positive) M. Popoff, Institut Pasteur, Paris, France
S. enterica serovar Agona SARB 1 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Anatum SARB 2 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Brandenburg SARB 3 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Cholerasuis SARB 4 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Decataur SARB 8 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Derby SARB 9 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Dublin SARB 12 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Dublin ID 1662K (Vi positive) M. Popoff, Institut Pasteur, Paris, France
S. enterica serovar Duisburg SARB 15 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Emek SARB 20 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Enteritidis SARB 16 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar S. Gallinarum SARB 21 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Haifa SARB 22 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Heidelberg SARB 23 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Indiana SARB 25 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Infantis SARB 26 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Miami SARB 28 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Montevideo SARB 30 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Muenchen SARB 32 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Newport SARB 36 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Panama SARB 39 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Paratyphi A SARB 42 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Paratyphi C SARB 48 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Paratyphi C ID 32K (Vi positive) M. Popoff, Institut Pasteur, Paris, France
S. enterica serovar Pullorum SARB 51 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Reading SARB 53 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Rubislaw SARB 54 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Saintpaul SARB 56 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Senftenberg SARB 59 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Stanley SARB 60 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Stanleyville SARB 61 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Typhi SARB 63 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Typhi SARB 64 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Typhi TY2 Imperial College Collection, London, United Kingdom
S. enterica serovar Typhi CT18 Imperial College Collection, London, United Kingdom
S. enterica serovar Typhimurium SARC1 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Typhimurium SL1344 Imperial College Collection, London, United Kingdom
S. enterica serovar Typhimurium LT2 Imperial College Collection, London, United Kingdom
S. enterica serovar Typhisuis SARB 70 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Wien SARB 71 Salmonella Genetic Stock Centre, Calgary, Canada
S. enterica serovar Wien SARB 72 Salmonella Genetic Stock Centre, Calgary, Canada

Oligonucleotides and PCR conditions.

Table 2 shows the PCR oligonucleotides designed to investigate the structure of SPI-7 from various serovars of S. enterica. PCR was performed by using an Expand high-fidelity PCR kit (Roche, St. Albans, United Kingdom) for fragments smaller than 10 kb and DNA polymerase XL PCR kits (Perkin-Elmer, Branchburg, N.J.) for PCR fragments larger than 10 kb. The locations of the oligonucleotides in SPI-7 of S. enterica serovar Typhi strain CT18 are shown in Table 2. Where possible, the PCR primers used to amplify the PCR fragments were chosen to give some degree of overlap between the fragments generated. This allowed production of contiguous segments covering much of the SPI-7 region for the Salmonella serovars examined. Oligonucleotides pairs were designed for PCR by using the MacVector 7.1 program (Accelrys Ltd., Cambridge, United Kingdom). These oligonucleotides were based on DNA sequences within genes when possible. Genome sequences from other S. enterica serovars were obtained from Washington University, St. Louis, Mo. (http://genome.wustl.edu/projects/bacterial/).

TABLE 2.

Oligonucleotides used to obtain and analyze the regions of SPI-7 from S. enterica serovars Dublin and Paratyphi C by using PCR fragments

Target DNA covered by PCR oligonucleotide pairs Primera Primer sequence (5′-3′)
phoN-cut3A SB001 (F) GCATATCACTCTGCTTTGCCCGAACTC
SB002 (R) GCCGATACTACGCTGGAAATGGTGGC
tRNApheU region-cut3A DE101 (F) CGCAAAGTGTGCTGGGTCATTCAAAC
DE102 (R) AGGCAGATGGAACTGGTGCTGGAAGG
tviA-tRNApheU DE032 (F) CCAATTCTTTTCTCCAGCGATACATAGTC
DE033 (R) GCTCAGTCGGTAGAGCAGGGGATT
samB-vexE DE076 (F) GCCCTCAAACTGCCGCCATTCCTTAGAC
DE079 (R) GCGACTGTTGCTGACTTAGCCGAG
traC-samA CS100 (F) CCACGCCTCTTTATTGCCGAAGCC
CS101 (R) CCATAGCATCCATTCCTGAAGCAC
traG-traC DE063 (F) GAGACCTATGCCGAGAAGTTACTGAC
DE064 (R) CTCAAGCTCACTGCACTGATACTCTC
traG region DE105 (F) TCAGTGCCCGTTATAATGCAGTCG
DE106 (R) CCGCAGAAGCAACTTCAGAATCTG
pilNa-traG DE065 (F) CAGATGAACCGCCAGGTCAGGCTC
DE066 (R) AGCGTTATTTCCTTCTCCCCCAAG
phoN-pilNa CS104 (F) TTGCCGAAGCAGTAGCGTAGTAGC
CS105 (R) GCTGAATCCAGAATAGATACCGACG
rci-pilR (primers designed to confirm S. enterica serovar Paratyphi C deletion in this region) CS106 (F) GATGTGAACGGAAAAAAACGGGACGCAC
CS107 (R) ATCTCTGATGGTCAGTGGGGACGGGGC
cut3A/tRNApheU-int2 (primers used to analyze S. enterica serovars Typhi and Dublin/Paratyphi C in this region) CS108 (F) TTTTATCCCGACCCAACCCATCCTATTC
CS109 (R) ATCAGTCCTCATCGTTTCAGACACACGC
viaB operon region 1-yjhP (STY4650)-tviD DE028 (F) GCCACACTATTTTCGCCCCTGCCAGGA
DE029 (R) GCTCATCCTGAGAACCCGCCAGACTG
viaB operon region 2-tviD-helD (STY4664) DE026 (F) GCCATGAGTCTGAAGCCAGGAGGAATT
DE027 (R) GCTGGAACCGTCATTCTATCCCGTAGT
samA-samB (primer pair designed to confirm the presence or absence of the SopE phage) DE078 (F) TCCCATAGCATCCATTCCTGAAGCACT
DE079 (R) GCCTCAAACTGCCGCCATTCCTTAGA
a

F, forward; R, reverse.

Sequencing, DNA alignment, BLASTP analysis, and annotation.

Genomic DNA of S. enterica serovar Paratyphi C and S. enterica serovar Dublin for use in PCRs were prepared by using the method of Hull et al. (26). DNA from the various Salmonella serovars examined were routinely prepared by using the cetyltrimethylammonium bromide method for chromosomal DNA (3). PCR-generated DNA fragments obtained by using oligonucleotides based on SPI-7 sequences from different DNA templates were randomly fragmented by sonication, and DNA that was approximately 1.5 kb long was gel purified and cloned into pUC18. The cloned DNA was sequenced in both directions by using pUC18-based primers. The coverage was at least threefold for all sequences. The sequences were assembled by using GAP4 (6) to produce contiguous sequences for each PCR product. These PCR-generated fragments were eventually aligned to cover most of the SPI-7 regions by using MacVector 7.1 alignment tools (Accelrys Ltd.). Analysis of SPI-7 sequences was performed with ARTEMIS (43) and the BLASTP database search program (1). For some of the work described here we used preliminary sequence data obtained from the DOE Joint Genome Institute (http://www.jgi.doe.gov/JGI_microbial/html/index.html).

Nucleotide sequence accession number.

The nucleotide sequence of the C. freundii recJ-tviA region has been deposited in the GenBank database under accession number AY282413.

RESULTS

Characterization of the SPI-7 region of S. enterica serovar Typhi.

Vi polysaccharide expression in S. enterica serovar Typhi is associated with the viaB operon encoded on a pathogenicity island known as SPI-7 (37). SPI-7 is a 134-kb DNA insertion in duplicated tRNApheU sequences between positions 4409574 and 4543073 on the S. enterica serovar Typhi strain CT18 chromosome (20). The intact tRNApheU sequence (positions 4543073 to 4543148) is 75 bp long, while the truncated tRNApheU sequence (positions 4409519 to 4409574) lacks the first 20 nucleotides. The viaB operons from two Vi-positive S. enterica strains, S. enterica serovar Typhi strain CT18 (37) and S. enterica serovar Typhi strain Ty2 (21), and a Vi-positive isolate of C. freundii (accession number AF316551) have been sequenced previously.

The phoN gene, STY4519, defines the left boundary of SPI-7 and is adjacent to the truncated tRNApheU and oriT remnant. The 12-kb DNA segment upstream of this truncated tRNApheU (including ΔoriT and the region between STY4503 and STY4519) probably represents a region horizontally transferred earlier, as originally proposed by Groisman et al. (19), since this small gene cluster is present in all Salmonella subspecies 1 strains tested by Porwollik et al. in microarrays, while it is absent from a variety of enteric bacteria examined, including Escherichia coli K-12 and O157:H7 (40).

The gene content of SPI-7 can be divided into distinct regions. Lying between the truncated tRNApheU and STY4536 (ssb) (Fig. 1A) are a number of genes that are likely to encode functions related to conjugal transfer of DNA, DNA replication, or transposition. For example, STY4536 (ssb) encodes a putative single-stranded DNA binding protein, and STY4533 (topB) encodes a putative topoisomerase B. From STY4537 to STY4553 (pilK) (Fig. 1B) are several previously characterized genes encoding a type IVB pilus system with a role in attachment to eukaryotic cells (50). The type IVB pilus system may originally have served as a mating pair formation cluster for a conjugative plasmid or conjugative transposon and is similar to the related gene cluster of the R64 plasmid (49).

FIG. 1.

FIG. 1.

Detailed annotation of six regions of SPI-7 from S. enterica serovar Typhi strain CT18. (A) phoN-ssb region, including the truncated tRNA and the adjacent phoN gene along with STY4517 and STY4518, which show homology to genes present in SPI-7. (B) pil locus, which encodes a type IVB pilus system and extends from the pilL gene to the pilK gene. (C) STY4553 to STY4596. This region harbors a number of genes that show identity to related genes involved in DNA transfer. (D) SopE bacteriophage, a P2-type bacteriophage located within the samA gene. The CDSs of this phage are shaded to highlight the region. The genes represented by black arrows represent the tail region; the genes represented by arrows with black stripes encode the lysis proteins of the phage; the genes represented by dark grey arrows encode capsid or head functions; the genes represented by light grey arrows (STY4629 to STY4632) are genes of foreign origin and have a lower G+C content than the surrounding genes; the genes represented by arrows with light grey stripes encode the regulatory functions of the phage. The cos site is located between STY4532 and STY4533 at coordinates 4499001 to 4499019 and has the sequence GGCGTGGCGGGGATACGAG. (E) viaB operon encoding the genes required for production and export of the Vi polysaccharide. Note the presence of the IS1 element. (F) STY4663-cutA3 region encoding putative integrases (shaded), a 16-bp duplication of a section of tRNApheU, and the tRNApheU-cut3A SPI-7 junction. Note that the STY4667 and STY4668 genes are highly homologous to the STY4517 and STY4518 genes, respectively, located near phoN and are highlighted to emphasize this. The numbers associated with genes are the STY numbers used in the Sanger Institute annotation of the S. enterica serovar Typhi strain CT18 genome (accession number AL62783). The numbers above the regions indicate locations (in base pairs) on the S. enterica serovar Typhi strain CT18 chromosome, as indicated by the Sanger Institute annotation.

The region from STY4553 (pilK) to STY4598 (samB) (Fig. 1C) is predicted to encode at least 24 hypothetical proteins, as well as a number of genes with significant matches as determined by BLASTP analysis with DNA transfer genes, such as STY4554 (traE), STY4562 (traG), and STY4573 (traC). Finally, STY4592 (ardC) exhibits similarity to a protein with a putative role in the conjugative process itself as a DNA escorting protein in the incW plasmid, pSa (4). A gene encoding an ArdC homologue has also been found in the symbiosis island of Mesorhizobium. loti (46). This region, along with the pil locus and the region located between STY4539 (pilL) and the truncated tRNApheU, hint that there is an integrated functional role for the entire locus in conjugation and DNA transfer. This region in SPI-7 is possibly derived from a conjugative plasmid with similarity to R64.

A bacteriophage encoding the SPI-1 effector protein, SopE (Fig. 1D), is inserted in the samA gene (32). Analysis of the SopE phage DNA sequence showed significant DNA sequence and coding sequence (CDS) similarity to the available sequence of phage Retron Ec67. That Retron Ec67 phage belongs to the P2/186 family of bacteriophages (13) and was originally isolated from E. coli strain CL-1 (25). Detailed analysis of the SopE phage sequence has revealed that many of the promoter sites regulating lysogenic and lytic functions are still present (14), which indicates that the phage may still be capable of excision from SPI-7. Additionally, the attachment site for the SopE phage has been determined to be duplications on either side of the phage insertion (attR at coordinates 4507385 to 4507393 and attL at coordinates 4473831 to 4473839) (34). A lexA binding site, characteristic of phage 186, has also been found in the SopE phage at coordinates 4499689 to 4499708. This site plays a role in 186 prophage induction (27). Four CDSs with low G+C contents are located adjacent to the cos site (cohesive ends) of the SopE phage, a site where foreign DNA is commonly inserted into phages of the P2 family (33). After the viaB operon (Fig. 1E) and between STY4663 and STY4680 are many genes whose functions are unknown. The genes encoding two candidate integrases, STY4666 and STY4680, were identified within this region of SPI-7 (Fig. 1F). The integrase encoded adjacent to the 3′ tRNApheU, STY4680 (int), shows similarity to integrases encoded by the P4 family of bacteriophages (54% identity over 416 amino acids to a P4-like integrase identified in E. coli CFT073, for example). The gene encoding the other candidate integrase, STY4666 (int2), which is nearer the viaB locus, is more similar to the gene encoding an integrase found in Actinobacillus actinomycetemcomitans. A 16-bp duplication of the last bases of tRNApheU, alongside the STY4666 gene, is indicative of further recombination events in S. enterica serovar Typhi strain CT18.

Two genes adjacent to STY4666 (int2), STY4667 and STY4668, are also worthy of note since they are highly similar to genes found just outside the truncated 5′ tRNApheU (Fig. 1A and Table 3), STY4517 and STY4518, respectively. Additionally, in Shigella flexneri plasmid pWR100 (9), genes with identity to STY4667 and STY4668 are present as similar duplications on either side of a known Shigella pathogenicity region (between the gene duplications of orf46/47 and orf85a/85b in pWR100) (16, 47). Genes similar to STY4667 and STY4668 have also been found to be associated with other well-characterized horizontally transferred DNA, including SPI-1 (36).

TABLE 3.

Selected genes showing synteny between SPI-7 and X. axonopodis pv. citri (accession number AE011859) or R. metallidurans (accession number NZ_AAA101000352)

Gene(s) Location (coordinates) Product size (amino acids) Function % Amino acid identity(ies) (range)a
tRNApheU (truncated) 4409519-4409574
STY4521 4409652-4410686 344 parB-like partition protein 41 (286 aa to XAC2205), 39 (289 aa to Reut4191)
STY4523 4412040-4413839 599 Hypothetical 31 (580 aa to XAC2206), 30 (600 aa to Reut4193)
STY4526 4415107-4415664 185 Hypothetical 32 (173 aa to Reut4194), 28 (165 aa to XAC2207)
STY4528 4415909-4417252 447 Hypothetical 32 (405 aa to Reut4195), 29 (399 aa to XAC2208)
STY4529 4417838-4418617 259 Hypothetical 30 (163 aa to XAC2209), 29 (227 aa to Reut4196)
STY4530 4418728-4420722 664 topB topoisomerase B 50 (675 aa to XAC2212), 49 (677 aa to Reut4199)
STY4534 4421390-4421839 149 Hypothetical 38 (136 aa to Reut4221), 33 (128 aa to XAC2236)
STY4535 4421920-4422138 72 Hypothetical 56 (65 aa to XAC2217)
STY4536 4422151-4422687 178 ssb single-stranded DNA-binding protein 60 (174 aa to pWWO Ssb), 68 (162 aa to P1 phage Ssb-p1)
STY4539 4423868-4425112 414 pilL membrane-located open reading frame 46 (126 aa to XAC2253), 38 (165 aa to Reut4232)
STY4554 4437266-4438141 291 traE (putative) 23 (219 aa to R64 plasmid open reading frame TraE)
STY4557 4438648-4439562 304 Hypothetical membrane protein 25 (170 aa to Reut4233)
STY4558 4439581-4440321 246 Hypothetical 40 (227 aa to XAC2255), 37 (237 aa to Reut4234)
STY4559 4440435-4440986 183 Hypothetical 44 (141 aa to XAC2256), 42 (148 aa to Reut4235)
STY4560 4441022-4441522 166 Hypothetical 41 (160 aa to XAC2257), 39 (153 aa to Reut4236)
STY4561 4441532-4442101 189 Hypothetical 51 (213 aa to NP_511181 [R46 plasmid])
STY4562 4442121-4444211 696 traG-like (DNA transfer region-associated gene) 65 (541 aa to XAC2259), 61 (709 aa to Reut4237), 26 (630 aa to TraG-like open reading frame in R27 plasmid)
STY4563 4444208-4444966 252 Hypothetical 42 (234 aa to XAC2260), 42 (228 aa to Reut4238)
STY4564 4445185-4445457 90 Hypothetical 42 (82 aa to Reut4247), 39 (81 aa to XAC2269)
STY4565 4445457-4445696 79 Hypothetical membrane-spanning domains 29 (77 aa to Reut4248)
STY4566 4445726-4446088 120 Hypothetical membrane-spanning domains 37 (107 aa to XAC2270), 35 (89 aa to Reut4249)
STY4567 4446098-4446472 124 Hypothetical 30 (90 aa to XAC2271)
STY4568 4446469-4447122 217 Hypothetical exported protein 54 (219 aa to Reut4250)
STY4569 4447122-4448021 299 Hypothetical 50 (255 aa to Reut4252), 44 (292 aa to XAC2272)
STY4570 4448011-4449489 492 Hypothetical exported protein 38 (513 aa to XAC2273)
STY4571 4449482-4449925 147 Hypothetical lipoprotein 47 (127 aa to Reut4254)
STY4572 4449922-4450341 139 Hypothetical 50 (70 aa to XAC2274), 45 (108 aa to Reut4255 [all N-terminal end only])
STY4573 4450338-4452761 807 traC-like (putative Dtr protein) 52 (817 aa to XAC2274), 51 (815 aa to Reut4255), 20 (354 aa to Virb4 Dtr protein from Helicobacter pylori)
STY4575 4453457-4453849 392 Hypothetical 36 (113 aa to XAC2282), 36 (125 aa to Reut4258)
STY4576 4453846-4454814 323 Hypothetical 49 (320 aa to XAC2283), 48 (320 aa to Reut4259)
STY4577 4454829-4456259 476 Hypothetical 41 (402 aa to XAC2284), 40 (439 aa to Reut4260)
STY4579 4456592-4458097 501 Hypothetical membrane protein 45 (499 aa to XAC2286), 42 (510 aa to Reut4262)
STY4583 4459393-4460640 415 ibrA regulatory protein (previously ybdN) 85 (40 aa to IbrA from EcoR-9), 84 (415 aa to IbrA [Z1203 and Z1643] from O157:H7 EDL933)
STY4584 4460652-4461266 204 IbrB regulatory protein (previously designated ybdM) 78 (203 aa to IbrB from EcoR-9), 78 (204 aa to IbrB [Z1204 and Z1644] from O157:H7 EDL933)
STY4585 4461477-4462019 180 Hypothetical 74 (178 aa to STY4858), 74 (178 aa to STM4502)
STY4592 4467675-4469624 649 ardC (DNA escorting protein anti- restriction) 34 (282 aa to ArdC from Mesorhizobium loti symbiosis island)
STY4593 4469696-4470604 302 Hypothetical 33 (279 aa to STY4594)
STY4594 4470678-4471577 299 Hypothetical 33 (279 aa to STY4593)
STY4595 4471619-4471978 119 Hypothetical 41 (105 aa to XAC2221), 36 (74 aa to Reut4219), 38 (76 aa to Orf23 of plasmid pCTX-M3)
STY4599-STY4645 4473830-4507389 SopE phage (S. enterica serovar typhi only)
STY4630 4496774-4497838 354 Hypothetical within A-T-rich region of SopE phage 35 (363 aa to S060 of SXT element from Vibrio cholerae)
STY4631 4497835-4498899 354 Hypothetical within A-T-rich region of SopE phage 40 (356 aa to S061 of SXT element from Vibrio cholerae)
STY4649 4508645-4509535 296 Putative 3-methyltransferase 75 (288 aa to STY4856)
STY4651-STY4662 4510583-4524679 ViaB operon
STY4664 4525533-4527044 503 helD DNA helicase 39 (472 aa to Orf193 of plasmid Rts1)
STY4665 4527028-4528617 529 Hypothetical 29 (451 aa to XAC2196), 28 (449 aa to Reut2852)
STY4666 4528781-4529794 337 int2 integrase 35 (265 aa to integrase from Actinobacillus actinomycetemcomitans)
Truncated tRNApheU 4529973-4529988
STY4667 4530221-4530514 97 Similar open reading frames often associated with transposon elements 98 (97 aa to STY4517), 98 (97 aa to STM4317), 26 (96 aa to Rv0918 of Mycobacterium tuberculosis)
STY4668 4530511-4530999 162 As for STY4667 92 (162 aa to STY4518), 92 (162 aa to STM4318), 50 (156 aa to Rv0919)
STY4673 4537312-4537533 73 ner-like Mu DNA binding 66 (68 aa to Ner open reading frame of Mu phage)
STY4680 4541654-4542913     (complement) 419 int integrase 54 (416 aa to P4-like prophage integrase from Escherichia coli CFT073)
tRNApheU (full length, 75 bp) 4543148-4543073
a

aa, amino acids. The designations of the CDSs of X. axonopodis pv. citri begin with XAC, and the designations of the CDSs of R. metallidurans begin with Reut.

Comparison of the viaB-associated DNA found in Vi-positive S. enterica serovar Dublin and S. enterica serovar Paratyphi C and identification of the viaB insertion site in the C. freundii genome.

Relatively few S. enterica serovars or isolates express Vi polysaccharide, and for those that do it is not known whether the Vi locus is also harbored on an SPI-7-related element. To address this question, the regions flanking the viaB operon were obtained by PCR from S. enterica serovar Paratyphi C Vi-positive strain 32K and S. enterica serovar Dublin Vi-positive strain 1622K by using PCR primers based on the SPI-7 region of S. enterica serovar Typhi (Table 2). PCR fragments generated from both S. enterica serovar Paratyphi C and S. enterica serovar Dublin by using these primers were subjected to DNA sequencing. Both S. enterica serovar Paratyphi C strain 32K and S. enterica serovar Dublin strain 1622K were found to encode the viaB operon associated with significant stretches of DNA showing similarity to SPI-7 (Fig. 2). Additionally, the SPI-7 regions of both S. enterica serovar Dublin and S. enterica serovar Paratyphi C, like that in S. enterica serovar Typhi, were inserted in duplicated tRNApheU sites and were over 99.5% identical to the equivalent regions of S. enterica serovar Typhi strain CT18. S. enterica serovar Typhi strain CT18 contains an additional 15 kb of DNA located in the region bordered by the STY4678 integrase gene (int) near tRNApheU and the gene encoding the other integrase STY4666 (int2) compared to the sequences of both S. enterica serovar Dublin and S. enterica serovar Paratyphi C. Consequently, the corresponding PCR product generated from S. enterica serovar Paratyphi C or S. enterica serovar Dublin was smaller (5.8 kb) than the 14-kb PCR product amplified from S. enterica serovar Typhi (Fig. 1). PCR analysis also demonstrated that SPI-7 of S. enterica serovar Paratyphi C and SPI-7 of S. enterica serovar Dublin lack the SopE phage that disrupts the samAB locus of SPI-7 of S. enterica serovar Typhi. The DNA sequence encoding the type IVB pilus cluster is intact in S. enterica serovar Dublin, but a deletion is present in S. enterica serovar Paratyphi C strain 32K between the STY4552 rci and STY4547 pilS genes (Fig. 2). Loss of these genes would result in possibly deleterious effects on pilus assembly. The remaining DNA sequences showing similarity to the S. enterica serovar Typhi SPI-7 sequence are virtually identical (over 99%) to each other in the three serovars.

FIG. 2.

FIG. 2.

SPI-7 of S. enterica serovar Typhi and the related SPI-7 regions of other S. enterica serovars, showing the sites of the PCR primer pairs used in relation to the S. enterica serovar Typhi strain CT18 sequence and the resulting products obtained, as indicated by dashed lines. (A) SPI-7 region from S. enterica serovar Typhi with the areas of interest labeled, including the gene clusters encoding the viaB exopolysaccharide, the SopE bacteriophage, and the type IVB pilus locus. (B) SPI-7 region in S. enterica serovar Dublin strain 1622K, which is notably lacking the region between int1 and int2 and also the SopE bacteriophage. (C) SPI-7 in S. enterica serovar Paratyphi C isolate 32K. (D) tRNApheU region in S. enterica serovar Typhimurium strain LT2 harboring a gene resembling merR at the phoN (upstream) end of this tRNA. (E) Intact tRNApheU as found, for example, in S. enterica serovar Enteritidis, with no additional DNA inserted between the cutA3 and phoN genes.

In C. freundii, the viaB operon is inserted at a different tRNA locus, tRNAglyU, based on our analysis of the flanking DNA (GenBank accession number AY282413). The gene order 4 kb upstream of this tRNAglyU site resembles that in a number of previously sequenced enteric bacteria, including S. enterica serovar Typhi strain CT18. The presence of the C. freundii viaB gene adjacent to tRNAglyU is of interest as this is the site where the P. aeruginosa SG17M genetic island is inserted, in contrast to the sequenced strain P. aeruginosa PAO1 (28); additionally, significant parts of this island show remarkable synteny with gene clusters in SPI-7, as described in detail below and shown in Fig. 3.

FIG. 3.

FIG. 3.

Synteny between sections of SPI-7 and related regions in the genomes of other bacteria. The diagram was constructed by using The Sanger Institute programs Artemis and ACT in conjunction with TBLASTX protein-versus-protein analysis. The synteny among S. enterica serovar Typhi strain CT18 SPI-7, X. axonopodis pv. citri, and P. aeruginosa SG17M gene island PAGI-3 is illustrated. The accession numbers are AL62783, AE008923, and AF440524, respectively. The grey shading indicates CDSs exhibiting 25 to 44% amino acid identity, and the black boxes indicate CDSs with 45 to 70% amino acid identity. The gene designations for traC, traG, and parB are putative designations with respect to SPI-7 in S. enterica serovar Typhi. The cargo genes of the P. aeruginosa SG17M island are not shown, but the gene designations are SG01 to SG47. The term cargo gene is used in the context suggested by Larbig et al. (28) and refers to the variable region on the gene islands that include a range of metabolic enzyme gene clusters in P. aeruginosa and viaB in S. enterica serovar Typhi. The cargo regions occupy similar locations in both islands with respect to the putative Dtr regions and the integrase gene adjacent to one of the tRNA duplications and a parB-like gene at the other end.

Analysis of SPI-7 indicates the presence of regions of synteny with bacteria of soil and plant origin, including Xanthomonas axonopodis pv. citri, Burkholderia fungorum, and P. aeruginosa.

In the original annotation, a number of regions of S. enterica serovar Typhi strain CT18 SPI-7 yielded few matches in database searches (37). These regions were reexamined by using the ARTEMIS program to discover if any new data would shed light on their origin and function. The presence of duplicated tRNApheU sites on either side of SPI-7 hinted that this pathogenicity island may have originally been a mobile element similar to 100-kb conjugative transposon CTnscr94, identified in S. enterica serovar Senftenberg strain 5494-57, which may also be located within tRNApheU (22). From database searches, it became evident that a large number of genes from both saprophytic bacteria and phytobacteria showed homology and were syntenic with specific regions in SPI-7 (Fig. 3 and Table 3).

A large number of these matches were to genes of unknown function in the region between the STY4597 (samB) and STY4552 (rci) genes of SPI-7. The close synteny extended to the region lying between STY4539 (pilL) and STY4521, the first gene downstream of the truncated tRNApheU-phoN junction (Fig. 1A). The soil bacteria and phytobacteria which have DNA sequences which exhibit synteny with regions of SPI-7 include the plant-adapted organism X. axonopodis pv. citri (12) and, to a lesser extent, Xylella fastidosa (15, 45), as well as Pseudomonas fluorescens, B. fungorum, and Ralstonia metallidurans (also called Ralstonia eutrophus). Significantly, gene islands recently identified in two isolates of P. aeruginosa (28) also showed matching synteny with these same regions in SPI-7. These isolates were obtained from a cystic fibrosis patient (P. aeruginosa strain C) and an aquatic environment (P. aeruginosa strain SG17M) (42). Similar synteny with SPI-7 was also observed with Haemophilus somnus strain 129PT (a bacterium found in the respiratory or genitourinary tract of cattle).

Figure 3 shows the extensive synteny of these regions from X. axonopodis pv. citri and P. aeruginosa SG17M with S. enterica serovar Typhi SPI-7, while Table 3 presents the synteny analysis data. For X. fastidosa, the synteny observed with SPI-7 matched that described by Larbig et al. for the P. aeruginosa SG17M and C islands (28).

Distribution of DNA insertions in tRNApheU in other S. enterica serovars that do not express Vi exopolysaccharide.

In order to ascertain the frequency of insertions occurring at the tRNApheU site in a range of Vi-negative S. enterica strains, DNA prepared from 30 different serovars were analyzed by using oligonucleotide primers designed to generate PCR products across the site. The isolates selected were from SARB (Table 1). Initially, PCR primers were designed by using DNA sequences in the phoN gene (primer SB001) and the cut3A gene (primer SB002) at the 5′ and 3′ ends of SPI-7, respectively. Two serovars, S. enterica serovar Typhimurium and S. enterica serovar Saintpaul, both yielded 3.7-kb PCR products. This result supports multilocus enzyme electrophoresis data which showed that S. enterica serovar Typhimurium and S. enterica serovar Saintpaul are very closely related (44). S. enterica serovar Typhimurium strain LT2 harbors a single complete tRNApheU with the addition of a gene related to merR adjacent to and downstream of the phoN gene. The PCR products described here were the predicted size when they were compared to the previously published S. enterica serovar Typhimurium strain LT2 sequence (31).

The following four Vi-negative S. enterica isolates yielded no detectable PCR product: SARB 1 (S. enterica serovar Agona), SARB 9 (S. enterica serovar Derby), SARB 59 (S. enterica serovar Senftenberg), and SARB 61 (S. enterica serovar Stanleyville). To detect any possible insertion at this site, PCR primers based on DNA sequences internal to SPI-7 were used to try to amplify each region of SPI-7 separately. The results were negative, as were the results obtained with Southern probes for specific loci within SPI-7. Further analysis of these four isolates with PCR primers specific for a sequence farther away from the tRNA eventually suggested that the initial negative PCR was due to sequence variation at the tRNApheU-phoN junction and thus to failure of the PCR primers to bind. This possibility is being investigated further in order to clarify these observations. Twenty-four serovars yielded PCR products that were approximately 2 kb long, indicating that there was no insertion at the tRNApheU locus. Interestingly, one of these 24 serovars, represented by SARB 64, had previously been reported to be Vi positive (8). In our hands, however, serological analysis confirmed that the SARB 64 isolate described here was indeed Vi negative. Consequently, it is possible that the entire SPI-7 locus was precisely deleted during lab storage.

DISCUSSION

In this study we confirmed that the viaB operon in Vi-positive S. enterica serovars, including S. enterica serovar Typhi, S. enterica serovar Paratyphi C, and S. enterica serovar Dublin, is encoded on a common but mosaic pathogenicity island termed SPI-7. The viaB operon is of particular interest for several reasons. The ability to express Vi polysaccharide has an unusual and restricted distribution in S. enterica. In addition, purified Vi polysaccharide is currently used as a component antigen in human typhoid vaccines, and consequently the basis of acquisition and stability of the viaB operon is of immense practical interest. The viaB operon of S. enterica serovar Typhi strain CT18 is characterized by the presence of an intact IS1 element (Fig. 2E). Analysis of this IS1 element (see annotation data in accession number AL62783) revealed that it lies between the normally overlapping tviD and tviE genes (STY4659 and STY4656, respectively), leading to disruption of the Shine-Dalgarno ribosome binding site for tviE that is encoded within the last 13 bases of tviD (21). An 8-bp duplication is present on either side of the IS1 inverted repeats, as expected. An IS1 element has also been found to insert within a hot spot of the viaB region of C. freundii isolates and is responsible for the Vi-negative negative phenotype of such isolates (35), and it is possible that the Vi-negative phenotype which we observed for S. enterica serovar Typhi strain CT18 was also due to the presence of this IS1 element. In C. freundii the Vi phenotype is reversible, hinting that the IS1 element can be excised under some circumstances, and this may also be the case for Salmonella strains like S. enterica serovar Typhi strain CT18.

Sequencing of the viaB-associated DNA from Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin strains showed that these S. enterica serovars harbor an SPI-7 related element with at least 99% similarity at the DNA and protein levels but lack the SopE phage. The SopE phage may have been acquired by S. enterica serovar Typhi after the basic SPI-7 element was acquired. There were only a small number of other deletions or insertions that were evident from this comparison. At this time we do not know if these deletions or insertions are unique to the Vi-positive S. enterica serovar Paratyphi C or S. enterica serovar Dublin strains which we sequenced. S. enterica serovar Typhi strain CT18 possesses a unique 15-kb region upstream of the viaB operon which is thought to represent a further integration event and may explain the presence of two integrase genes in S. enterica serovar Typhi SPI-7. The integrase gene nearest the intact tRNApheU boundary of S. enterica serovar Typhi SPI-7 shows homology to the P4 phage family of integrase genes. The intact integrase gene (STY4666) that is approximately 15 kb into SPI-7 is present in all three serovars and is most similar to an integrase gene from A. actinomycetemcomitans. From these data we surmised that the original SPI-7 was most similar to that found in S. enterica serovar Dublin and that there was a common source of SPI-7 for all three serovars. The fact that the DNA sequences of the common regions of SPI-7 are so highly conserved suggests that SPI-7 acquisition by the three serovars was a relatively recent event. The very marked differences among the G+C contents of the regions of SPI-7 described here support the concept that the regions are mosaics, which were formed by serial acquisition of DNA. The G+C content of the viaB locus is 44% (Fig. 1E), and the G+C content of the region between the viaB locus and the intact tRNApheU is 47% (Fig. 1F). These regions in particular may have been acquired in the pathogenicity island later.

Our detailed analysis of the overall gene complement of SPI-7 provides additional evidence that SPI-7 was originally obtained by horizontal transfer, perhaps in the form of a conjugative transposon. The type IVB pilus (50) could have originally constituted the mating pair formation system for a conjugative transposon or plasmid, but many expected genes required for DNA transfer were not identified previously. We now believe that these genes may be located between the previously uncharacterized region bordered by samB and rci of SPI-7. The regions on each side of the pil locus contain both a putative DNA transfer system and genes potentially directly associated with single-stranded DNA during the conjugative process, such as ssb,ardC, and topB homologues. Putative traC and traG genes have been identified in the region that lies between samB and rci, as well as the hypothetical genes mentioned above. It is of interest that in the transfer region of the Bacteriodes conjugative transposon, CTnDOT (7), putative functions could be assigned to only two genes, traC and traG. This finding may be related to particular important domains, such as ATPase activity and the site of the gene products within the conjugative machinery of the host bacteria (e.g., as coupling proteins linking Dtr and Mpf functions in the conjugative apparatus).

The presence of other genes, such as samAB and parB, supports the putative origin of SPI-7 as a conjugative transposon that evolved from a conjugative plasmid by loss of plasmid replication genes (i.e., repA) and other genes for plasmid maintenance. The presence of intact samAB genes is of interest since these genes encode DNA repair enzymes, which are frequently found on plasmids, such as pLT, the virulence plasmid of S. enterica serovar Typhimurium strain LT2 (31). Putative DNA repair genes have also been found in the conjugative integrating element R391 (5) and in the SG17M island (radC) (28) of P. aeruginosa.

We also obtained evidence that many of the SPI-7 gene products encoded on either side of the pil locus exhibit significant levels of sequence similarity to predicted proteins from a number of soil bacteria and phytobacteria, including B. fungorum, R. metallidurans, P. fluorescens, P. putida plasmid pWWO, and P. aeruginosa, as well as the plant pathogen X. axonopodis pv. citri and its close relative X. fastidosa. The apparent similarity extends past the gene sequences as analysis revealed that many of the genes showing homology to SPI-7 were in clusters, which also shared a high level of conservation of gene order and orientation with SPI-7 genes. The similarity of many SPI-7 genes to genes from soil bacteria and phytobacteria raises the possibility that SPI-7 and viaB may have originated from such sources. These bacteria produce a wide range of exopolysaccharides, some of which are encoded on plasmids (18). Enteric bacteria could encounter some of the bacteria containing genes that exhibit gene synteny with SPI-7 in the gut via contaminated food (food containing soil residue, for example). It is known, for example, that the soil bacterium B. fungorum is capable of surviving and growing in an acid environment (10) and thus may be able to survive in the acidic contents of the stomach. Whatever the source is, the potential transmissibility of these islands is emphasized by their insertions in tRNA sites such as tRNApheU for SPI-7 and tRNAglyU for P. aeruginosa SG17M and in viaB for C. freundii. In these specific cases, an integrase is located near the tRNA site. This is also the case for the symbiosis island of M. loti that is located at a tRNAphe site (17, 46). It is known that phage integrases use tRNA genes as chromosomal attachment sites, and it is likely that these phage-derived integrases may perform the same function.

In summary, the synteny of SPI-7 with bacteria from soil and plants indicates that the SPI-7 regions may have a specific function related to DNA transfer, mobilization, and possible stabilization of the island and hence to the conservation seen. A common distant ancestor can also be postulated for these regions based upon the extensive synteny observed. This example of horizontal transfer in conjunction with recent studies with the ubiquitously adapted organism P. aeruginosa (28) underlies the diverse interactions possible between bacteria from environmental and enteric sources. The ongoing sequencing of saprophytic bacteria and phytobacteria will undoubtedly reveal more examples of such genetic exchange. Experiments are under way to examine the pilus locus of SPI-7 to assess if it can still act as a mating pore formation structure for any putative conjugative transposon. Further evidence that SPI-7 may be a functional element is provided by the observation that in S. enterica serovar Typhi strain SARB 64 SPI-7 is absent even though this isolate was originally described as a Vi-positive strain (8). The SARB 64 isolate which we have is Vi negative but is also definitely an S. enterica serovar Typhi isolate. We are currently investigating the genome of this organism in more detail using DNA microarrays.

The fact that Vi production is associated with a relatively unstable and potentially mobilizable element may have some consequences for the utility of Vi as an antigenic component of human typhoid vaccines. Vi production appears to have been a relatively recent acquisition by S. enterica. Interestingly, S. enterica serovar Typhi harbors mutations in three genes, wcaA, wcaD, and wcaK, associated with colonic acid biosynthesis, and Vi production may have replaced this function in S. enterica serovar Typhi. However, if sufficient immunological pressure is exerted by Vi vaccination, it is possible that Vi could be lost by S. enterica serovar Typhi or that Vi could be replaced by an alternative but immunologically distinct capsular locus which could replace the SPI-7-associated viaB locus. Although this may seem unlikely, it seems prudent to monitor the Vi status of primary S. enterica serovar Typhi isolates and the stability of the viaB locus during Vi vaccination trials.

Acknowledgments

This work was supported by The Wellcome Trust and the BBSRC.

We thank Ken Sanderson, Salmonella Genetic Stock Centre, Calgary, Canada, and M. Y. Popoff, Pasteur Institute, Paris, France, for generous advice and for supplying the strains used in this study. Ian Dodd of the University of Adelaide gave us invaluable advice concerning all aspects of bacteriophage biology. We also thank the DOE Joint Genome Institute for sequence data for the saprophytic bacteria mentioned in this paper (http://www.jgi.doe.gov/JGI_microbial/html/index.html)

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