Composition, acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7

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 tRNA(pheU) 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 tRNA(pheU) 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 tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-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.

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 tRNA^pheU, and the tRNA^pheU-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.

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) tRNA^pheU region in S. enterica serovar Typhimurium strain LT2 harboring a gene resembling merR at the phoN (upstream) end of this tRNA. (E) Intact tRNA^pheU as found, for example, in S. enterica serovar Enteritidis, with no additional DNA inserted between the cutA3 and phoN genes.

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.