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. 2004 May;186(10):3214-23.
doi: 10.1128/jb.186.10.3214-3223.2004.

Salmonella Enterica Serovar Typhi Strains From Which SPI7, a 134-kilobase Island With Genes for Vi Exopolysaccharide and Other Functions, Has Been Deleted

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Salmonella Enterica Serovar Typhi Strains From Which SPI7, a 134-kilobase Island With Genes for Vi Exopolysaccharide and Other Functions, Has Been Deleted

Satheesh Nair et al. J Bacteriol. .
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Abstract

Salmonella enterica serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between pheU and 'pheU (truncated), two genes for tRNA(Phe). SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage. Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion. In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative. We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons. Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single pheU gene; microarrays show that all SPI7 genes are deleted. Two of the strains produce amplicons which have A derived from pheU at bp 27, while five have C derived from 'pheU at this position; thus, the position of the crossover which results in the deletion can be inferred. The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the viaB operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained. We propose that these deletions occurred during storage following isolation.

Figures

FIG. 1.
FIG. 1.
The genes in the region of SPI7 on the chromosome of serovar Typhi and their deletions. See the work of Pickard et al. (31) for a detailed map and annotations of gene function. (A) Vi-positive SPI7+ strain. The genes shown include the left junction, viaB operon, the right junction of SPI7 and genes to the “left” side of SPI7. The island is inserted between an intact 75-bp pheU gene (black arrow) for tRNAPheU and truncated ′pheU (striped arrow). Genes STY4465 and STY4664 are emphasized as they include the junction points of the deletions detected in TYT1669. The primers used for multiplex PCR and target sites are shown; the predicted amplicons (aroC and L-Junc, etc.) are shown as thick horizontal lines with their predicted sizes in base pairs. The map is not to scale. (B) Structure predicted if SPI7 is deleted by recombination between ′pheU and pheU. The predicted amplicons tRNA and tRNA Plus are illustrated as thick lines. (C) Schematic drawing of serovar Typhi TYT1669 which shows a partial deletion of SPI7 and regions on the ”left“ side of SPI7. Based on microarray data, primers were designed for both multiplex PCR and sequencing. The primers 4464F and 4664R result in the amplicon 1669Junc (2,251 bp); nested primers 1669seqF and -R were used to determine sequence of this amplicon and reveal the fusion of the STY4465 and STY4664.
FIG. 2.
FIG. 2.
Complete digestion of DNA of strains of serovar Typhi with endonuclease I-CeuI, separation by PFGE and staining with ethidium bromide. Fragment designations are given on the right for SPI7 positive serovar Typhi Ty2 (lane 10) and CT18 (lane 1), which are used as controls, and on the left for the SPI7 negative serovar Typhi strains (lanes 2 to 7, and lane 9). The I-CeuI D (134 kb), E (149 kb) and F (42 kb) fragments are not shown, as these fragments were identical sizes for all the strains studied. Lane 2, strain T202; lane 3, strain T104; lane 4, strain SARB64; lane 5, strain R1962; lane 6, strain 415Ty; lane 7, strain 421Ty; lane 9, strain TYT1669. One of the strains used in the study, strain 26.003, was not shown in the figure but it had the same banding pattern as the other 6 strains in lanes 2 to 7. The sizes of the fragments are shown in kilobases (kb). The λ concatemer marker is shown in lane 8 (M) though not very visible. All the lanes shown are from the same gel run. Note that I-CeuI B(717 kb) and I-CeuI G′ (ca. 706) are difficult to separate.
FIG. 3.
FIG. 3.
Multiplex PCR of serovar Typhi. Lanes 2 to 8 represent SPI7-negative strains that clearly show the absence of tviB gene (868 bp), left (1,689 bp) and right (377 bp) junctions of SPI7. The 1,275-bp amplicon seen in these SPI7-negative strains indicates the deletion between the phoN and tRNAPheU genes. Lane 9 is serovar Typhi Ty2 used as a SPI7-positive control strain, hence the amplification of the right and left junction of SPI7 and the tviB gene. Lane 10 depicts serovar Typhi TYT1669 that harbors a partial SPI7 deletion. TYT1669 has the right junction of SPI7, but a deletion between STY4664 (last gene present of the island) and STY4464 (first gene present after the left flank deletion). The 2,251-bp product is an amplification between STY4464 and STY4664 due to the 175-kb deletion between the 2 genes. The 639-bp aroC gene was produced in all the strains and acts as a control for Salmonella DNA. Lane 1, 100-bp marker (Amersham); lane 11, λ HindIII marker (Amersham).
FIG.4.
FIG.4.
Comparative genome hybridization of nine S. enterica serovar Typhi strains versus serovar Typhi CT18. Genes are plotted in order of their position on the Typhi CT18 genome. The 10 regions of more than one gene deleted from other Typhi strains are indicated. The narV gene partially deleted from strains Ty2, T202, T104 and TYT1669 is marked by an asterisk.
FIG. 5.
FIG. 5.
Nucleotide sequence analysis of deletions in serovar Typhi. (A) The sequence of an SPI7+ strain is shown at the top, in the gene order phoN, ′pheU, (SPI7), pheU, yjdC; this is based on published sequence of the genome. Under this it the sequence of the tRNA Plus amplicon (Fig. 1B) produced from seven strains with precise deletions of SPI7. Two of the strains have A in the pheU gene at bp 27 (postulated to be due to crossover X in region I), and five have C due to crossover Y in region II. (B) The sequence of the N-terminal part of gene STY4465 and of the N-terminal part of gene STY4664 is shown. This sequence was determined from amplicon 1669 Junc (Fig. 1C). The sequences which are deleted from the strain TYT1669 are shown in lowercase type, inferred from published genomic sequences; the sequences which are included in the junction are in boldface uppercase type; the G and C at the junction points are shown in shading. The deletion in the strain is inferred to be 175 kb; the sequence of the N-terminal portion of the resulting gene is shown at the bottom of the figure.

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