Analysis of the hypervariable region of the Salmonella enterica genome associated with tRNA(leuX)

Abstract

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region.

FIG. 1.

Comparisons of sequenced E. coli and Salmonella genomes reveal seven different organizations of tRNA^leuX-adjacent genes. Predicted intact open reading frames and pseudogenes (marked with a cross) are shown as arrows indicating the direction of transcription. The Salmonella serovar Typhimurium LT2 STY4854-like pseudogene is shown as a cross without an arrow because it has lost its start codon. The boundaries of each island are marked with thick vertical lines. Genes shaded with spots are common to two or more of the islands shown and therefore lie outside of the tRNA^leuX-adjacent islands as defined in this study. Genes of related origin or function are shaded similarly, as indicated in the key. The complete fully annotated genome sequences analyzed are those of E. coli nonpathogenic K-12 (strain MG1655 accession number NC_000913) (9) and pathogenic O157:H7 substrain RIMD 0509952 (28) (accession number NC_002695), Salmonella serovar Typhi CT18 (40) (accession number NC_003198), and Salmonella serovar Typhimurium LT2 (35) (accession number NC_003197). Gene numbers assigned by the sequencing projects are indicated for LT2 (35) and CT18 (40). Partially sequenced or fully sequenced, but unannotated, genomes that were also compared are as follows: S. enterica subsp. I serovars Enteritidis (strain PT4) and Paratyphi A (strain ATCC 9150) and S. bongori (stain 12419) (www.sanger.ac.uk/Projects/Salmonella and www.genome.wustl.edu). The genes and pseudogenes shown for Salmonella serovar Paratyphi A are predicted to be present by comparison with Salmonella serovar Typhi CT18. The incomplete Salmonella serovar Paratyphi A genome sequence contains many additional frameshifts and is missing regions between contigs compared with CT18, but as these features remain to be verified, they are not represented here.

FIG. 2.

Microarray comparison of Salmonella serovar Typhi CT18 SPI-10 with different salmonellae. The microarray data image was constructed by using GeneSpring software. Functional groups into which the SPI-10 genes can be divided, the direction of their transcription (depicted by arrows for each gene), and the G-C content of this mosaic island (noted in red) are shown at the top. Each row of data is the result of challenging the microarray with 40 different Salmonella isolates, which are labeled along the right hand side and are described in Table 1. Each column represents a specific gene either within (STY4821 to STY4852) or on either side of (STY4820 and STY4853-56) the Salmonella serovar Typhi CT18 tRNA^leuX island. The color scheme for the present/conserved or absent/diverged nature of the genes is displayed (bottom). The darkest blue corresponds to those genes that are considered absent/divergent with the highest degree of certainty. The brightest yellow corresponds to genes that are assigned present/conserved with the highest degree of certainty. Those regions that are orange are genes where hybridization with test DNA is higher than that of reference DNA. Grey indicates missing data. It can be seen that data for different strains of the same serovar are generally very similar, with one clear exception: Salmonella serovar Typhi strain KT516 (marked with an asterisk) does not contain an intact CT18-like P4 phage (STY4821 to STY4834).

FIG. 3.

(a) PCR analysis confirms the absence of the CT18 P4 phage in Salmonella serovar Typhi KT516. The primers used for these analyses are as follows (see also Table S1 in the supplemental material): lane 1, primer pair 1-3; lane 2, primer pair 4-5; lane 3, primer pair 6-7; lane 4, primer pair 8-9; lane 5, primer pair 10-11; lane 6, primer pair 12-13; lane 7, primer pair 14-15. The CT18 genome numbers for each gene (or genes) amplified and the five PCR products that lie within the CT18 P4 phage (STY4821-24 and STY4826-27) are noted above the gel. PCR analysis of Salmonella serovar Typhi KT516 confirms the observation, made using microarrays (Fig. 2), that this strain does not contain an intact CT18-like P4 phage, whereas transposase STY4848 and the helicase-containing region (STY4849 and STY4851) are present. (b) Circularized ST46 phage DNA was detected by PCR with genomic DNA from Salmonella serovar Typhi strains BRD948, CVD908, or CT18 but not with KT516, which is missing ST46. The CT18 genome numbers for each gene (or genes) amplified are noted above the gel. Whether the primers were designed to amplify chromosomal and/or circular phage DNA products is indicated. The primers used are detailed in Materials and Methods.

FIG. 4.

Examples of Southern blots showing that STY4822 is Salmonella serovar Typhi specific and that the Salmonella serovar Typhimurium SPI-10 genes tested (STM4493 and STM4496-98) are only shared with three other serovars: Salmonella serovar Derby (not shown here), Salmonella serovar Saintpaul, and Salmonella serovar Stanleyville. Data were only included in the final analysis (summarized in Table 2) where a clear aroC control band was visible (with the exception of Salmonella serovar Typhi BRD948, which is an aroC deletion mutant). Strain names and serovars are noted above each panel, and the probes used are indicated at the side. (a) Four Salmonella serovar Typhi strains tested contain STY4822, whereas strains of Salmonella serovar Typhimurium, Salmonella serovar Paratyphi A, Salmonella serovar Paratyphi B, and Salmonella serovar Paratyphi C do not. (b) STY4822 is present in the control Salmonella serovar Typhi CT18, but it is not detected in 11 SARB collection strains tested here. Salmonella serovar Typhimurium LT2 tRNA^leuX-adjacent genes, on the other hand, are shared with Salmonella serovar Stanleyville (STM4493 and STM4496-98) and Salmonella serovar Saintpaul (STM4496-98), and are shown in lanes that are boxed.

FIG. 5.

Examples of PCR analysis of Salmonella serovar Saintpaul SARB56 and Salmonella serovar Stanleyville SARB61 compared with Salmonella serovar Typhimurium LT2. Strain names are noted below each panel. The LT2 genome numbers for each gene (or genes) amplified are noted above each panel. (i) PCR with primers STM4492_for and STM4495_rev (see Table S1 in the supplemental material) reveal that SARB61 and LT2 contain a similarly sized DNA fragment between STM4492 and STM4495. SARB56, on the other hand, contains only a short DNA fragment between STM4492 and STM4495. (ii) PCR with primers STM4490_for and STM4492_rev (see Table S1 in the supplemental material) revealed a similarly sized DNA fragment between the STM4490-like and STM4492-like genes of SARB56 and SARB61, which is larger than the equivalent region in LT2. (iii and iv) Schematic depiction of the conclusions drawn from Southern blotting (Table 2 and Fig. 4), PCR (panels i and ii), and sequencing data comparing SARB56 and SARB61 tRNA^leuX islands with that of Salmonella serovar Typhimurium LT2. STM4495 lies adjacent to STM4492 in SARB56, and a novel insertion is present between STM4490 and STM4491 in both SARB56 and SARB61 (containing one predicted gene shown here in black). Genes that were detected by Southern blotting (Table 2) are shown in gray.

FIG. 6.

Examples of PCR comparison of tRNA^leuX island genes from LT2 with Salmonella serovar Derby strains SARB9 and SARB10. The LT2 genome numbers for each gene (or genes) amplified are noted above each panel and numbers for genes that were not detected by PCR or where insertions were found to be present compared with LT2 are underlined. The primers used for the PCR analyses shown in panels (i to iii) are as follows (see also Table S1 in the supplemental material): lane 1, primer pair 16-17; lane 2, primer pair 16-19; lane 3, primer pair 20-22; lane 4, primer pair 21-25; lane 5, primer pair 24-26; lane 6, primer pair 24-27; lane 7, primer pair 24-28; lane 8, primer pair 29-30; lane 9, primer pair 29-31. (i) Salmonella serovar Derby SARB9 contains genes similar to LT2 genes STM4490 to STM4499, but STM4488- and STM4489-like genes were not detected. (ii) Salmonella serovar Typhimurium LT2 controls for PCRs shown in panels i and iii. (iii) None of the LT2 tRNA^leuX island genes (STM4488 to STM4498) were initially detected in Salmonella serovar Derby SARB10 by PCR. STM4499 (data not shown) and STM4487, which lie on either side of the island, were both present. (iv) PCR with a long-range DNA polymerase with primers STM4487_for and STM4488_rev (see Table S1 in the supplemental material) revealed a larger DNA fragment between STM4487 and STM4488 in SARB10 than in the equivalent region in LT2. (v and vi) Schematic depiction of the gene arrangements in SARB9 and SARB10 supported by PCR data, such as that shown in panels i to iv, Southern blotting (Table 2 and Fig. 4), and sequencing of the SARB10 insertion between tRNA^leuX and STM4488 (amplified with primers tRNA_for and STM4488_rev). The sequences that lie between tRNA^leuX and STM4490 in SARB9 and between STM4488 and STM4499 in SARB10 remain unknown (shown as question marks). Genes detected by Southern blotting (Table 2) are shown in grey.