Comparative genome analysis of the high pathogenicity Salmonella Typhimurium strain UK-1

Abstract

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines.

Figure 1. Genome atlas of Salmonella enterica serovar Typhimurium UK-1.

(A) The chromosome. Base pairs are indicated outside the outer circle. The circles represent the following (from outside to inside): Circle 1 shows the distribution of predicted ORFs in the leading and lagging strands (see details in the color legend for Circle 1). Circle 2 shows the UK-1 pseudogenes (black, single circle). Circle 3 shows the phage regions in UK-1 (Cyan, single circle). Circle 4 shows the genomic islands predicted by IslandViewer (red, single circle). Circle 5 displays the GC content of the genome (red: high GC content, purple: low GC content). Circle 6 displays GC skew ([G+C]/[G−C]) plot. (B) The UK-1 plasmid pSTUK-100 genome. Base pairs are indicated outside the outer circle. From outside to inside: genes predicted in the plasmid genome (two circles; all ORFs are shown in grey since there were no unique genes found in the pSTUK-100 genome.), pseudogene(s) identified in pSTUK-100 (black, single circle), GC content of the plasmid genome (red: high GC content, purple: low GC content), and GC Skew Plot. For the GC content and GC skew analysis, we applied a sliding window of 1,000 bp with an overlap of 500 bp. The atlas was created using GenomeViz software .

Figure 2. Phylogenetic relationship of the five S. Typhimurium strains.

(A) The phylogenetic tree was inferred with ML method based on the conserved genomic sequences. The S. Typhimurium strains are rooted to S. Typhi Ty2. The upper-left subtree shows the phylogenetic relationship of the five strains in a smaller scale. The relationship was supported by the bootstrapping values shown on the subtree. The distance (marked in red) based on the number of SNPs was also presented on the phylogenetic tree. The right panel shows the complete genome alignment of the five strains generated in MAUVE . The regions conserved among all genomes are colored in purple and the regions conserved among subsets of the genomes are colored differently. If the areas contain sequence elements not aligned, those are marked in white. Regions that are not colored indicate no detectable homology among the five genomes in MAUVE. The distinguished phages and phage remnants are marked on the alignment (black: detected among all of the five strains, red: detected in a subset of strains). (B) Comparison of the lengths of genomes, phages, and genomes excluding phage regions among the five S. Typhimurium strains. Length of phages is displayed on the second Y-axis due to the relatively small value of phages in contrast to the whole genome size. (C) Alignment of the UK-1 Gifsy-1 sequence segment harboring the two UK-1 unique genes with sequences from the other four S. Typhimurium strains. The sequence alignments were generated in MAUVE. The color scheme used for the alignment is described in Fig. 2A. The predicted genes in these regions are shown with red solid arrays. Each gene name is indicated with the strain name (UK indicates UK-1, STM indicates LT2, 14- indicates 14028s, MW indicates D23580, and SL indicates SL1344) followed by its locus number obtained from each of the annotation files. The two UK-1 unique genes are marked in red in the UK-1 genome.

Figure 3. SNPs detected in the UK-1 strain with respected to the four S. Typhimurium genomes.

(A) Pair-wise comparison of the synonymous and nonsynonymous SNPs among the five sequenced S. Typhimurium strains. (B) The distribution of UK-1 genes containing SNPs. The inner pie chart shows the number of genes carrying SNPs grouped by COG category . For each group, the distribution of genes is shown in the outer pie charts, which describe the number of reference strains that contribute to the UK-1 SNPs. The legend of the inner pie chart is shown at the left of the pie charts. The legend of the outer pie charts is shown in the upper-right corner.

Figure 4. Two CRISPRs detected in the five S. Typhimurium strains.

(A) Genetic map of the two CRISPR/cas systems present in Salmonella Typhimurium UK-1. 17 cas genes were detected around the two CRISPRs. Three core cas genes are noted with red triangles. (B) Overview of the two CRISPR loci in the five S. Typhimurium strains. The repeats are shown as dark diamonds. Spacers are shown as colored rectangles. In each CRISPR, spacers with identical sequence in the studied genomes are shown in the same color. The white rectangles indicate the strain specific spacers.