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. 2018 May 18;9:877.
doi: 10.3389/fmicb.2018.00877. eCollection 2018.

Genome-Wide Comparative Functional Analyses Reveal Adaptations of Salmonella Sv. Newport to a Plant Colonization Lifestyle

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Free PMC article

Genome-Wide Comparative Functional Analyses Reveal Adaptations of Salmonella Sv. Newport to a Plant Colonization Lifestyle

Marcos H de Moraes et al. Front Microbiol. .
Free PMC article

Abstract

Outbreaks of salmonellosis linked to the consumption of vegetables have been disproportionately associated with strains of serovar Newport. We tested the hypothesis that strains of sv. Newport have evolved unique adaptations to persistence in plants that are not shared by strains of other Salmonella serovars. We used a genome-wide mutant screen to compare growth in tomato fruit of a sv. Newport strain from an outbreak traced to tomatoes, and a sv. Typhimurium strain from animals. Most genes in the sv. Newport strain that were selected during persistence in tomatoes were shared with, and similarly selected in, the sv. Typhimurium strain. Many of their functions are linked to central metabolism, including amino acid biosynthetic pathways, iron acquisition, and maintenance of cell structure. One exception was a greater need for the core genes involved in purine metabolism in sv. Typhimurium than in sv. Newport. We discovered a gene, papA, that was unique to sv. Newport and contributed to the strain's fitness in tomatoes. The papA gene was present in about 25% of sv. Newport Group III genomes and generally absent from other Salmonella genomes. Homologs of papA were detected in the genomes of Pantoea, Dickeya, and Pectobacterium, members of the Enterobacteriacea family that can colonize both plants and animals.

Keywords: comparative genomics; pan-genome; plant-microbe interactions; tomato; vegetable safety.

Figures

FIGURE 1
FIGURE 1
Phylogenetic and comparative genomic analysis of the sv. Typhimurium and sv. Newport genomes. (A) Phylogenetic tree for 1,597 genomes of sv Typhimurium and sv Newport isolates, constructed using SNPs and minimal evolution over raw distance. Colors represent the major clades identified and dots represent branches with bootstrap values higher than 0.85. iTOL v4 (Letunic and Bork, 2016) was used to visualize the tree. (B) Principal Coordinate Analysis of the gene presence and absence profile of the same genomes. Salmonella sv. Typhimurium strains are represented in purple, sv. Newport Group II isolates in red and sv. Newport Group III strains in green. The arrow indicates strain C4.2 of sv. Newport, recovered from a tomato-linked outbreak of human salmonellosis and used in this study.
FIGURE 2
FIGURE 2
Salmonella core, shell and accessory genomes. (A) Number of genes per number of Salmonella sv. Newport Group II, sv. Newport Group III and sv. Typhimurium genomes. Many genes were found in only one or a few genomes (peaks on the left of each plot). The core genes, present in almost all genomes of each group, are represented by the peak on the right of each plot. (B) Venn diagram representing the shared and unique elements of Salmonella core genes (shared by >95% of all members of each group) among the groups studied.
FIGURE 3
FIGURE 3
Change in abundance of Salmonella C4.2 loci after screening in tomatoes. Relative abundance of transposon insertions in loci after the incubation was compared to the initial inoculum. The position of each locus on the y-axis represents log2(Fold Change) of relative abundance and on the x-axis represents a physical position on the Salmonella chromosome. Loci with a significant change in abundance (FDR < 0.1) are shown in green when shared between S. Newport C4.2 and S. Typhimurium ATCC 14028 and purple when unique to S. Newport C4.2. Loci with an FDR > 0.1 are shown in gray. Arrows point to loci and operons targeted for further experiments.
FIGURE 4
FIGURE 4
Comparison of functions required for colonization of tomatoes between S. Newport C4.2 and S. Typhimurium ATCC 14028. S. Newport C4.2 metabolic pathways involved in tomato colonization are shown in purple, S. Typhimurium ATCC 14028 metabolic pathways involved in tomato colonization are shown in orange, and required pathways shared by both strains are shown in green. Pathways were identified as required for tomato colonization if the corresponding mutants were less represented in the output pool [log2(FC) < -0.5, FDR < 0.1]. Key metabolic steps are shown as shaded boxes. KEGG Orthology terms for the protein sequences corresponding to the underrepresented mutants were retrieved using BlastKOALA and mapped using KEGG Mapper. False color overlays were imposed in Adobe Photoshop 2014.
FIGURE 5
FIGURE 5
Competitive fitness of isogenic mutants involved in amino acid biosynthesis. Approximately 103 CFUs of a mix of the wild type and an isogenic mutant (1:1) were inoculated into each tomato, followed by 7-day incubation at 22°C. The Competitive Index CI was calculated using the formula (MUTout:WTout)/(MUTin:WTin). The replicates were obtained from six tomato fruits with three inoculation sites per fruit. Each dot represents a single replicate and the black bar represents the mean log2(CI) of all replicates. Statistical significance was established by comparing CI’s using ANOVA and Tukey’s post hoc test. Asterisks represent strains that are statistically different from the neutral mutant ISG7 (P-value < 0.05).
FIGURE 6
FIGURE 6
Competitive fitness of isogenic mutants of genes unique to S. Newport C4.2. The Competitive Index CI was calculated using the formula (MUTout:WTout)/(MUTin:WTin) and inoculation and statistical analysis was done as described before. (A) CI of isogenic mutants for peg.4638, peg.4639, and peg.4640. (B) CI of isogenic mutants for papA and the complemented isogenic mutant ΔpapA phoN::papA.
FIGURE 7
FIGURE 7
Comparison of growth and colony morphology between S. Newport C4.2 and its papA mutant. (A) Growth of strains in LB medium at 250 rpm and 37°C in the presence and absence of 0.5 mM paraquat. Three independent replicates were used per condition. (B) Colony morphology of S. Newport C4.2 and its papA mutant on Congo Red indicator plates. The smooth surface and edges show the absence of the rough and dry phenotype.

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