Fig 1.
pyrE encodes fifth enzyme in pyrimidine biosynthesis.
(A) Genetic context of pyrE (STM3733) in Salmonella Typhimurium. (B) De novo pyrimidine synthesis and salvage pathways in bacteria. Pathways that produce UMP, the precursor of all pyrimidines, are shown. The six steps of the de novo pyrimidine biosynthesis pathway and their corresponding enzymes, genes in parentheses are shown. Double arrowheads indicate that the activity is capable of both conversions.
Table 1.
Strains used in this study.
Fig 2.
ΔpyrE mutant colonizes 4-day-old chicks poorly in competitive infection with wild type.
(A) Fifteen 4-day-old chicks were infected orally with 1x109 CFU of equal mixture of ΔpyrE and isogenic wild type, HA431 (HA420 ΔphoN::KanR). At day 1, 3, and 9 post-infection, 5 chicks were humanely euthanized, ceca (C), cecal tonsil (CT), bursa (B), spleen (S), and liver (L) were excised, homogenized in PBS, and plated on LB agar plate supplemented with kanamycin and XP to enumerate the colony forming units (CFU) for ΔpyrE and wild type respectively. (B) Return of wild type copy of pyrE in trans restores colonization to the level of wild type during competitive infection with wild type, HA877 (HA420 ΔphoN::NalR pWSK29) and either HA1301(ΔpyrE::KanR pWSK29) (open trianges) or HA1293 (ΔpyrE::KanR pWSK29::STM3733) (filled trianges) at day 9 post-infection. Infections were performed as described in (A). Data are shown as the ratio of mutant to wild type recovered from each tissue normalized to the input ratio and converted logarithmically. Horizontal bars denote the mean, and error bars denote the standard deviation. Statistical significance was determined by using a Student’s 2-tail t-test with p < 0.05 (*), p< 0.001 (**).
Fig 3.
ΔpyrE mutant grows poorly in M9 minimal media.
Growth was tested in vitro by individual growth (A) or competitive growth with wild type (B). (A) Stationary phase cultures of the ΔpyrE mutant (open square) and wild type, HA431 (HA420 ΔphoN::KanR) (dark grey triangle) were sub-cultured in LB (solid line) or M9 broth (dotted line) supplemented with kanamycin for 24 hours. Overnight cultures were washed in M9 broth twice prior to sub-culturing in M9 broth. At the indicated time points, CFU in each culture was enumerated after serial dilution and plating. (B) Overnight cultures of ΔpyrE mutant and the wild type (HA431) were washed twice with M9 broth and mixed at 1:1 ratio in M9 broth. Resulting mixture was sub-cultured in M9 broth supplemented with kanamycin at 37°C for 24h. At the indicated time points the CFU of both strains was enumerated by serial dilution and plating. Dataare shown as ratio of mutant to wild type recovered normalized by the input ratio, and converted logarithmically. Statistical significance was determined by using a Student’s two-tail t-test with p< 0.05 (*) and error bars denote standard error. Experiments were performed on three independent occasions.
Fig 4.
Growth defect of ΔpyrE mutant in M9 media is restored by uracil supplementation.
(A) Overnight cultures of a ΔpyrE mutant and the wild type, HA431 (HA420 ΔphoN::KanR) were washed in M9 minimal broth twice and 3 μl of washed culture was spotted on M9 media with (Uracil+) or without uracil supplementation (Uracil-) (20 mg/L). Plates were incubated overnight at 37°C. This experiment was performed in triplicate and representative photographs are shown. (B-C) Bacteria (ΔpyrE mutant: filled square, or the wild type wild type, HA431 (HA420 ΔphoN::KanR): filled circle) from overnight cultures were washed in M9 broth, and sub-cultured in un-supplemented M9 broth with (solid line) or in M9 broth with uracil supplementation at 20mg/ml (dashed line). At the indicated time points, aliquots were taken for absorbance measurement at 570nm (B), or were serially diluted in PBS and plated on LB supplemented with kanamycin and XP to enumerate CFU (C). Bars indicate mean and error bars indicate standard deviation. All experiments were performed on three separate occasions.
Fig 5.
ΔpyrE mutant is able to colonize and persist in mice and calves in the levels similar to wild type.
(A) Bovine ligated ileal loops were infected with 1x109 CFU of a 1:1 mixture of the ΔpyrE mutant and wild type, HA877 (HA420 ΔphoN::NalR pWSK29) by intraluminal injection. After 12 hours post-infection loops were excised and the amount of luminal fluid was determined as a gross measure of inflammation. Luminal fluid (F), mucus (M) and tissue (T) were collected and processed for CFU determination. Infection was performed in 3 different animals. (B) Four CBA/J mice were infected with 1x109 CFU of a 1:1 mixture of the ΔpyrE mutant and wild type, HA431 (HA420 ΔphoN::KanR) by gavage.) Feces were collected every three days, and the CFU of each strain was determined by serial dilution and plating. (C) At 41 days post-infection, the cecum (CC), Peyer’s patches (PP), mesenteric lymph nodes (MLN), spleen (S), and liver (L) were excised, and processed as described in Fig 1. Data is shown as the ratio of mutant to wild type recovered, normalized to the input ratio, and converted to logarithmically. The median is indicated. Statistical significance was determined by using a Student’s two-tail t-test with p < 0.05.
Fig 6.
Colonization defect of ΔpyrE mutant in chicks is rescued by feeding uracil-supplemented diet.
(A) Schematic of the experimental design. Chicks were divided into 2 groups after hatching and fed either our regular chick diet (no added uracil, or uracil -) or the same diet supplemented with uracil (uracil +). At day 4 post-hatch, chicks in both groups were weighed and orally infected with 1 x 109 CFU of a 1:1 mixture of the ΔpyrE mutant and wild type, HA431 (HA420 ΔphoN::KanR). (B) At day 3 post-infection (corresponds to 7 days of age), chicks from both groups were weighed and five chicks from each group were euthanized. (C) The remaining chicks being fed our regular chick diet were maintained on this diet for the duration of the experiment (uracil -/-, filled circles). The remaining chicks in uracil supplemented group were sub-divided into 2 groups: One group (uracil +/+, filled triangles) was fed a uracil-supplemented diet for the duration of our study, and the other group was returned to our regular chick diet (uracil +/-, no added uracil from days 3–9 post infection, filled squares). At day 9 post-infection, all three groups of chicks were weighed and euthanized. For all groups and all time points, ceca (C) cecal tonsil (CT) and Bursa of Fabricius (B) were collected and processed as described previously. Data are shown as ratio of mutant to wild type in the infected tissue, normalized to the input ratio and converted logarithmically. Statistical significance was determined by using a Student’s 2-tailed t-test with p < 0.05 (*), p< 0.01 (**), p< 0.001 (***), NS: not significant. Error bars denote standard error.