Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters

Intracellular free iron concentrations of an S. Typhimurium fur mutant overexpressing STM3944. (A) Strains JV119 and JV120 were grown in LB to an OD₆₀₀ of ∼1.0, and desferrioxamine-chelatable free iron concentrations were determined by EPR spectroscopy. Representative EPR spectra for an S. Typhimurium fur mutant containing the pBAD-Cm vector (JV119) or pBAD-Cm::stm3944 (JV120) normalized to cell density are shown. (B) Quantitation of intracellular free iron concentrations. Data are means ± 1 standard deviation from three independent experiments. *, concentration significantly different from that of the wild type (P = 0.01, Student's t test).

Iron-sulfur cluster activity in wild-type and mutant S. Typhimurium strains under basal conditions and following H₂O₂ stress. (A) Basal iron-sulfur cluster enzyme activity was assayed in wild-type and cyaY, yggX, cyaY yggX, and iscA mutant strains. Aconitase (Acn), serine deaminase (Sda) and NADH dehydrogenase I (NdhI) activities were measured in mutant strains grown in LB to an OD₆₀₀ of ∼1.0 and normalized to wild-type activity. Data shown are means ± 1 standard deviation from at least three independent experiments. *, activity significantly different from that of the wild type (P < 0.05, Student's t test); #, activity significantly different from that of the wild-type, cyaY mutant, or yggX mutant strain (P < 0.05, Student's t test). (B) Relative Sda, Acn, and NdhI activity was assayed following 15 min exposure to 0.5, 4, or 8 mM H₂O₂. For each assay, activity was normalized to the enzymatic activity in the absence of H₂O₂, which is displayed as 100%.

Reactivation of aconitase activity following damage by H₂O₂. Strains grown in LB to an OD₆₀₀ of ∼1.0 were treated with 4 mM H₂O₂ for 15 min. Spectinomycin and the iron chelator DTPA were included to block new protein synthesis and to inhibit iron transport from the extracellular medium, respectively. Catalase was added (at 0 min) to terminate the H₂O₂ stress, and aconitase reactivation was measured at the indicated time intervals after the termination of H₂O₂ stress. Activities were normalized to the activity of the untreated control (prior to H₂O₂ exposure), which was set to 100%. (A) Aconitase reactivation in wild-type S. Typhimurium, the cyaY mutant, the yggX mutant, and the cyaY yggX mutant. (B) Aconitase reactivation in wild-type S. Typhimurium, the cyaY mutant containing empty vector pBAD-Cm, and the cyaY mutant containing pBAD-Cm::cyaY. (C) Aconitase reactivation in wild-type S. Typhimurium, the yggX mutant containing empty vector pBAD-Kan, and the yggX mutant containing pBAD-Kan::yggX. Data shown are means ± 1 standard deviation from three independent experiments. *, reactivation levels significantly different from that for the wild type (P < 0.05, Student's t test); #, reactivation significantly different from that for the wild-type, cyaY mutant, and yggX mutant strains (P < 0.05, Student's t test).

Relative nitric oxide resistance of wild-type and mutant S. Typhimurium strains. Growth of the wild type and the cyaY, yggX, and cyaY yggX mutant strains was monitored in the presence of the NO· donor Sper/NO. Strains were inoculated at a density of 10⁶ CFU ml⁻¹ in M9 minimal medium plus 0.2% gluconate with (open symbols) or without (closed symbols) 750 μM Sper/NO. Growth was monitored in a Bioscreen C microplate reader with agitation at 37°C. Data shown are means ± 1 standard deviation from at least three independent experiments.

Virulence of wild-type S. Typhimurium and isogenic mutant derivatives in a murine model of systemic infection. (A) Seven-week-old C3H/HeN mice (n = 10) were injected intraperitoneally with 1 × 10³ to 2 × 10³ CFU of wild-type or isogenic mutant strains. Mice were monitored daily for signs of illness, and moribund animals were euthanized. Data are representative of those from at least two or more reproducible, independent experiments. P values were calculated using the log-rank Mantel-Cox test. (B) Seven-week-old C3H/HeN mice (n = 5) were injected intraperitoneally with 2 × 10³ total CFU of a 1:1 mixture of wild-type and isogenic mutant bacteria. All animals were euthanized at 8 days postinfection, and the competitive index of the remaining mutant bacteria to the remaining wild-type bacteria was calculated for both livers and spleens. The cyaA, yggX, and iscA mutants each displayed a statistically significant (P < 0.05) competitive disadvantage in the liver compared to the wild type, though the yggX defect (confidence interval = 0.72) was minor compared to the cyaY (confidence interval = 0.19) and iscA (confidence interval = 0.41) mutations. In the spleen, only cyaY and iscA mutants displayed statistically significant (P < 0.05) competitive disadvantages of 0.41 and 0.39, respectively. P values were calculated by the Wilcoxon rank-sum test.