FIG 7.

The (p)ppGpp⁰ strain has a strong Mn requirement due to elevated ROS generation. (A to C) Growth of E. faecalis (p)ppGpp⁰ (A) and OG1RF wild-type (B) strains in complete, Mn-depleted, or Fe/Mn-depleted FMC medium in the presence or absence of 1 mM reduced glutathione. (C) Growth of the (p)ppGpp⁰ strain in metal-depleted FMC medium in the presence of oxidized glutathione. (D) Quantification of SOD activity in OG1RF and (p)ppGpp⁰ strains. Cells were grown to an OD₆₀₀ of ∼0.3 in FMC medium depleted of Fe or Mn or both and were harvested by centrifugation. Cells were then lysed, serially diluted, and tested for SOD activity using the cytochrome c, xanthine, xanthine oxidase method. (E) Transcriptional expression of sodA in response to Mn depletion. The E. faecalis OG1RF wild-type and (p)ppGpp⁰ strains were grown in complete, Mn-depleted, or Fe-and-Mn-depleted FMC medium to the mid-exponential-growth phase, and transcript levels of sodA were determined by qRT-PCR. The bar graphs show averages and standard deviations of results from three independent experiments performed in triplicate. Differences among strains and under different conditions were compared via Student's t test (P ≤ 0.05). (F) H₂O₂ production in OG1RF and (p)ppGpp⁰ strains. Cells were grown to an OD₆₀₀ of ∼0.3 in FMC medium depleted of Fe or Mn or both and were harvested by centrifugation. Then, cells were washed in NaPO₄ buffer, mixed with Amplex red solution, and incubated for 30 min before H₂O₂ determination. The graphs show averages and standard deviations of results from at least three independent experiments. Differences seen with the same strain under different conditions (*) or between parent and (p)ppGpp⁰ strains under the same condition (#) were compared (P ≤ 0.05). NT, not tested.