Figure 2. Involvement of RNAIII and rot-dependent pathways in agr-mediated protection from H₂O₂-mediated killing.

Cultures were grown for 1 hr following dilution from overnight cultures to early log phase (OD₆₀₀∼0.15) and then treated with 20 mM H₂O₂ for 60 min before determination of percent survival by plating and enumeration of colonies. (A) Wild-type LAC (WT, BS819), Δagr mutant (BS1348), and ΔrnaIII mutant (GAW183). (B) Δrot and Δagr Δrot double mutant (BS1302). (C) Wild-type (WT) strain Newman (NM, BS12), Δagr mutant (BS13), and ΔRNAIII mutant (BS669). (D) Overexpression of rot. Rot was expressed from a plasmid-borne wild-type rot (pOS1-Plgt-rot, strain VJT14.28). Data represent the mean ± SD. from biological replicates (n=3).

Figure 2—figure supplement 1. Deficiency of downstream global regulators does not differentially affect agr-mediated protection from H₂O₂-mediated cell death.

The effect of (A) sigB, (B) mgrA, and sae on survival in the presence or absence of agr during treatment with H₂O₂ was measured. Cells were grown to early log phase (OD₆₀₀∼0.15) and treated with 20 mM of H₂O₂ for 60 min. Data represent the mean ± SD. from biological replicates (n=3). Bacterial strains were BS819 (LAC) and BS12 (Newman, NM) for wild-type (WT) and BS1348 (LAC) and BS13 (NM) for the agr, and BS1435-36, BS1280, BS1282, and BS1246, BS1518 for sigB, sae and mgrA mutants, respectively. The genes tested were either part of known two-component systems or SarA protein-family regulatory circuits involved in virulence gene expression. They are all downstream/epistatic to agr-RNAIII (reviewed in Bronesky et al., 2016). Mutations in sigB, mgrA, and sae showed little or no effect with respect to the protective agr-mediated phenotype. These results support the idea that rot is the primary regulator pathway that protects the wild-type from H₂O₂-mediated killing.