Figure 3. Agr-mediated protection from H2O2 stress is uncoupled from agr activation kinetics.
(A) Assay design. An ΔagrBD deletion mutant (GAW130) was complemented in trans by the autoinducing product (AIP) of AgrBD in an ΔrnaIII (GAW183) mutant that produces AIP endogenously; AgrC activation in the ΔagrBD strain leads to downstream activation of RNAIII. The agrBD strain, engineered in-frame to avoid polar effects on downstream genes agrC and agrA, senses but does not produce an autoinducer. The ΔrnaIII mutant, constructed by replacement of rnaIII with a cadmium resistance cassette (rnaIII::cadA), produces autoinducer but lacks RNAIII, the effector molecule of agr-mediated phenotypes with respect to H2O2. (B) Trans-activation demonstrated by hemolysin activity on sheep blood agar plates. Bottom of figure shows zone of clearing (hemolysin activity) after mixing 108 ΔagrBD CFU with an equal number of ΔrnaIII. Zone of clearance is a consequence of AgrC receptor activation in trans by AIP produced by the ΔrnaIII mutant. (C) Absence of trans-activation with short-term culture. The wild-type strain RN6734 (WT, BS435), ΔrnaIII (GAW183), ΔagrBD (GAW130), and ΔrnaIII and ΔagrBD mutants were mixed 1:1 immediately before growth from overnight culture. Overnight cultures were diluted (OD600∼0.05) into fresh Tryptic Soy Broth (TSB) medium, mixed, and grown to early log phase (OD600∼0.15) when they were treated with 20 mM H2O2 for 60 min and assayed for percent survival by plating. (D) Kinetics of killing by H2O2. Survival assays employing ΔrnaIII and ΔagrBD mixtures, performed as in panel C, but grown from early exponential (1 hr, OD600∼0.15) through late log (5 hr, OD600∼4) phase in TSB. Cultures were treated with H2O2 (20 mM for 1 hr) at the indicated time points. (E) Proportion of mixed population for panel D represented by each mutant after incubation. The ΔagrBD mutant contained an erythromycin-resistance marker to distinguish the strains following plating of serial dilutions on TS agar with or without erythromycin (5 μg/). Data represent the mean ± SD. from biological replicates (n=3). (F) Trans-activation during long-term culture. The wild-type strain RN6734 (WT, BS435), ΔrnaIII (strain GAW183), ΔagrBD (strain GAW130), and ΔrnaIII and ΔagrBD mutants mixed 1:1 prior to overnight culture. Survival assays employing ΔrnaIII and ΔagrBD mixtures, performed as in panel C. (G) Kinetics of killing by H2O2. Survival assays employing ΔrnaIII and ΔagrBD mixtures, performed as in panel D. Cultures were treated with H2O2 (20 mM for 1 hr) at the indicated time points. (H) Proportion of mixed population for panel G represented by each mutant after incubation, performed as in panel E. Data represent the mean ± SD. from biological replicates (n=3).
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