Figure 1. agr protects from killing by H₂O₂ throughout the growth cycle.

(A) Effect of culture growth phase. Overnight cultures of S. aureus LAC wild-type (WT, BS819) or Δagr (BS1348) were diluted (OD₆₀₀∼0.05) into fresh TSB medium and grown with shaking from early exponential (1 h, OD₆₀₀∼0.15) through late log (5 h, OD₆₀₀∼4) phase. At the indicated times, early (undiluted) and late exponential phase cultures (diluted into fresh Tryptic Soy Broth (TSB) medium to OD₆₀₀∼0.15) were treated with H₂O₂ (20 mM). After 60 min, aliquots were removed, serially diluted, and plated for determination of viable counts. Percent survival was calculated relative to a sample taken at the time of H₂O₂ addition. (B) Kinetics of killing by H₂O₂. Wild-type and Δagr mutant strains were grown to early exponential (OD₆₀₀∼0.15) and treated with 20 mM H₂O₂ for the times indicated, and percent survival was determined by plating. (C) Effect of H₂O₂ concentration on survival. Cultures prepared as in panel B were treated with the indicated peroxide concentrations for 60 min prior to plating and determination of percent survival. (D) Complementation of agr deletion mutation. Cultures of wild-type (WT) cells (BS819), Δagr mutant (BS1348), and complemented Δagr mutant carrying a chromosomally integrated wild-type operon (pJC1111-agrI) were treated with 20 mM H₂O₂ for 60 min followed by plating to determine percent survival. Data represent the means ± SD. from biological replicates (n=3).

Figure 1—figure supplement 1. Correlation of growth phase and agr expression.

(A) Growth curves. Overnight cultures of S. aureus LAC wild-type (WT, BS819) or Δagr mutant (BS1348) were diluted (OD₆₀₀∼0.05) in fresh TSB medium and growth was monitored by measuring the optical density at 600 nm (OD₆₀₀). (B) Tests of agrP3 promoter activity. S. aureus LAC wild-type (WT, BS819) containing agrP3-lux (SaPI1 attC::agrP3-lux; strain BS1222) or control containing a promoterless lux gene within the attC site (SaPI1 attC::pGYLux, strain BS999) grown as in (A) for the indicated times. agrP3 activity (relative luminescence units [RLU]) was assayed at the indicated times (see Materials and methods). Data represent the means ± SD. from biological replicates (n=3).

Figure 1—figure supplement 2. Correlation of lag-time and agr-mediated protection from H₂O₂-mediated killing.

Overnight cultures of S. aureus LAC wild-type (WT, BS819) and Δagr mutant (BS1348), grown for the indicated times following dilution to fresh medium, were treated with H₂O₂ (20 mM for 60 min) (Figure 1A). Data represent the means ± SD. from biological replicates (n=3). Survival of Δagr mutant cells was unchanged up to the 40 min time point, and then it dropped sharply. The sharp drop coincided with the time to first division (i.e. the lag time), as evidenced by an increase in colony-forming units (CFUs) at the 40 min time point in the absence of treatment (B and C). In contrast to results with the Δagr strain, survival of the wild-type strain gradually decreased throughout the experiment (A). Increased lag-time is associated with tolerance to lethal stress owing to a delay in growth when switched to a new environment (Fridman et al., 2014). Thus, our observations suggest that a subpopulation of Δagr mutant cells remains longer in a dormant state, decreasing the lethality of H₂O₂. The differential effect of the lag time on the wild-type and Δagr mutant cultures was absent during exponential growth (40 min). These results suggest that agr contributes to at least two forms of protection from H₂O₂-mediated killing: tolerance by a transient lag state and tolerance during growth phase. To focus on the latter form, assays involving cultures after overnight growth were grown for ~65 min (OD₆₀₀∼0.15).

Figure 1—figure supplement 3. Extended lag phase and decreased growth rate and yield of an Δagr mutant.

(A) Growth curves. S. aureus LAC wild-type (WT, BS819) and Δagr mutant (BS1348) cultures were grown in chemically-defined medium supplemented with 0.5% Casamino acids and 14 mM glucose (CDMG CAS) for the indicated times following 1000-fold dilution of overnight cultures grown in TSB. Growth of diluted cultures was monitored for 15 hr every 40 min by measuring the OD₆₀₀ using an Agilent LogPhase 600 Microbiology Reader (Santa Clara, CA). (B) Lag times. Data in panel A were used to determine lag times by extrapolation of the linear portion of the growth curve. Growth rates (µ, h⁻¹) calculated from five biological replicates are displayed in panel (B). Data are mean ± SD. Statistical significance was calculated with Student’s two-tailed t-test (****p≤0.0001).

Figure 1—figure supplement 4. Agr-mediated protection from H₂O₂-mediated killing among diverse S. aureus strains.

Laboratory strains LAC, RN6734, Newman (NM, BS12), MW2 (BS450), and clinical isolates BS39 and 126 a with agr deficient mutant derivatives were compared for survival following treatment with 20 mM of H₂O₂ for 60 min. In this experiment, overnight cultures were diluted in Tryptic Soy Broth (TSB) and grown to early log phase (OD₆₀₀∼0.15). Percent survival was determined relative to samples taken at the time of peroxide treatment. Some mutants were created by transduction of marker-disrupted alleles (LAC, RN6734, Newman, MW2) while others were naturally occurring (BS40, 127) (see Table 1). Data represent the mean ± SD. from biological replicates (n=3). The data show that peroxide lethality varies among strains, but in each case, deletion of agr increases killing.