Figure 7. Rot-mediated up-regulation of H₂O₂-stimulated genes relative to those in an agr mutant.

Genes shown are those up-regulated in a Δagr Δrot double mutant (BS1302) relative to that observed with the Δagr strain (BS1348). H₂O₂ treatment was for 30 min. Peroxide concentrations for Δagr (2.5 mM H₂O₂) and Δagr Δrot (10 mM H₂O₂) were determined to achieve ~50% cell survival [see Methods and Figure 7—figure supplement 1]. RNA-seq data are from three independent cultures. Heatmap colors indicate expression z-scores. See Supplementary file 3 for supporting information.

Figure 7—figure supplement 1. Normalization of the lethal concentration of H₂O₂ with wild-type and Δagr strains.

Overnight cultures were diluted into fresh Tryptic Soy Broth (TSB) medium and grown to early log phase (OD₆₀₀=0.15 to achieve sufficient colony forming units (CFU) for RNA-seq). These cultures were treated with the indicated concentrations of H₂O₂ for 30 min prior to measurement of survival by plating. Data represent the mean ± SD. from biological replicates (n=3). Bacterial strains were BS819 for WT and BS1348 for the agr mutant. To focus RNA-seq analysis on lethal rather than cell death responses, we sought to reduce H₂O₂ concentrations and thereby lethality to achieve ~50% (dotted line) cell survival, normalized to wild-type and Δagr mutant strains. Survival of the Δagr mutant with H₂O₂ for 30 min at a concentration of 2.5 mM closely approximated 50% survival of the wild-type with 10 mM H₂O₂, providing a basis for choice of concentrations and treatment time for RNA-seq analysis.