Fig 3.

Validation of Tn-seq using single-gene deletion mutants. (A) S. pneumoniae WT and mutant strains were grown in a 96-well plate in brain heart infusion media for 6 hours at 37°C in the presence of 5% CO₂. Absorbance measurements (OD₆₂₀) were taken every 30 min, following which the media-alone sample values were subtracted. Data are presented as the mean ± SD from at least three independent experiments, the y-axis is log₁₀ scale. (B–F) S. pneumoniae strains (B) WT, (C) ΔahpD, (D) ΔtrxA, (E) ΔsodA, and (F) ΔspxB (OD₆₂₀ 0.02) were incubated in the presence or absence of 800 µM HOSCN ± catalase (CAT, 20 µg/mL) for 90 min in Hank’s balanced salt solution buffer, pH 6.8. Viable bacteria were determined by counting colony forming units and expressed relative to those before incubation (t ₀). Data are presented as the mean ± SD from at least three independent experiments, with symbols designating matching replicate data. A significant difference to the respective control was determined by paired t-tests and is indicated by * for P < 0.05. (D–F) Schematic representation of the function of proteins TrxA, SodA, and SpxB, in reducing oxidized thiols in proteins, detoxifying superoxide, and metabolizing pyruvate, respectively. The schematic representations were created with Biorender.com.