FIG 5 .

CupA’s ability to reduce Cu²⁺ is necessary to prevent copper stress. (A) Maximum rate of CupA Cu²⁺ reduction and release of Cu¹⁺, as determined by BCS, for recombinant CupA and various mutations of the Cu¹⁺-binding domain, as described by Fu et al. (14). Each error bar represents the standard error of the mean of three experiments. The rate was averaged over a 5-s interval. (B) V_max and K_m of wild-type CupA and variants as determined by curve fits to a Michaelis-Menten model. (C) CopY Cu²⁺ reduction and release of Cu¹⁺ over time, as determined by BCS, for recombinant CupA and various mutations of the Cu¹⁺-binding domain at a 312.5 µM Cu²⁺ concentration. Each error bar represents the standard error of the mean of three experiments. (D) B_max and T₅₀ of wild-type and mutant forms of CupA as determined by curve fits to a hyperbola one-site model fit. (E) Endpoint growth of S. pneumoniae TIGR4, the ΔcupA mutant, the ΔcupA mutant with the empty pABG5 vector, and the ΔcupA mutant complemented with wild-type and various mutant forms of cupA at 150 µM Cu²⁺ in THYB at pH 6.5 at 37°C. *, P < 0.01 compared to the ΔcupA mutant complemented with wild-type cupA on the pABG5 vector in a Student t test. Each strain, except TIGR4, contains pABG5. Data are representative of three independent experiments with three replicates. (F) Zone of inhibition of S. pneumoniae TIGR4, the ΔcupA mutant, the ΔcupA mutant with the empty pABG5 vector, and the ΔcupA mutant complemented with wild-type and various mutant forms of cupA on TSA blood agar plates incubated overnight at 37°C. Zones were measured as the distance from the edge of the disc containing 10 µl of 1 M Cu²⁺. *, P < 0.01 compared to the ΔcupA mutant complemented with wild-type cupA on the pABG5 vector; **, P < 0.01 compared to the ΔcupA mutant complemented with wild-type cupA on the pABG5 vector and the ΔcupA mutant with the empty pABG5 vector in a Student t test. Data are representative of three independent experiments with six replicates each. N/A, not available.