Figure 3: CopZ is required for Cu tolerance in R. capsulatus.

(A) WT R. capsulatus and the ΔcopZ strain, complemented with plasmid-borne copies of different CopZ variants when indicated were grown on MPYE medium until they reached the exponential phase. They were subsequently diluted in fresh medium to an OD₆₃₀ 0.6 and diluted with sterile MPYE medium in a 96-well plate. 5 μl of the 10³-10⁶ diluted samples were spotted onto pre-warmed MPYE agar plates supplemented with different concentrations of CuSO₄ and were analyzed after approx 24h incubation. (B) As in (A) but different R. capsulatus single and double mutants were analysed. Note, that the plate shown on the lower panel (wt and ΔccoA strain) was incubated only for 16 h for preventing the enrichment of revertants of the ΔccoA strain (Ekici et al., 2014). (C) Copper uptake was measured in whole cells by incubating cells with ⁶⁴CuCl and measuring Cu content in whole cells by scintillation counting. The values shown represent the mean of three independent experiments and the standard deviation is indicated by error bars.