Figure 6
From: Cu Transport by the Extended Family of CcoA-like Transporters (CalT) in Proteobacteria
The RfnT-like CalT proteins do not restore the cbb3-Cox defect of R. capsulatus ΔccoA mutant. (A) R. capsulatus ΔccoA mutant (SE8), the ΔccoA mutant expressing either R. capsulatus CcoA (positive control) or the CalT from O. anthropi (CalT-O), R. palustris (CalT-R) or A. tumefaciens (CalT-A) were stained using the NADI procedure (Methods), which detects cbb3-Cox activity in R. capsulatus colonies via the development of a blue color. Note that cbb3-Cox activity was detected only in the presence of CcoA, and not the CalT-O, -R or -A proteins. (B) In vitro cbb3-Cox activities in chromotophore membranes of R. capsulatus SE8 strain expressing either CcoA or CalT-O, -R or -A. Cox assays were initiated by adding 30–150 μg of DDM-dispersed chromatophore membrane proteins to the assay buffer containing 0.1% DDM and 50 μM of reduced cyt c. Decrease in absorbance at 550 nm resulting from oxidation of cyt c was recorded. Addition of 0.1 mM KCN stopped immediately cyt c oxidation confirming the specificity of the cbb3-Cox activity. The cbb3-Cox activity determined for SE8/CcoA strain was 462 nmoles of cyt c oxidized/min/mg protein and set as 100%. Note that the R. capsulatus SE8 strains expressing CalT-O, -R or -A proteins have no cbb3-Cox activity. Mean values of at least three independent measurements with corresponding error bars are shown, and statistical analysis was performed using the Student’s t test, with p < 0.01 as the level of significance between CcoA (*) and the other strains. (C) Whole-cell Cu content measured by ICP-MS for wild-type R. capsulatus (WT), CcoA−, (ΔccoA) or a CcoA– strain expressing either CcoA or CalT-A. Each bar represents the mean values of four individually digested and independently measured samples from each strain and statistical analysis was performed using the Student’s t test, with p < 0.01 as the level of significance between CcoA (*) and the other strains.
