Efflux of chromate by Pseudomonas aeruginosa cells expressing the ChrA protein

The ChrA protein of Pseudomonas aeruginosa plasmid pUM505 confers resistance to chromate. Using an in vitro system, we reported [Alvarez, A.H. et al. (1999) J. Bacteriol. 181, 7398–7400] that chromate resistance is based on energy-dependent efflux of chromate. It is shown here that ChrA determines in vivo efflux of 51CrO42− as well. Chromate-loaded cell suspensions of P. aeruginosa strain PAO1 harboring recombinant plasmid pEPL1, which expresses the ChrA protein, showed accelerated efflux of 51CrO42− as compared to the plasmidless chromate-sensitive derivative. After a 10-min loading, about 40% of 51CrO42− was lost from resistant cells in 15 min. Chromate efflux by resistant cells showed a typical saturation kinetics with an apparent Km of 82±11 μM chromate and a Vmax of 0.133±0.009 nmol chromate min−1 (mg protein)−1. Oxyanions sulfate and molybdate inhibited chromate efflux in a concentration-dependent fashion, whereas arsenate and ortho-vanadate had no significant effect on chromate release. Inhibition of chromate extrusion by valinomycin, nigericin, and carbonyl cyanide m-chlorophenylhydrazone, but not by oligomycin or dicyclohexylcarbodiimide, indicated that chromate efflux was driven by the membrane potential.