Efflux of chromate by Pseudomonas aeruginosa cells expressing the ChrA protein

Abstract

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 51CrO(4)(2-) as well. Chromate-loaded cell suspensions of P. aeruginosa strain PAO1 harboring recombinant plasmid pEPL1, which expresses the ChrA protein, showed accelerated efflux of 51CrO(4)(2-) as compared to the plasmidless chromate-sensitive derivative. After a 10-min loading, about 40% of 51CrO(4)(2-) was lost from resistant cells in 15 min. Chromate efflux by resistant cells showed a typical saturation kinetics with an apparent K(m) of 82+/-11 microM chromate and a V(max) 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.