Chromate efflux by means of the ChrA chromate resistance protein from Pseudomonas aeruginosa

Abstract

Everted membrane vesicles of Pseudomonas aeruginosa PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more (51)CrO(4)(2-) than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent K(m) of 0.12 mM chromate and a V(max) of 0. 5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential.

FIG. 1

(A) Chromate uptake by everted membrane vesicles of PAO1 (○) and PAO1(pCRO616) (□). Membrane vesicles (0.15 mg/ml) were suspended in phosphate buffer with 200 μM ⁵¹CrO₄²⁻. After incubation at 25°C in the presence of 2 mM NADH, ⁵¹CrO₄²⁻ uptake was measured. Uptake by vesicles from PAO1(pCRO616) was assayed in the absence of NADH (■). (B) Kinetics of chromate uptake by vesicles of PAO1(pCRO616). The uptake conditions were as described above, except that various concentrations of chromate were tested and aliquots were filtered after a 10-min incubation. The solid line represents the best fit to the Michaelis-Menten (hyperbolic) equation. The inset shows a linear transformation of the Michaelis-Menten equation by using the Hanes-Woolf plot (14). Data are representative of three assays in duplicate.