Transporter-MediatedUptake of 2-Chloro- and 2-Hydroxybenzoate by Pseudomonashuttiensis StrainD1

Abstract

ABSTRACT We investigated the mechanisms of uptake of 2-chlorobenzoate (2-CBa) and 2-hydroxybenzoate (2-HBa) by Pseudomonas huttiensis strain D1. Uptake was monitored by assaying intracellular accumulation of 2-[UL- ring - 14 C]CBa and 2-[UL- ring - 14 C]HBa. Uptake of 2-CBa showed substrate saturation kinetics with an apparent K m of 12.7 ± 2.6 μM and a maximum velocity ( V max ) of 9.76 ± 0.78 nmol min −1 mg of protein −1 . Enhanced rates of uptake were induced by growth on 2-CBa and 2-HBa, but not by growth on benzoate or 2,5-di-CBa. Intracellular accumulations of 2-CBa and 2-HBa were 109- and 42-fold greater, respectively, than the extracellular concentrations of these substrates and were indicative of uptake mediated by a transporter rather than driven by substrate catabolism (“metabolic drag”). Results of competitor screening tests indicated that the substrate range of the transporter did not include other o -halobenzoates that serve as growth substrates for strain D1 and for which the metabolism was initiated by the same dioxygenase as 2-CBa and 2-HBa. This suggested that multiple mechanisms for substrate uptake were coupled to the same catabolic enzyme. The preponderance of evidence from tests with metabolic inhibitors and artificial electrochemical gradients suggested that 2-CBa uptake was driven by ATP hydrolysis. If so, the 2-CBa transporter would be the first of the ATP binding cassette type implicated in uptake of haloaromatic acids.