Open AccessTransporter-MediatedUptake of 2-Chloro- and 2-Hydroxybenzoate by Pseudomonashuttiensis StrainD1
Author(s) -
Alice Yuroff,
Grzegorz Sabat,
William J. Hickey
Publication year - 2003
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.69.12.7401-7408.2003
Subject(s) - intracellular , transporter , extracellular , metabolism , biochemistry , catabolism , enzyme kinetics , substrate (aquarium) , efflux , biology , enzyme , chemistry , active site , gene , ecology
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-14C]CBa and 2-[UL-ring-14C]HBa. Uptake of 2-CBa showed substrate saturation kinetics with an apparent Km of 12.7 +/- 2.6 micromoles and a maximum velocity (Vmax) 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.