Transporter-mediated uptake of 2-chloro- and 2-hydroxybenzoate by Pseudomonas huttiensis strain D1.
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.
Yuroff
A S
AS
Center for Molecular and Environmental Toxicology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Sabat
G
G
Hickey
W J
WJ
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000332:Aerobiosis; D000693:Anaerobiosis; D002723:Chlorobenzoates / Q000378:metabolism*; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D011549:Pseudomonas / Q000254:growth & development / Q000378:metabolism*; D020156:Salicylic Acid / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002723:Chlorobenzoates; D003470:Culture Media; D000255:Adenosine Triphosphate; C060415:2-chlorobenzoic acid; D020156:Salicylic Acid