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Applied and Environmental Microbiology, December 2003, p. 7401-7408, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7401-7408.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Transporter-Mediated Uptake of 2-Chloro- and 2-Hydroxybenzoate by Pseudomonas huttiensis Strain D1

A. S. Yuroff,^1, G. Sabat,^2, and W. J. Hickey^1,2*

Center for Molecular and Environmental Toxicology,¹ Department of Soil Science, University of Wisconsin—Madison, Madison, Wisconsin²

Received 17 March 2003/ Accepted 8 September 2003

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 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.

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* Corresponding author. Mailing address: Department of Soil Science, University of Wisconsin—Madison, Madison, WI 53706-1299. Phone: (608) 262-9018. Fax: (608) 265-2595. E-mail: wjhickey{at}wisc.edu.

Present address: Department of Pathobiological Sciences, University of Wisconsin—Madison, Madison, WI 53706-1299.

Present address: Biotechnology Center, University of Wisconsin—Madison, Madison, WI 53706-1299.

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Applied and Environmental Microbiology, December 2003, p. 7401-7408, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7401-7408.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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