Characterization of the phthalate permease OphD from Burkholderia cepacia ATCC 17616

Abstract

The ophD gene, encoding a permease for phthalate transport, was cloned from Burkholderia cepacia ATCC 17616. Expression of the gene in Escherichia coli results in the ability to transport phthalate rapidly into the cell. Uptake inhibition experiments show that 4-hydroxyphthalate, 4-chlorophthalate, 4-methylphthalate, and cinchomeronate compete for the phthalate permease. An ophD knockout mutant of 17616 grows slightly more slowly on phthalate but is still able to take up phthalate at rates equivalent to that of the wild-type strain. This means that 17616 must have a second phthalate-inducible phthalate uptake system.

FIG. 1

Dendrogram showing the relationship of the 17616 ophD gene product to selected transport proteins. Permeases involved in aromatic acid transport fall into three families as defined by Pao et al. (16): the ACS family, the AAHS family, and the MHS family. The ACS family includes the phthalate transporter OphD from B. cepacia 17616 (this work), the putative phthalate transporter Pht1 from P. putida NMH102-2 (15), the putative tartrate transporters (TtuB) from A. vitis AB3 (20) and AB4 (7), the putative p-hydroxyphenylacetate permease HpaX from E. coli (17), and the putative transporter B2246 from E. coli (2). The MHS family includes the 4-methylphthalate transporter MopB from B. cepacia Pc701 (19), the citrate transporters CitH from Klebsiella pneumoniae (24) and CitA from Salmonella typhimurium (22), the α-ketoglutarate transporter KgtP from E. coli (21), the proline/betaine transporter ProP from E. coli (8), and the dicarboxylate transporter PcaT from P. putida PRS2000 (GenBank accession no. U48776). The AAHS family includes the p-hydroxybenzoate and protocatechuate transporter PcaK from P. putida (14), the putative aromatic acid transporter PcaK from Acinetobacter sp. strain ADP1 (12), the benzoate transporter BenK from Acinetobacter sp. strain ADP1 (5), the putative vanillate transporter VanK from Acinetobacter sp. strain ADP1 (GenBank accession no. AF009672), the muconate transporter MucK from Acinetobacter sp. strain ADP1 (25), and the 2,4-dichlorophenoxyacetate transporter TfdK from Ralstonia eutropha (13). The amino acid sequences were aligned with the Pileup program of the Genetics Computer Group package (9), the alignment was confirmed by visual inspection, and the phylogenetic tree was calculated with the PAUP program by using the minimal-distance method.

FIG. 2

Phthalate uptake by IPTG-induced E. coli JM109 containing the cloned 17616 ophD (■), the cloned DBO1 orf1/ophD (●), or the vector pALTER-Ex1 (▴). Error bars, standard deviation from three independent experiments.

FIG. 3

Phthalate uptake by B. cepacia 17616 (wild type) following growth on phthalate (■) or p-hydroxybenzoate (●) and by 17616-CZ1 (ophD knockout mutant) following growth on phthalate (▴). Error bars, standard deviation from three independent experiments.