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Department of Microbiology, Monash University, Clayton, Victoria 3168, Australia
2Author for correspondence: Christopher P. Saint. Tel: +61 8 259 0378. Fax: +61 8 259 0228. e-mail: chris.saint@sawater.sa.gov.au
ABSTRACT
We have determined the entire nucleotide sequence of a 4.4 kbp fragment of pMOP, a plasmid involved in 4-methylphthalate catabolism in Burkholderia cepacia (formerly Pseudomonas cepacia) Pc701. Two complete ORFs were identified and termed mopA and mopB. mopB encodes a 4-methylphthalate permease which is a member of a superfamily of symport proteins found in both prokaryotes and eukaryotes. Functionality was assigned to MopB by detailed analysis of the predicted amino acid sequence, resulting in the identification of 12 hydrophobic membrane-spanning domains and motifs associated with this class of protein. An assay was developed to demonstrate MopB function in substrate uptake. Of 4-methylphthalate, 4-hydroxyisophthalate, benzoate, p-toluate and phthalate, only uptake of 4-methylphthalate and phthalate was demonstrated, suggesting that two carboxyl groups in the ortho position are essential for substrate recognition. The predicted protein MopA showed significant levels of homology to reductase proteins implicated in aromatic and aliphatic catabolism, and contained motifs recognized as binding the ADP and flavin moieties of FAD/NAD. Northern hybridization experiments determined that mopA and mopB are cotranscribed, but expression was only seen in cells grown on 4-methylphthalate and not in cells grown on closely related structural analogues, including phthalate. mopA and mopB may be situated at the 3'-terminus of a cistron about 10 kbp in size. The isolation and characterization of a 4-methylphthalate permease gene may lead to the identification of other permeases involved in bacterial biodegradation processes and possibly the construction of strains with enhanced degradative abilities.
Present address: The Cooperative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, Bolivar, SA5108, Australia.
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