Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum

Nitrogen Fixation Research Group, School of Biological and Environmental Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia ABSTRACT Mutants of Rhizobium leguminosarum bv. viciae MNF300 and R. leguminosarum bv. trifolii WU95 unable to accumulate 4-hydroxybenzoate lack 4-hydroxybenzoate hydroxylase. The capacity of these mutants to take up and grow on 4-hydroxybenzoate was restored by a 2.0 kb Eco RI- PstI DNA fragment. This contained only one ORF which had over 60% DNA sequence similarity with the structural gene for 4-hydroxybenzoate hydroxylase ( pobA ) from Pseudomonas spp. and Acinetobacter. Reported effects of metabolic inhibitors and substrate analogues on the apparent uptake of 4-hydroxybenzoate have now been shown to be due to their direct effect on 4-hydroxybenzoate hydroxylase. We propose that uptake of 4-hydroxybenzoate is via a metabolic ‘drag’ mechanism dependent on the activity of the pobA gene product. * Author for correspondence: Andrew R. Glenn. Tel: +61 9 360 6158. Fax: +61 9 310 3505. e-mail: arglenn@csuvax.csu.murdoch.edu.au Keywords: Rhizobium leguminosarum , aromatic substrate uptake, 4-hydroxybenzoate, sequence of pobA