Cloning, characterization, and expression of genes encoding Pseudomonas putida salicylate hydroxylase and Pseudomonas cepacia salicylate hydroxylase and m-hydroxybenzoate-6-hydroxylase

A Pseudomonas cepacia DNA fragment containing the gene encoding a functional salicylate hydroxylase was cloned into pRO 2317 and expressed in P. aeruginosa PAO. The P. cepacia DNA fragment in the clone is 7.5 kb in size and its physical map has been constructed. The P. aeruginosa PAO containing the clone can grow at the expense of salicylate as the sole carbon source whereas the parental P . aeruginosa PAO can grow at the expense of catechol but not salicylate as the sole carbon source because of its deficiency of salicylate hydroxylase. Salicylate hydroxylase activities were detected in crude lysates prepared from cells of P. aeruginosa PAO containing the clone. The cloned salicylate hydroxylase was also identified by antibodies against purified P. cepacia salicylate hydroxylase. The gene for P. cepacia m-hydroxybenzoate-6-hydroxylase was cloned into pRO 1727 and expressed in P. aeruginosa PAO. The P. cepacia DNA fragment in the clone is 1.8 kb in size and its physical map has been constructed. The P. aeruginosa PAO containing the clone can be grown on m-hydroxybenzoate as the sole carbon source. However, the parental P. aeruginosa PAO can grow at the expense of gentisate but not m- hydroxybenzoate as the sole carbon source because of its deficiency of in-hydroxybenzoate-6-hydroxylase. Crude lysates prepared from cells of P. aeruginosa PAO containing the clone exhibited m-hydroxybenzoate-6-hydroxylase activities. The cloned enzyme was further detected by immunoprecipitation using polyclonal antibodies against purified P. cepacia m- hydroxybenzoate-6-hydroxylase. The gene for P. putida salicylate hydroxylase was subcloned into pRO 2317 and expressed in P. aeruginosa PAO. The two salicylate hydroxylases from P. putida and P. cepacia did not exhibit any homologies with respect to DNA cross hybridization and immunoblot with polyclonal antibodies against P. cepacia salicylate hydroxylase.