Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida.
The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.
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Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5500.
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D003001:Cloning, Molecular / Q000379:methods; D015342:DNA Probes; D005798:Genes, Bacterial; D007120:Immunochemistry; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011995:Recombination, Genetic
D015342:DNA Probes; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase