CLONING AND CHARACTERIZATION OF EXTRADIOL AROMATIC RING-CLEAVAGE DIOXYGENASES OF PSEUDOMONAS-AERUGINOSA JI104

We have cloned multiple extradiol aromatic ring-cleavage dioxygenase ( EDO) genes from a gene library of Pseudomonas aeruginosa JI104, which is a benzene degrader isolated from soil near a gasworks. Southern hyb ridization analysis revealed that P. aeruginosa JI104 possessed three homologous catechol 2,3-dioxygenase (C230) genes. Nucleotide sequences of the cloned C230 genes, xylE(JI104-1, 2, 3) were almost identical t o that of the archetypal C230 gene (xylE(TOL)), which is carried on th e TOL plasmid, pWWO. We also cloned another EDO gene, bphC(JI104,) the product of which showed less activity for catechol than did XylE(JI10 4), but higher activity for 2,3-dihydroxy biphenyl. The nucleotide seq uence of bphC(JI104) was identical to that of bphC(KF707) (2,3-dihydro xybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes KF707). T he substrate specificities of the four EDOs of P. aeruginosa JI104 mer e markedly different from each other. Although XylE(JI104-1) and XylE( TOL) were 94% homologous, the specificities of the gene products for 4 -chlorocatechol were extremely different. Results of a study of the ch imeric enzymes composed of XylE(JI104-1) and XylE(TOL) N- and C-termin al regions showed that the difference in the specificity for 4-chloroc atechol was dependent on the C-terminal amino acid sequences. All of t he isofunctional homologous EDOs in P. aeruginosa JI104 seem to have b een derived from a common ancestor and evolved into the present forms in which each EDO is involved in a different degradation pathway and t hey all coexist in one strain.