IDENTIFICATION OF THE METABOLIC INTERMEDIATES OF PHTHALATE BY TN5 MUTANTS OF PSEUDOMONAS-TESTOSTERONI AND ANALYSIS OF THE 4,5-DIHYDROXYPHTHALATE DECARBOXYLASE GENE

Phthalate-, 4,5-dihydro-4,5-dihydroxyphthalate (DDP)-, 4,5-dihydroxyph thalate (DHP)-, and protocatechuate-accumulating mutants of Pseudomona s testosteroni M4-1 were isolated by transposon insertion mutagenesis. From the DDP-accumulating, transposon-inserted mutant strain M4-122, the 4,5-dihydroxyphthalate decarboxylase gene which is involved in pht halate metabolism was cloned and its nucleotide sequence determined. T he structural gene, designated phtD, was 990 bp, corresponding to a pr otein of 330 amino acid residues (calculated molecular weight 37,200). The phtD gene was expressed in Escherichia coli by its own promoter l ocated upstream of the phtD open reading frame. The promoter shows hig h homology with the E. coli sigma(70) consensus sequence. The putative amino acid sequence has 77.6% homology with that of the pht5 gene fro m Pseudomonas putida. The nucleotide sequence of the upstream region o f the phtD gene has high homology with that of pht1, the putative posi tive regulator for the other pht genes of P. putida. The deduced amino acid sequence of the upstream region of the phtD gene also shows high similarity with that of Pht1. From these results, the genes of P. tes tosteroni and P. putida involved in phthalate metabolism would seem to have evolved from the same origin but diversified to compose differen t gene orders in different organisms.