CHARACTERIZATION AND EXPRESSION OF THE PLASMID-BORNE BEDD GENE FROM PSEUDOMONAS-PUTIDA ML2, WHICH CODES FOR A NAD(-DEPENDENT CIS-BENZENE DIHYDRODIOL DEHYDROGENASE())

The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the b ed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD(+)- dependent dehydrogenase (bedD) required to convert benzene into catech ol. Analysis of the nucleotide sequence upstream of the benzene dioxyg enase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp se quence identical to the inverted repeat of IS26, In vitro translation analysis showed bedD to code for a polypeptide of ca, 39 kDa, Both the nucleotide and the deduced amino acid sequences show significant iden tity to sequences of glycerol dehydrogenases from Escherichia coli, Ci trobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistanc e cassette was unable to utilize benzene for growth, The bedD gene pro duct was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenas e, The dehydrogenase encoded by bedD was overexpressed in Escherichia coli and purified. It was found to utilize NAD(+) as an electron accep tor and exhibited higher substrate specificity for cis-benzene dihydro diol and 1,2-propanediol compared with glycerol, Such a medium-chain d ehydrogenase is the first to be reported for a Pseudomonas species, an d its association with an aromatic ring-hydroxylating dioxygenase is u nique among bacterial species capable of metabolizing aromatic hydroca rbons.