A LOCUS OF PSEUDOMONAS-PICKETTII DTP0602, HAD, THAT ENCODES 2,4,6-TRICHLOROPHENOL-4-DECHLORINASE WITH HYDROXYLASE-ACTIVITY, AND HYDROXYLATION OF VARIOUS CHLOROPHENOLS BY THE ENZYME

Pseudomonas pickettii DTP0602 utilizes 2,4,6-trichlorophenol (2,4,6-TC P) as a sole source of carbon and energy. 2,4,6-TCP is dechlorinated a nd converted to 2,6-dichlorohydroquinone by a primarily attacking enzy me of catabolism. The genes encoding the enzyme were cloned using a tr ansposon tagging strategy in Escherichia coil and Pseudomonas putida. A kanamycin-resistant strain derived from P. pickettii DTP0602 by Tn5 insertion, which was named DTP6251, had less dechlorinase activity of 2,4,6-TCP than the parent strain, and 2,6-dihydroquinone accumulated i n the culture broth of this mutant. A DNA fragment containing Tn5 toge ther with its flanking region was isolated from strain DTP6251. Deleti on and subcloning analysis of the fragment showed that a 3.5-kb region flanked by Tn5 was essential for dechlorinase activity. Two open read ing frames denoted hadA and hadB were located in the region: hadA span ned 1,554 nucleotides and encoded a polypeptide with a deduced molecul ar mass of 58,540; hadB spanned 591 nucleotides and encoded a polypept ide with a deduced molecular mass of 21,167., A set of promoter sequen ces (sigma(54) recognition sequence; -GG-...-GC- at positions -24 and -12) was found upstream of hadA. Two polypeptides were produced when t his region was expressed under the control of the tac and trc promoter s in E. coil. HadA was a chlorophenol-4-hydroxylase that hydroxylated various chlorophenols other than 2,4,6-TCP at position 4 to yield corr esponding p-dihydroquinones. HadA seemed to be a flavoprotein because FAD and NADH were required for its hydroxylation activity in vitro. Ev en though both hadA and hadB were essential for expression of hydroxyl ase activity in vivo, the mixture consisting of HadA and NADH (and/or FAD) expressed hydroxylase activity in vitro. The product of the hadB gene (i.e., HadB) was not essential for expression of dechlorinase act ivity in vitro.