THE ATZB GENE OF PSEUDOMONAS SP STRAIN ADP ENCODES THE 2ND ENZYME OF A NOVEL ATRAZINE DEGRADATION PATHWAY

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encodi ng the first metabolic step for the degradation of the herbicide atraz ine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transform ation of hydroxyatrazine to N-isopropylammelide. The product was ident ified by use of high-performance liquid chromatography, mass spectrome try, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of p MD1 was used to determine that atzB was located 8 kb downstream of atz A. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce pla smid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstr ating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP , (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) cod on usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamin ation of the s-triazine substrate melamine. The atzA and atzB genes ca talyze the first two steps of the metabolic pathway in a bacterium tha t rapidly metabolizes atrazine to carbon dioxide, ammonia, and chlorid e.