CLONING, CHARACTERIZATION, AND EXPRESSION OF A GENE REGION FROM PSEUDOMONAS SP STRAIN ADP INVOLVED IN THE DECHLORINATION OF ATRAZINE

We previously identified a Pseudomonas sp. strain, ADP, which rapidly metabolized atrazine in liquid culture, agar plates, and soils (R, T. Mandelbaum, D. L. Allan, and L. P. Wackett, Appl. Environ. Microbiol, 61:1451-1457, 1995). In this study, we report the cloning and partial characterization of a gene region from Pseudomonas sp. strain ADP that encodes atrazine degradation activity. A 22-kb EcoRI genomic DNA frag ment, designated pMD1, was shown to encode atrazine dechlorination act ivity in Escherichia coli DH5 alpha. Atrazine degradation was demonstr ated by a zone clearing assay on agar medium containing crystalline at razine and by chromatographic methods. A gene conferring the atrazine- clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragmen t in pACYC184, designated pMD4, and was expressed in E. coli. This res ult and random Tn5 mutagenesis established that the 1.9-kb AvaI fragme nt was essential for atrazine dechlorination. High-pressure liquid and thin-layer chromatographic analyses were used to rigorously establish that E. coli containing pMD4 degraded atrazine and accumulated hydrox yatrazine. Hydroxyatrazine was detected only transiently in E. coli co ntaining pMD1. This is consistent with the idea that hydroxyatrazine i s the first metabolite in atrazine degradation by Pseudomonas sp. stra in ADP. A 0.6-kb ApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine-degrad ing bacteria isolated in Switzerland and Louisiana. These data suggest that genes encoding atrazine hydrolysis to hydroxyatrazine are widesp read in nature and contribute to the formation of hydroxyatrazine in s oil, a reaction previously attributed solely to abiotic processes.