Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine

M. L. De Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, M. J. Sadowsky

Research output: Contribution to journalArticlepeer-review

111 Scopus citations

Abstract

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. 61:14511457, 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 fragment, designated pMD1, was shown to encode atrazine dechlorination activity in Escherichia coil DH5α. Atrazine degradation was demonstrated by a zone- clearing assay on agar medium containing crystalline atrazine and by chromatographic methods. A gene conferring the atrazine-clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragment in pACYC184, designated pMD4, and was expressed in E. coli. This result and random Tn5 mutagenesis established that the 1.9-kb AvaI fragment 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 hydroxyatrazine. Hydroxyatrazine was detected only transiently in E. coli containing pMD1. This is consistent with the idea that hydroxyatrazine is the first metabolite in atrazine degradation by Pseudomonas sp. strain ADP. A 0.6-kbApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine- degrading bacteria isolated in Switzerland and Louisiana. These data suggest that genes encoding atrazine hydrolysis to hydroxyatrazine are widespread in nature and contribute to the formation of hydroxyatrazine in soil, a reaction previously attributed solely to abiotic processes.

Original languageEnglish (US)
Pages (from-to)3373-3378
Number of pages6
JournalApplied and environmental microbiology
Volume61
Issue number9
DOIs
StatePublished - 1995

Fingerprint Dive into the research topics of 'Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine'. Together they form a unique fingerprint.

Cite this