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Applied and Environmental Microbiology, August 2000, p. 3134-3141, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of S-Triazine Herbicide Metabolism by a Nocardioides sp. Isolated from Agricultural Soils

Edward Topp,1,* Walter M. Mulbry,2 Hong Zhu,1 Sarah M. Nour,1 and Diane Cuppels1

Agriculture and Agri-Food Canada, London, Ontario, Canada N5V 4T3,1 and Soil Microbial Systems Laboratory, ARS/U.S. Department of Agriculture, Beltsville, Maryland 207052

Received 30 December 1999/Accepted 11 May 2000

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified as Nocardioides sp. strains. None of the isolates mineralized [ring-U-14C]atrazine. There was no hybridization to genomic DNA from these strains using atzABC cloned from Pseudomonas sp. strain ADP or trzA cloned from Rhodococcus corallinus. S-Triazine degradation was studied in detail in Nocardioides sp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H218O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end product N-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have a Km for atrazine of 25 µM and a Vmax of 31 µmol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 µM EDTA but was inhibited by 100 µM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizing Nocardioides sp. widely distributed in agricultural soils degrades a range of s-triazine herbicides by means of a novel s-triazine hydrolase.

* Corresponding author. Mailing address: Agriculture and Agri-Food Canada, 1391 Sandford St., London, Ontario, Canada N5V 4T3. Phone: (519) 457-1470, ext. 235. Fax: (519) 457-3997. E-mail: toppe{at}em.agr.ca.

Applied and Environmental Microbiology, August 2000, p. 3134-3141, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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