Characterization of S-triazine herbicide metabolism by a Nocardioides sp. isolated from agricultural soils

Abstract

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-(14)C]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 H(2)(18)O, 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 K(m) for atrazine of 25 microM and a V(max) of 31 micromol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 microM EDTA but was inhibited by 100 microM 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.

FIG. 1

Structures of s-triazine compounds used in this study.

FIG. 2

Phylogenetic tree based on the 16S ribosomal DNA sequence data showing the relationships of strains C157 and C190 with the most closely related bacteria identified in the GenBank database. Included in the analysis are Actinoplanes utahensis, Aeromicroblum fastidiosum, N. simplex, N. plantarum, Nocardioides albus, N. jensenii, S. lividans, and R. globerulus as an outlier. The bar indicates 0.01 substitutions per nucleotide position.

FIG. 3

Disposition of radioactivity during metabolism of [ring-U-¹⁴C]atrazine by whole cells of strain C190. Radioactivity is expressed as the area integrated under peaks.

FIG. 4

Mass spectra of an N-ethylammelide analytical standard (a) and of the end product of atrazine metabolism recovered from culture filtrates of Nocardioides strain C190 incubated in H₂¹⁶O (b) or in H₂¹⁸O (c).

FIG. 5

Proposed pathway of atrazine metabolism by Nocardioides sp. strain C190. Metabolite I is hydroxyatrazine, and metabolite II is N-ethylammelide.

FIG. 6

SDS-PAGE gel of purified triazine hydrolase. The numbers correspond to the molecular masses (in kilodaltons) of protein standards.