Arthrobacter aurescens TC1 metabolizes diverse s-triazine ring compounds

Abstract

Arthrobacter aurescens strain TC1 was isolated without enrichment by plating atrazine-contaminated soil directly onto atrazine-clearing plates. A. aurescens TC1 grew in liquid medium with atrazine as the sole source of nitrogen, carbon, and energy, consuming up to 3,000 mg of atrazine per liter. A. aurescens TC1 is metabolically diverse and grew on a wider range of s-triazine compounds than any bacterium previously characterized. The 23 s-triazine substrates serving as the sole nitrogen source included the herbicides ametryn, atratone, cyanazine, prometryn, and simazine. Moreover, atrazine substrate analogs containing fluorine, mercaptan, and cyano groups in place of the chlorine substituent were also growth substrates. Analogs containing hydrogen, azido, and amino functionalities in place of chlorine were not growth substrates. A. aurescens TC1 also metabolized compounds containing chlorine plus N-ethyl, N-propyl, N-butyl, N-s-butyl, N-isobutyl, or N-t-butyl substituents on the s-triazine ring. Atrazine was metabolized to alkylamines and cyanuric acid, the latter accumulating stoichiometrically. Ethylamine and isopropylamine each served as the source of carbon and nitrogen for growth. PCR experiments identified genes with high sequence identity to atzB and atzC, but not to atzA, from Pseudomonas sp. strain ADP.

FIG. 1.

Specificity of s-triazine utilization by A. aurescens strain TC1. Substrates were added at a concentration of 200 mg per liter and used as the sole source of nitrogen. (A) Growth of strain TC1 on substrates where the chlorine substituent of atrazine was replaced with fluoro, methylmercapto, mercapto, O-methyl, cyano, hydrogen, azido, and amino functional groups; (B) growth of strain TC1 where the N-isopropyl group of atrazine was replaced with OH, NHCH₂CH₃, and NHC(CH₃)₂CN functional groups; (C) growth of strain TC1 on substrate variants containing symmetrical N-alkyl substituents.

FIG. 2.

Influence of different concentrations of atrazine on growth of A. aurescens TC1. Strain TC1 was grown in minimal medium using atrazine as a sole source of carbon, nitrogen, and energy. (A) Culture grown with 500 mg of atrazine per liter (□) and culture initially grown on 500 mg of atrazine per liter and receiving an additional 500 mg of atrazine per liter at the time indicated by the arrow (▪); r² = 0.90. (B) Linear correlation of growth of A. aurescens TC1 on several concentrations of atrazine; r² = 0.99.

FIG. 3.

Utilization of N-alkyl amines as a source of C and N by A. aurescens TC1. Cells were grown in minimal medium containing 7.5 mM N-isopropylamine plus 15 mM NH₄NO₃ (A), 7.5 mM N-isopropylamine (B), 7.5 mM ethylamine plus 15 mM NH₄NO₃ (C), and 7.5 mM ethylamine (D).

FIG. 4.

Atrazine metabolite(s) detected in stationary-phase cultures of A. aurescens TC1. UV absorbance and radioactivity as a function of HPLC retention time are shown for culture supernatants of stationary-phase A. aurescens TC1 grown in minimal medium containing cold and ring-labeled [¹⁴C]atrazine. (A) HPLC elution profile at 195 nm; (B) corresponding radioactivity.

FIG. 5.

Metabolites produced by A. aurescens strain TC1 during growth on atrazine. (A) HPLC elution profile of metabolites from culture supernatant at late exponential growth phase. Atrazine and hydroxyatrazine were eluted with the medium front. (B) MS of atrazine and metabolic intermediates produced by strain TC1 in culture filtrates sampled during exponential through stationary growth phase. The relative ion count for each compound is presented as a ratio of the ion count at each time point divided by the minimum ion count observed over the time course. •, atrazine; ○, hydroxyatrazine; ▪ (inset), N-isopropylammelide.

FIG. 6.

PCR amplification of A. aurescens TC1 total genomic DNA with primers for the atzB and atzC genes from Pseudomonas sp. strain ADP. Lanes: A, negative control (atzB primers and buffer); B, atzB primers with genomic DNA from A. aurescens TC1; C, atzB primers with genomic DNA from Pseudomonas sp. strain ADP; D, negative control (atzC primers and buffer); E, atzC primers with genomic DNA from A. aurescens TC1; F, atzC primers and genomic DNA from Pseudomonas sp. strain ADP.