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.s-Triazine rings are common scaffolds for the synthesis of industrial chemicals; they are found in pesticides, plastic resins, dyes, and explosives (20). s-Triazine herbicides are widely used in modern agriculture, where they kill susceptible plants by coordinating to the quinone-binding protein in photosystem II, thereby inhibiting photosynthetic electron transfer (19). Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-s-triazine) is one of the most widely used herbicides in the United States for the control of broadleaf weeds in corn, sorghum, and sugarcane (1). Other s-triazine herbicides include ametryn, atratone, cyanazine, prometryn, and simazine.The fate of s-triazine compounds in the environment depends on the metabolic activities of soil microorganisms (7,14). Since 1994, a number of laboratories have independently isolated, by enrichment culture, several genera of gram-negative bacteria capable of atrazine dechlorination (4,16,22,31,33,35,36,38). Subsequent to dechlorination, metabolism of hydroxyatrazine liberates nitrogen to sustain bacterial growth. More recently, gram-positive bacteria from the genera Nocardioides (32) and Arthrobacter (22) have been shown to grow on atrazine, the former using atrazine as the sole source of carbon and nitrogen for growth. Streptomyces strain PS1/5 was also shown to metabolize several s-triazine herbicides in the presence of additional carbon and nitrogen in the growth medium (29).It is now established that bacteria metabolize melamine and the triazine herbicides such as atrazine via enzyme-catalyzed hydrolytic reactions (6,11,12,35). The enzymatic basis of atrazine mineralization has been most extensively studied in Pseudomonas sp. strain ADP (3,8,16,17...

Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.

The TrzN protein, which is involved in s-triazine herbicide catabolism by Arthrobacter aurescens TC1, was cloned and expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified via nickel column chromatography. The purified TrzN protein was tested with 31 s-triazine and pyrimidine ring compounds; 22 of the tested compounds were substrates. TrzN showed high activity with sulfur-substituted s-triazines and the highest activity with ametryn sulfoxide. Hydrolysis of ametryn sulfoxide by TrzN, both in vitro and in vivo, yielded a product(s) that reacted with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) to generate a diagnostic blue product. Atrazine chlorohydrolase, AtzA, did not hydrolyze ametryn sulfoxide, and no color was formed by amending those enzyme incubations with NBD-Cl. TrzN and AtzA could also be distinguished by reaction with ametryn. TrzN, but not AtzA, hydrolyzed ametryn to methylmercaptan. Methylmercaptan reacted with NBD-Cl to produce a diagnostic yellow product having an absorption maximum at 420 nm. The yellow color with ametryn was shown to selectively demonstrate the presence of TrzN, but not AtzA or other enzymes, in whole microbial cells. The present study was the first to purify an active TrzN protein in recombinant form and develop a colorimetric test for determining TrzN activity, and it significantly extends the known substrate range for TrzN.

The environmental fate of agricultural chemicals is dependent upon their metabolism by microorganisms. s-Triazine herbicides figure prominently in chemical weed control. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-s-triazine) is one of the most widely used s-triazine herbicides for the control of broadleaf weeds in corn (50). Bacteria that metabolize and use s-triazine herbicides as their sole source of nitrogen for growth have only recently been isolated and characterized (9,27,34,46,48,49,52). Metabolism of the s-triazine herbicide atrazine has been extensively studied by using Pseudomonas sp. strain ADP (27) (Fig. 1). Metabolism is initiated via a dechlorination reaction catalyzed by atrazine chlorohydrolase (AtzA) (16,17). In the second reaction of the degradation pathway, AtzB catalyzes the hydrolysis of hydroxyatrazine to yield Nisopropylammelide (8). The third metabolic step utilizes Nisopropylammelide isopropylaminohydrolase (AtzC) to hydrolytically remove N-isopropylamine and generate cyanuric acid (38). Nearly identical genes encoding these enzymes have been found in a wide variety of gram-negative and gram-positive bacteria (9,15,34,36,49) and have been localized to a recently sequenced catabolic plasmid, pADP-1, in Pseudomonas sp. strain ADP (30). While atrazine metabolism is relatively rare among microorganisms, cyanuric acid can be metabolized by many soil bacteria (12,13,18,22). Thus, the advent of bacterial atrazine catabolism is thought to have required the relatively recent evolution of three new enzymes: AtzA, AtzB, and AtzC (41).Interestingly, AtzA, AtzB, and AtzC have all been identified as members of the amidohydrolase protein superfamily (38). Amidohydrolases are distributed throughout the three domains of living organisms: Eubacteria, Archaea, and Eucarya. Members of the superfamily catalyze the hydrolysis of amides or the CON bond of amines (19). Moreover, superfamily members are responsible for key steps in different metabolic pathways, such as purine-pyrimidine metabolism and the degradation of histidine and cytosine (19), and include cytosine deaminase, urease, adenosine deaminase, phosphotriesterase, and ammelide aminohydrolase. These and other amidohydrolase superfamily members have been found to contain mononuclear or binuclear metal centers (19). This has shifted attention to looking for putative metal-coordinating amino acids during sequence analysis of newly identified members of the amidohydrolase superfamily.AtzC shows only modest sequence identity, 27%, to the cytosine deaminase CodA (38). However, sequence analyses of the 35 amino acids that are proposed to reside near or at the four residues which are ligands to a putative metal center show that AtzC and cytosine deaminase have 61% overall sequence identity and 85% sequence similarity. CodA is active with bound