Pseudomonas sp. strain ADP is the model strain for studying bacterial degradation of the s-triazine herbicide atrazine. In this work, we focused on the expression of the atzDEF operon, involved in mineralization of the central intermediate of the pathway, cyanuric acid. Expression analysis of atzD-lacZ fusions in Pseudomonas sp. strain ADP and Pseudomonas putida showed that atzDEF is subjected to dual regulation in response to nitrogen limitation and cyanuric acid. The gene adjacent to atzD, orf99 (renamed here atzR), encoding a LysR-like regulator, was found to be required for both responses. Expression of atzR-lacZ was induced by nitrogen limitation and repressed by AtzR. Nitrogen regulation of atzD-lacZ and atzR-lacZ expression was dependent on the alternative factor N and NtrC, suggesting that the cyanuric acid degradation operon may be subject to general nitrogen control. However, while atzR is transcribed from a N -dependent promoter, atzDEF transcription appears to be driven from a 70 -type promoter. Expression of atzR from a heterologous promoter revealed that although NtrC regulation of atzD-lacZ requires the AtzR protein, it is not the indirect result of NtrC-activated AtzR synthesis. We propose that expression of the cyanuric acid degradation operon atzDEF is controlled by means of a complex regulatory circuit in which AtzR is the main activator. AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system.

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas ؊ mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas ؊ mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is a herbicide of the s-triazine family used for broad-leaf weed control in both crop and noncrop lands. Its widespread use and high mobility in soil have led to its frequent detection in surface water and groundwater at concentrations exceeding the maximum levels allowed (21,22,30,37). The high incidence of atrazine-contaminated water and the increasing concern about the toxicological and ecotoxicological properties of atrazine (3,6,16,17) have boosted research directed toward bioremediation of atrazine-polluted sites.A few laboratories have reported the isolation of bacteria with the ability to utilize atrazine, achieving in some cases the complete mineralization of the herbicide (see reference 29 and references therein). The best-characterized atrazine-mineralizing bacterial strain is Pseudomonas sp. strain ADP (23), which uses atrazine as the sole nitrogen source by means of a catabolic pathway comprising six enzymatic steps (25,40). The complete degradative pathway is encoded in the 108-kbp conjugative catabolic plasmid pADP-1, which was recently sequenced (25). The atzA, atzB, and atzC genes, responsible for the conversion of atrazine to cyanuric acid, are harbored at three distant positions within a large (Ͼ40 kbp) unstable region in pADP-1. Loss of one or more of these genes is the cause of the frequent appearance of Atr Ϫ (unable to utilize atrazine) mutants in nonselective medium (10). The genes involved in the s-triazine ring cleavage and ammonium release are clustered at a different location in pADP-1, to form the atzDEF operon (25). The atzA, atzB, and atzC genes have been shown to be widespread and plasmid borne in a number of independent isolates from different parts of the world (9, 10, 31, 39, 40).The influence of nitrogen compounds on the efficiency of atrazine catabo...

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