Regulation of thePseudomonassp. Strain ADP Cyanuric Acid Degradation Operon

Abstract

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.

Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

“…1,4-Dioxane was used as internal reference and integration standard (assigned as ␦ ϭ 66.6 ppm relative to an external tetramethylsilane reference). Initial 13 C NMR results were confirmed using a Varian Inova 600 MHz NMR spectrometer with a 5-mm Auto-X Dual Broadband probe held at 25°C.…”

“…Initially, broadband proton-decoupled 13 C NMR spectra were acquired at 20°C and 100.5 MHz on a Varian Unity Inova 400 MHz NMR spectrometer equipped with a Nolorac 4 Nuc probe and operating under VnmrJ 2.2D software. 1,4-Dioxane was used as internal reference and integration standard (assigned as ␦ ϭ 66.6 ppm relative to an external tetramethylsilane reference).…”

“…The atzD gene in Pseudomonas sp. strain ADP is part of an operon that encodes the catabolism of the s-triazine compound cyanuric acid (13,26). Cyanuric acid catabolism releases ammonia to support cell growth.…”

New aspects on atrazine biodegradation

“…AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system. The mechanism for NtrC mediated activation of atzDEF expression has been not yet identified (García-González et al, 2005). The effect of nitrogen on the herbicide degradation pathway is strongly relevant to the use of this strain in bioremediation, since many agricultural fields are rich in nitrogen owing to routine fertilization.…”

“…The atzDEF operon resides in a contiguous cluster adjacent to the orf99, a potencial LysR-type transcriptional regulator (Martínez et al, 2001). In order to analyze the regulation of the expression of atzDEF genes, atzD-lacZ and atzR-lacZ transcriptions fusions were obtained, and the β-galactosidase assay was performed by García-González et al (2005). Expression of the cyanuric acid degradation atzDEF operon is specifically induced by cyanuric acid.…”