| Cyanuric acid is one of the intermediates of atrazine biodegradation. Atrazine has been detected in ground and surface waters because of its widespread usage in the world. Few strains are known for their ability to degrade and mineralize atrazine. Some of the atrazine-catabolizing bacteria harbor atzD and trzD genes encoding for cyanuric acid amidohydrolase. In this study, 25 new strains having the ability to mineralize cyanuric acid were isolated from soil sample exposed to atrazine spill. Based on the sequence of their 16S rRNA gene, the 25 new strains were represented by bacteria in seven genera. The best BLAST hits were to: Alcaligenes sp., Tetrathiobacter kashmirae, Klebsiella sp., Ochrobactrum anthropi, Agrobacterium tumefaciens, Gordonia sp., and Achromobacter sp. Among these genera, Alcaligenes sp. and Tetrathiobacter kashmirae represented the majority of the strains, and Tetrathiobacter sp., Ochrobactrum sp., Gordonia sp., and Achromobacter sp. genera were previously unknown to have the ability to metabolize cyanuric acid.; Studies done using the polymerase chain reaction indicated the presence of trzD only among these new cyanuric acid-catabolizing bacteria, while atzD was completely absent. By sequencing, it was found that trzD is highly conserved among these new strains. By PCR, other genes encoding for atrazine and cyanuric acid degradation were found absent except atzC encoding N-isopropylammelide isopropylamiohydrolase. Surprisingly, the PCR results indicated the absence of genes (atzE, atzF, trzE and trzF) essential for mineralizing biuret and allophanate in the pathway of cyanuric acid degradation. However, strains were still able to degrade cyanuric acid. Therefore, there is a high possibility to find a new set of genes that are encoding for biuret and allophanate catabolism in the newly isolated strains.; Using 14C-labeled-cyanuric acid as a source of nitrogen by resting cells and cell free extract indicated that cyanuric acid was completely mineralized by newly isolated strains. HPLC analysis for the byproducts of cyanuric acid degradation indicated that allophanate, not urea, is the metabolite of biuret catabolism. Growth of new strains on equimolar amount of nitrogen from cyanuric acid and ammonium chloride indicated a full usage and corporation of the nitrogen of cyanuric acid into microbial mass. |
