Cyanuric acid is an industrial chemical produced during the biodegradation of s-triazine pesticides. The biodegradation of cyanuric acid has been elucidated using a single model system, Pseudomonas sp. strain ADP, in which cyanuric acid hydrolase (AtzD) opens the s-triazine ring and AtzEG deaminates the ring-opened product. A significant question remains as to whether the metabolic pathway found in Pseudomonas sp. ADP is the exception or the rule in bacterial genomes globally. Here, we show that most bacteria utilize a different pathway, metabolizing cyanuric acid via biuret. The new pathway was determined by reconstituting the pathway in vitro with purified enzymes and by mining more than 250,000 genomes and metagenomes. We isolated soil bacteria that grow on cyanuric acid as a sole nitrogen source and showed that the genome from a Herbaspirillum strain had a canonical cyanuric acid hydrolase gene but different flanking genes. The flanking gene trtB encoded an enzyme that we show catalyzed the decarboxylation of the cyanuric acid hydrolase product, carboxybiuret. The reaction generated biuret, a pathway intermediate further transformed by biuret hydrolase (BiuH). The prevalence of the newly defined pathway was determined by cooccurrence analysis of cyanuric acid hydrolase genes and flanking genes. Here, we show the biuret pathway was more than 1 order of magnitude more prevalent than the original Pseudomonas sp. ADP pathway. Mining a database of over 40,000 bacterial isolates with precise geospatial metadata showed that bacteria with concurrent cyanuric acid and biuret hydrolase genes were distributed throughout the United States.IMPORTANCE Cyanuric acid is produced naturally as a contaminant in urea fertilizer, and it is used as a chlorine stabilizer in swimming pools. Cyanuric acid-degrading bacteria are used commercially in removing cyanuric acid from pool water when it exceeds desired levels. The total volume of cyanuric acid produced annually exceeds 200 million kilograms, most of which enters the natural environment. In this context, it is important to have a global understanding of cyanuric acid biodegradation by microbial communities in natural and engineered systems. Current knowledge of cyanuric acid metabolism largely derives from studies on the enzymes from a single model organism, Pseudomonas sp. ADP. In this study, we obtained and studied new microbes and discovered a previously unknown cyanuric acid degradation pathway. The new pathway identified here was found to be much more prevalent than the pathway previously established for Pseudomonas sp. ADP. In addition, the types of environment, taxonomic prevalences, and geospatial distributions of the different cyanuric acid degradation pathways are described here.

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