Fluoro-recognition: New in vivo fluorescent assay for toluene dioxygenase probing induction by and metabolism of polyfluorinated compounds.

The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.

Microwell Fluoride Screen for Chemical, Enzymatic, and Cellular Reactions Reveals Latent Microbial Defluorination Capacity for -CF3 Groups.

The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment. New discoveries will be greatly facilitated by the ability to screen many natural and engineered microbes in a combinatorial manner against large numbers of fluorinated compounds simultaneously. Here, we describe a low-volume, high-throughput screening method to determine defluorination capacity of microbes and their enzymes. The method is based on selective binding of fluoride to a lanthanum chelate complex that gives a purple-colored product. It was miniaturized to determine biodefluorination in 96-well microtiter plates by visual inspection or robotic handling and spectrophotometry. Chemicals commonly used in microbiological studies were examined to define usable buffers and reagents. Base-catalyzed, purified enzyme and whole-cell defluorination reactions were demonstrated with fluoroatrazine and showed correspondence between the microtiter assay and a fluoride electrode. For discovering new defluorination reactions and mechanisms, a chemical library of 63 fluorinated compounds was screened in vivo with Pseudomonas putida F1 in microtiter well plates. These data were also calibrated against a fluoride electrode. Our new method revealed 21 new compounds undergoing defluorination. A compound with four fluorine substituents, 4-fluorobenzotrifluoride, was shown to undergo defluorination to the greatest extent. The mechanism of its defluorination was studied to reveal a latent microbial propensity to defluorinate trifluoromethylphenyl groups, a moiety that is commonly incorporated into numerous pharmaceutical and agricultural chemicals. IMPORTANCE Thousands of organofluorine chemicals are known, and a number are considered to be persistent and toxic environmental pollutants. Environmental bioremediation methods are avidly being sought, but few bacteria biodegrade fluorinated chemicals. To find new organofluoride biodegradation, a rapid screening method was developed. The method is versatile, monitoring chemical, enzymatic, and whole-cell biodegradation. Biodegradation of organofluorine compounds invariably releases fluoride anions, which was sensitively detected. Our method uncovered 21 new microbial defluorination reactions. A general mechanism was delineated for the biodegradation of trifluoromethylphenyl groups that are increasingly being used in drugs and pesticides.

Unexpected Mechanism of Biodegradation and Defluorination of 2,2-Difluoro-1,3-Benzodioxole by Pseudomonas putida F1.

Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. IMPORTANCE There are more than 9,000 polyfluorinated compounds developed for commercial use, some negatively impacting human health, and they are generally considered to be resistant to biodegradation. Only a limited number of studies have identified microbes with enzymes sufficiently reactive to defluorinate difluoromethylene carbon groups. The present study examined one important group of commercial fluorinated chemicals and showed its rapid defluorination by a bacterium and its key enzyme, a Rieske dioxygenase. Rieske dioxygenases are common in environmental bacteria, and those closely resembling toluene dioxygenase from Pseudomonas putida F1 are candidates for biodegradative defluorination of the common 2,2-fluoro-1,3-benzodioxole (DFBD) moiety.

Cyanuric Acid Biodegradation via Biuret: Physiology, Taxonomy, and Geospatial Distribution.

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.