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.
Bibliographical noteFunding Information:
We thank Becky Parales for providing the recombinant E. coli strain pDTG602. We thank Thomas Niehaus for making available the Cary spectrophotometer. This project was partly funded by the MnDRIVE Industry and the Environment program and a grant from ExxonMobil Environmental and Property Solutions Company. We have no conflicts of interest to declare. L.P.W., K.G.A., and M.D.B. conceived and designed the experiments. M.D.B., K.G.A., and J.E.R. performed the experiments. M.D.B., K.G.A., J.E.R., and L.P.W. analyzed the data. M.D.B., K.G.A., and L.P.W. wrote the paper. All authors edited and approved the manuscript.
© 2022 American Society for Microbiology.
- Pseudomonas putida F1
- high throughput
- Biodegradation, Environmental
- Pseudomonas putida
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't