CLC^F, Not CrcB, Protects Pseudomonas putida ATCC 12633 During Intense Fluoride Stress

Fluoride is abundant in the Earth's crust but is rarely used by biological systems and is toxic inside cells. Most prokaryotic genomes contain genes encoding fluoride export channels, known as CrcB, or fluoride/proton antiporters, known as CLC^F. Prokaryotes rely on one type only. In this study, Pseudomonas putida ATCC 12633 that natively expressed a chromosomally-encoded CrcB was engineered with plasmids containing a CLC^F exporter gene. The addition of CLC^F and subsequent adaptive evolution made the cells resistant to > 500 mM sodium fluoride and able to degrade 150 mM 2-fluoropropionic acid and export 150 mM fluoride. In the absence of CLC^F and adaption, only 1 mM 2-fluoropropionic was degraded and culture density decreased. The adaption of multiple cell lines occurred uniformly. All increased their CLC^F gene copy and incurred mutations in the native CrcB. Two of those point mutations and a designed deletion were introduced into the unadapted wild-type strain and confirmed as CrcB knockouts. In total, the CLC^F antiporter was selected for under the conditions used and was necessary for biodegrading high concentrations of an organofluorine compound. Sustaining viability at high fluoride levels is relevant for engineering prokaryotes to biodegrade fluorinated chemicals and installing fluoride into compounds by biosynthesis.