Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes moonlighting transporter functions

Fluoride (F–) is the anionic form of fluorine, the 13th most abundant element in Earth crust, and it is toxic to many organisms above a threshold concentration. Environmental bacteria can withstand relatively high F– concentrations, but the only mechanism described so far is the CrcB dependent efflux. CrcB mediated export is the primary mechanism of F– tolerance in the model environmental bacterium Pseudomonas putida, yet spontaneous NaF-tolerant mutants arise in the absence of the CrcB transporter, showing that this is not the sole pathway of tolerance. We performed a genome wide search for factors affecting F– tolerance in P. putida. Screening >141,000 transposon mutants, we identified PP_3125, a Cro/cI type transcription regulator, as a determinant of high F– tolerance in a strain lacking the CrcB transporter. Proteomic and transcriptomic analyses revealed genes controlled by PP_3125, among which the benzoate transporter BenE-I was linked to the tolerance phenotype. These results show that transporters can adopt moonlighting functions, an aspect unexplored in bacteria. More broadly, the findings reveal alternative fluoride tolerance mechanisms in P. putida and define targets for engineering stress resilient microbial strains for industrial applications. Overall design: P. putida KT2440 (WT) or mutants derived (?crcB = DC; ?crcB ?PP_3125 = DD; ?PP_3125 = D3) grown in the presence (extra "F") or absence of NaF.