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

Fluoride (F-), the anionic form of fluorine and the 13th most abundant element in Earths crust, is toxic to most organisms above relatively low threshold concentrations. Environmental bacteria often tolerate elevated fluoride levels, but the only known resistance mechanism so far involves CrcB-mediated efflux. In the environmental bacterium Pseudomonas putida, CrcB export is the primary defense against fluoride stress. Yet, spontaneous NaF-tolerant mutants emerge even without this transporter, suggesting the existence of additional pathways. To uncover these mechanisms, we performed a genome-wide screen of over 141,000 transposon mutants. We identified PP_3125, a Cro/cI-type transcriptional regulator, as essential for high fluoride tolerance in a {Delta}crcB background. Transcriptomic and proteomic analyses revealed PP_3125-regulated genes, including the benzoate transporter BenE-I, which contributes directly to fluoride tolerance. These findings demonstrate that bacterial transporters can acquire moonlighting functions beyond their canonical roles and reveal previously unrecognized fluoride tolerance strategies in P. putida. Together, our results expand understanding of microbial adaptation to toxic ions and provide new targets for engineering stress-resilient strains for environmental and industrial applications. IMPORTANCEOur work identifies a new fluoride tolerance mechanism in Pseudomonas putida that functions independently of the well-characterized CrcB efflux system. We show that inactivation of transcriptional regulator, PP_3125, activates a transporter with an unexpected moonlighting role in fluoride tolerance, highlighting how bacteria can repurpose existing functions to survive environmental stress. This discovery deepens our understanding of microbial stress responses and suggests strategies to engineer robust microbial strains capable of thriving in fluoride-contaminated settings. Such strains could be valuable for bioremediation, sustainable bioprocessing, and other biotechnological applications where fluoride exposure limits microbial performance.