Role of the CrcB transporter of Pseudomonas putida in the multi-level stress response elicited by mineral fluoride.

The presence of mineral fluoride (F ) in the environment has both a geogenic and anthropogenic origin, and the halide has been described to be toxic in virtually all living organisms. While the evidence gathered in different microbial species supports this notion, a systematic exploration of the effects of F salts on the metabolism and physiology of environmental bacteria remained underexplored thus far. In this work, we studied and characterized tolerance mechanisms deployed by the model soil bacterium Pseudomonas putida KT2440 against NaF. By adopting systems-level omic approaches, including functional genomics and metabolomics, we gauged the impact of this anion at different regulatory levels under conditions that impair bacterial growth. Several genes involved in halide tolerance were isolated in a genome-wide Tn-Seq screening-among which crcB, encoding an F -specific exporter, was shown to play the predominant role in detoxification. High-resolution metabolomics, combined with the assessment of intracellular and extracellular pH values and quantitative physiology experiments, underscored the key nodes in central carbon metabolism affected by the presence of F . Taken together, our results indicate that P. putida undergoes a general, multi-level stress response when challenged with NaF that significantly differs from that caused by other saline stressors. While microbial stress responses to saline and oxidative challenges have been extensively studied and described in the literature, very little is known about the impact of fluoride (F ) on bacterial physiology and metabolism. This state of affairs contrasts with the fact that F is more abundant than other halides in the Earth crust (e.g. in some soils, the F concentration can reach up to 1 mg g ). Understanding the global effects of NaF treatment on bacterial physiology is not only relevant to unveil distinct mechanisms of detoxification but it could also guide microbial engineering approaches for the target incorporation of fluorine into value-added organofluorine molecules. In this regard, the soil bacterium P. putida constitutes an ideal model to explore such scenarios, since this species is particularly known for its high level of stress resistance against a variety of physicochemical perturbations.