The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis

Abstract

Fluorinated organic chemicals, such as per- and polyfluorinated alkyl substances (PFAS) and fluorinated pesticides, are both broadly useful and unusually long-lived. To combat problems related to the accumulation of these compounds, microbial PFAS and organofluorine degradation and biosynthesis of less-fluorinated replacement chemicals are under intense study. Both efforts are undermined by the substantial toxicity of fluoride, an anion that powerfully inhibits metabolism. Microorganisms have contended with environmental mineral fluoride over evolutionary time, evolving a suite of detoxification mechanisms. In this perspective, we synthesize emerging ideas on microbial defluorination/fluorination and fluoride resistance mechanisms and identify best approaches for bioengineering new approaches for degrading and making organofluorine compounds.

Fig. 1. As the 13th most abundant element in Earth’s crust, fluorine has interacted with living systems since the inception of cellular life.

Left, During the pre-Anthropocene era of life, covering ~3.8 billion years, fluorine in the form of fluoride anion (F⁻), derived largely from minerals, exhibited toxicity to cells and protocells by binding to Mg²⁺ and Ca²⁺ centers in enzymes or ribozymes. Fluoride export functions arose early in evolution. Today, most living things avoid fluorine, but a few rare plants and prokaryotes naturally evolved to biosynthesize fluoroacetate as a metabolic toxin to kill competitors and predators. Right, In the last 100 years and into the Anthropocene, humans have exposed the biosphere to a tsunami of inorganic and organic fluorine compounds. Of greatest concern are the large number of per- and polyfluorinated compounds (PFAS), such as perfluorooctanoic acid (PFOA) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX), shown at the top right. PFAS are persistent in the environment, raising human and ecosystem health concerns.

Fig. 2. Microbial responses to fluoride stress.

Pink spheres represent F⁻, orange triangles represent PO₄⁻, and gray spheres represent Ca²⁺. Most bacteria exhibit a subset of these responses. (1) Flouride export is the first line of defense against environmental fluoride, which usually enters the cell via weak acid accumulation at low pH (bottom right). Bacteria typically encode one of the two types of fluoride exporters: Fluc (crcB) or CLC^F. (2) Fluoride-responsive riboswitches are widespread among bacteria, upregulating the expression of genes involved in fluoride resistance. These bind fluoride as a Mg²⁺-fluoride complex. Other unknown mechanisms of gene regulation also exist. (3) Weak acid accumulation of fluoride reduces the proton-motive force and decreases the cytoplasmic pH, which cells counteract by expressing Na⁺/H⁺ antiporters. Fluoride-acclimated microbes exhibit enduring changes in pH homeostasis. (4) Various microbes overexpress inorganic pyrophosphatase, other phosphatases, and phosphate importers. This might be partly to surmount inhibition of phosphoryl transfer enzymes by fluoride, but it has also been shown that phosphate protects cells from fluoride stress. (5) Fluoride and divalent cations like Ca²⁺ and Mg²⁺ form poorly soluble complexes, which alters divalent metal ion homeostasis. Divalent cation transporters are overrepresented in operons with fluoride export proteins. (6) Fluoride inhibits several glycolytic enzymes, notably enolase, decreasing intermediates in lower glycolysis and the TCA cycle. Bacteria respond to this inhibition in various ways, including overexpression of glycolytic enzymes, metabolic shift to anaerobic fermentation, or pausing metabolism and growth. (7) As a consequence of the perturbations to oxidative metabolism and metal ion homeostasis, many microbes mount an oxidative stress response when fluoride levels are high. (8) Although less well understood as part of a natural fluoride response, some bacteria are able to synthesize minerals, such as fluorapatite (shown), with lattices that incorporate fluoride and effectively sequester this ion, intra- or extracellularly. (9) Many microbes exhibit changes in extracellular phenotypes like adhesion, biofilm formation, cell membrane structure and integrity, and polysaccharide export upon fluoride stress.

Fig. 3. Distribution of fluoride exporters among representative bacterial species.

Genomes are from a phylogenetically representative genomes set curated by the Joint Genome Institute (GEBA dataset, Genomic Encyclopedia of Bacteria and Archaea, bacterial genomes only). At right are the exporter distributions for four major bacterial phyla, with phylogenetic branch lengths according to ref. .