Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride

After fluoride exporters, the next-most common class of riboswitch-associated genes is Na+/H+ antiporters, which contribute to pH homeostasis in bacteria.

“Indeed, after fluoride exporters, the next-most common class of riboswitch-associated genes is Na+/H+ antiporters67, which contribute to pH homeostasis in bacteria94.”

The best-studied regulatory element is the fluoride riboswitch, which binds fluoride as a fluoride-magnesium complex and upregulates transcription of downstream genes.

“The best-studied regulatory element is the fluoride riboswitch, which binds fluoride as a fluoride-magnesium complex and upregulates the transcription of downstream genes67,89.”

The gene encoding pyrophosphatase is among the genes most frequently associated with fluoride riboswitches, and in various organisms, expression of pyrophosphatase, polyphosphatase, and phosphate importers has been functionally linked to fluoride resistance.

“The gene encoding pyrophosphatase is among the genes most frequently associated with fluoride riboswitches67, and in various organisms, including Enterobacter cloacae FRM95, Acidithiophilus ferredoxins96, and S. cerevisiae97, the expression of pyrophosphatase, polyphosphatase, and phosphate importers has been functionally linked to fluoride resistance.”

Baker et al. (2012) reported that eriC and crcB are fluoride resistance genes from P. syringae and E. coli.

“Baker et al. (2012) reported that ‘eriC’ and ‘crcB’ are identified as fluoride resistance gene from P. syringae and E. coli.”

The gene crcB acts as a putative F− ion transporter and helps bacteria reduce the toxic effect of F− on the bacterial cell.

“The gene crcB act as putative F– ion transporter and help bacteria to reduce the toxic effect of F– on the bacterial cell (Baker et al. 2012).”

Microbial organisms naturally exposed to toxic F− levels have developed fluoride-sensing RNA devices, such as riboswitches, to control expression of genes encoding proteins that relieve the deleterious effects of fluoride.

“Furthermore, microbial organisms naturally exposed to toxic F− levels have developed fluoride‐sensing RNA devices (e.g. riboswitches, harnessed as biosensors in this study) to control the expression of genes encoding proteins that relieve the deleterious effects of this anion (Baker et al., 2012).”

CrcB channels are highly selective for fluoride.

“While ClCF transporters can transport both chloride and fluoride ions or only fluoride, CrcB channels are highly selective for fluoride (6, 12).”

There are two distinct classes of fluoride export proteins: ClCF and CrcB (Fluc) channels.

“There are two distinct classes of fluoride export proteins: ClCF and CrcB (Fluc) channels (5, 6, 22).”

ClCF transporters can transport both chloride and fluoride ions or only fluoride.

“While ClCF transporters can transport both chloride and fluoride ions or only fluoride, CrcB channels are highly selective for fluoride (6, 12).”

CrcB functions as a passive ion channel and is considered a primary defense against fluoride toxicity in many bacteria, including Escherichia coli, Bacillus subtilis, and Pseudomonas species.

“CrcB functions as a passive ion channel and is considered a primary defense mechanism against fluoride toxicity in many bacteria, including Escherichia coli, Bacillus subtilis, and Pseudomonas species (5–7).”

If fluoride is formed in the cytoplasm, it would be directly toxic, inhibiting critical enzymes at concentrations as low as 70 μM.

“If formed in the cytoplasm, fluoride would be directly toxic, inhibiting critical enzymes at concentrations as low as 70 μM (23, 24).”

Nature has evolved effective proteins to protect against fluoride leaking into cells from mineral fluoride.

“Thus, nature has evolved effective proteins to protect against fluoride leaking into cells from mineral fluoride (24, 49).”

The expression of the Fluc channel in A. bakii is controlled by a transcriptional F− riboswitch located upstream of crcB1.

“The expression of the Fluc channel in A. bakii is controlled by a transcriptional F− riboswitch located upstream of crcB1 (fig. S8A) (24, 26).”

Genetic knockout of fluoride exporters causes fluoride hypersensitivity in multiple species, including oral microbiota, reducing the inhibitory concentration of fluoride to the tens-to-hundreds of micromolar range.

“Genetic knockout of fluoride exporters causes fluoride hypersensitivity in multiple species, including oral microbiota, reducing the inhibitory concentration of fluoride to the tens-to-hundreds of micromolar range (17–19).”

The fluoride retention in biofilms with ∆CLCF S. mutans mirrors intracellular fluoride accumulation measured in other microbes that lack fluoride exporters.

“This effect mirrors the intracellular fluoride accumulation that has been measured in other microbes that lack fluoride exporters (17, 20, 52).”

The high fluoride accumulation in biofilms with ∆CLCF S. mutans is due to fluoride accumulation in the cytoplasm of species that lack a fluoride exporter.

“We propose that this is due to fluoride accumulation in the cytoplasm of the species that lack a fluoride exporter, as has been shown to occur in other fluoride export-deficient bacteria (17, 20).”

First evidence of molecular mechanism for bacterial resistance to fluoride was discovery of widespread family of fluoride-responsive riboswitches, found upstream of genes predicted to encode halide-specific membrane channels.

“The first evidence of a molecular mechanism for bacterial resistance to fluoride was the discovery of a widespread family of fluoride-responsive riboswitches, found upstream of genes predicted to encode halide-specific membrane channels (170–172).”

When coupled to reporter system, fluoride riboswitch has been shown to have utility as sensitive and biocompatible fluoride sensor in living bacteria or in wholly in vitro systems.

“When coupled to a reporter system, the fluoride riboswitch has also been shown to have utility as a sensitive and biocompatible fluoride sensor that can be used in living bacteria or in wholly in vitro systems (170, 173).”

A wild-type E. coli strain was employed in which a fluoride-sensing riboswitch from Pseudomonas syringae controls the expression of a lacZ reporter gene in a fluoride concentration-dependent manner.

“Specifically, we employed a previously described wild-type (WT) E. coli strain in which a fluoride-sensing riboswitch from Pseudomonas syringae controls the expression of a lacZ reporter gene in a fluoride concentration-dependent manner (Baker et al., 2012; Nelson et al., 2014).”

Knockouts of genomic Fluc/FEX proteins increase fluoride sensitivity of bacteria including E. coli, B. subtilis, and S. sanguinis, and fungi including S. cerevisiae, C. albicans, N. crassa, and A. fumigatus.

“Knockouts of genomic Fluc/FEX proteins increase fluoride sensitivity of bacteria including Escherichia coli(14, 25), Bacillus subtilis(25, 64), and oral bacterium Streptococcus sanguinis(49), and fungi including Saccharomyces cerevisiae(3, 26), Candida albicans(26), Neurospora crassa(26), and Aspergillus fumigatus(65).”

It is now firmly established that Fluc proteins are F−-specific ion channels that protect E. coli and yeasts from F− toxicity.

“It is now firmly established that Fluc proteins are F−-specific ion channels that protect E. coli and yeasts from F− toxicity (Baker et al., 2012; Stockbridge et al., 2012, 2013; Li et al., 2013).”

Mitigation of fluoride stress is multi-faceted, with some bacteria sensing toxic fluoride with a riboswitch, producing enzymes less sensitive to fluoride, inducing fluoride-specific membrane export proteins, and carrying out other as-yet-to-be defined functions.

“Mitigation of fluoride stress has been shown to be multi-faceted, with some bacteria sensing toxic fluoride with a riboswitch, producing enzymes less sensitive to fluoride, inducing fluoride-specific membrane export proteins, and carrying out other as-yet-to-be defined functions (5, 8, 9).”

WT bacteria mount a gene expression response at intracellular fluoride concentrations around 60 μM.

“Congruent with this, WT bacteria mount a gene expression response at intracellular fluoride concentrations around 60 μM67, and extracellular concentrations as low as 200 μM68.”

Genes annotated as haloacid dehalogenases are often associated with fluoride riboswitches.

“In addition, genes annotated as haloacid dehalogenases are often associated with riboswitches67.”

The gene encoding chorismate mutase is one of the most common genes in fluoride exporter gene neighborhoods, and its expression is upregulated in fluoride-resistant S. mutans together with the fluoride exporters in the same operon.

“The gene encoding chorismate mutase, a key enzyme in the biosynthesis of aromatic amino acids, is one of the most common genes in fluoride exporter gene neighborhoods67, and its expression is upregulated in fluoride-resistant S. mutans together with the fluoride exporters in the same operon91.”

Enolase is often observed in operons controlled by fluoride riboswitches, and in fluoride-resistant E. cloacae FRM, enolase transcripts are upregulated 176-fold in response to fluoride.

“Enolase is often observed in operons controlled by fluoride riboswitches67, and in fluoride-resistant E. cloacae FRM, enolase transcripts are upregulated 176-fold in response to fluoride95.”

Formate hydrogen lyase is another of the most common fluoride riboswitch-associated enzymes.

“Formate hydrogen lyase, which oxidizes formic acid to produce ATP during anaerobic sugar fermentation, is another of the most common fluoride riboswitch-associated enzymes67.”

The most common proteins found in operons with fluoride riboswitches are fluoride exporters, although only ~15% of Flucs and ~30% of CLCFs are regulated by riboswitches.

“The most common proteins found in operons with these riboswitches are the fluoride exporters67, although only ~15% of Flucs and ~30% of CLCFs are regulated by riboswitches.”

New insights into the genes and mechanisms used by bacterial cells to overcome fluoride toxicity were revealed by the discovery of fluoride-responsive riboswitches that control the expression of genes conferring resistance to fluoride.

“New insights into the genes and mechanisms used by bacterial cells to overcome fluoride toxicity were revealed by the discovery of fluoride-responsive riboswitches that control the expression of a number of genes conferring resistance to fluoride (Baker et al., 2012).”

Numerous other genes presumably involved in mitigating fluoride toxicity are also associated with fluoride riboswitches.

“Numerous other genes presumably involved in mitigating fluoride toxicity are also associated with fluoride riboswitches (Baker et al., 2012).”

WT E. coli cultures can still grow at fluoride concentrations as high as 100 mM.

“which can still grow at concentrations as high as 100 mM (Baker et al., 2012).”

The most common gene associated with fluoride riboswitches, crcB (also called fex or fluc), codes for a fluoride-specific channel protein.

“The most common gene associated with fluoride riboswitches, termed crcB (also called fex, or fluc), codes for a fluoride-specific channel protein (Baker et al., 2012; Li et al., 2013; Stockbridge et al., 2013).”

Another commonly controlled gene, eriCF, encodes a fluoride-selective antiporter.

“Another commonly controlled gene, eriCF, encodes a fluoride-selective antiporter (Baker et al., 2012; Stockbridge et al., 2012).”

An E. coli strain in which the fluc gene was deleted (Δfluc) is approximately 200-fold more sensitive to fluoride.

“An E. coli strain in which the fluc gene was deleted (Δfluc) is ~200-fold more sensitive to fluoride (Baker et al., 2012).”

The deleterious phenotype resulting from fluc deletion in E. coli can be compensated by expressing an eriCF gene derived from another bacterium.

“Moreover, the deleterious phenotype resulting from fluc deletion in E. coli can be compensated by expressing an eriCF gene derived from another bacterium (Baker et al., 2012)”

The fluoride-sensing riboswitch is the most selective biosensor for fluoride currently known.

“Therefore, we designed a screen that utilized the most selective biosensor for fluoride currently known – a recently discovered fluoride sensing riboswitch (Baker et al., 2012).”

The proteins expressed from eriCF appear to be biologically equivalent to those encoded by fluc.

“indicating that the proteins expressed from eriCF appear to be biologically equivalent to those encoded by fluc.”

Fluc proteins are F--specific ion channels of the Fluc family.

“microorganisms export this anion through F--specific ion channels of the Fluc family1–4.”

Unicellular organisms counteract F- toxicity through F--exporting membrane transport proteins that keep cytoplasmic F- below inhibitory levels.

“Unicellular organisms directly exposed to environmental F- counteract the anion’s toxicity through the action of F--exporting membrane transport proteins that keep cytoplasmic F- below inhibitory levels1–4,8.”

Expression of F− export proteins in the plasma membrane undermines weak acid accumulation of fluoride.

“By providing a route for accumulating F− to exit the cell, expression of these F− export proteins in the plasma membrane simply undermines weak acid accumulation of fluoride (1, 14, 25, 26).”

The conserved crcB riboswitch motif, present in all domains of life, acts as a transcriptional "on-switch" upon fluoride ion binding.

“However, the existence of the microbial fluoride response was unknown until 2012, when Ronald Breaker and colleagues showed that the conserved crcB riboswitch motif (Figure 2d), present in all domains of life, acts as a transcriptional “on-switch” upon fluoride ion binding(25).”

Fluoride exporters from the CLCF and Fluc families are not necessarily associated with a riboswitch; some, including the E. coli Fluc channel, are instead constitutively expressed.

“Fluoride exporters from the CLCF and Fluc families are not necessarily associated with a riboswitch(1, 25); some, including the E. coli Fluc channel, are instead constitutively expressed (14).”

The riboswitch-associated CLC proteins cluster together in a single bacterial clade of the CLC phylogeny.

“The riboswitch-associated CLC proteins cluster together in a single bacterial clade of the CLC phylogeny(25).”

Thousands of fluoride-sensing riboswitches have been discovered in many diverse bacterial strains.

“On the first point, thousands of fluoride‐sensing riboswitches (Fig. 5) have been discovered in many diverse bacterial strains (Baker et al., 2012; Breaker, 2012).”

Proteins expressed via fluoride riboswitches include higher copy numbers of enolase, stress genes, DNA repair functions and hypothetical proteins.

“Proteins expressed via fluoride riboswitches include higher copy numbers of enolase, stress genes, DNA repair functions and hypothetical proteins.”