Fluoride resistance and transport by riboswitch-controlled CLC antiporters

Two unrelated classes of F−-detoxifying transporters have been described: the CLCF-type F−/H+ antiporter (a subclass of the CLC superfamily, including the EriCF transporter of Pseudomonas syringae) and the 'Fluc' family of small membrane proteins.

“Two unrelated classes of F−‐detoxifying transporters have been described thus far: (i) the CLCF‐type F−/H+ antiporter, a subclass of the widespread CLC (voltage‐gated chloride channel) superfamily of anion‐transport proteins (Stockbridge et al., 2012)”

ClCF transporters function as proton-fluoride antiporters, facilitating active extrusion of fluoride from the cell.

“ClCF transporters function as proton-fluoride antiporters, facilitating active extrusion of fluoride from the cell (22).”

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).”

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).”

S. mutans possesses two genes that encode F-/H+ antiporters from the CLC family, termed CLCF.

“S. mutans possesses two genes that encode F-/H+ antiporters from the CLC (chloride channel) family of anion channels and transporters, termed CLCF (22).”

Streptococcus mutans extrudes intracellular fluoride using a CLCF F-/H+ antiporter.

“S. mutans possesses two genes that encode F-/H+ antiporters from the CLC (chloride channel) family of anion channels and transporters, termed CLCF (22).”

The fluoride-binding motifs in 1289 and 1290 are distinct from the selectivity filter sequences of CLC chloride transporters.

“which are quite distinct from the selectivity filter sequences of CLC chloride transporters (22, 43).”

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).”

ΔFluc E. coli is hypersensitive to F− in pH-neutral media, and this effect is reversed by expressing F− exporters with a rescue plasmid.

“In previous work, a Fluc-knockout (ΔFluc) strain of E. coli was shown to be hypersensitive to F−, as assessed by growth in pH-neutral media, and this effect could be reversed by expressing F− exporters in the bacteria with a rescue plasmid (Breaker, 2012; Stockbridge et al., 2012).”

The induction of the membrane export protein CrcB is highly protective against fluoride.

“The induction of the membrane export protein-designated CrcB is shown to be highly protective against fluoride (9, 12, 13).”

Fluoride-specific CLCFs can be identified by three signature sequences: GNNLI/GMGLI in the N-terminal domain, GREGT/V at the transport machinery, and GEVTP in the C-terminal domain.

“Fluoride-specific CLCFs can be identified based on three signature sequences: the GNNLI/GMGLI in the N-terminal domain that defines ion selectivity, GREGT/V at the heart of the transport machinery, and the GEVTP sequence in the C-terminal domain that contributes fluoride-binding residues77–79.”

CLCFs are fluoride/proton antiporters that harness the proton gradient to expel fluoride from the cytoplasm.

“The CLCFs are fluoride/proton antiporters that harness the proton gradient to expel fluoride from the cytoplasm77.”

CLCFs are >80-fold selective for fluoride over chloride.

“Biochemical experiments have demonstrated that CLCFs are >80-fold selective for fluoride over chloride, the halide that is closest in size and the main biological anion77,78.”

Expression of CLCF has been demonstrated to confer resistance to fluoride toxicity, supporting hypothesis that F− efflux is its physiological function.

“Moreover, expression of CLCF has been demonstrated to confer resistance to fluoride toxicity, supporting the hypothesis that F− efflux is its physiological function (172).”

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).”

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.”

The CLCF family are secondary active transporters that harness the proton motive force to export F− in exchange for H+ import.

“These proteins are secondary active transporters that harness the proton motive force to export F− in exchange for H+ import(1).”

Fluoride export proteins belong to one of two families: the CLCF family and the Fluc/FEX family.

“These proteins belong to one of two families, each with a completely different fold, and that export F− via fundamentally different mechanisms.”

The CLCFs harness the proton gradient directly, by coupling fluoride export to proton import.

“The CLCFs harness the proton gradient directly, by coupling fluoride export to proton import(1).”

Nearly all microorganisms possess membrane proteins dedicated to the export of fluoride ion (F−).

“Nearly all microorganisms possess membrane proteins dedicated to the export of fluoride ion (F−).”

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).”

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 transporters harness the transmembrane proton gradient to export one F− in exchange for one H+, and the unitary transport rates of different characterized homologues range from ~400-1000 ions per second.

“The transporters harness the transmembrane proton gradient to export one F− in exchange for one H+, and the unitary transport rates of different characterized homologues ranges from ~400–1000 ions per second (1, 50).”

CLCF representatives from both gram-negative and gram-positive bacteria protect Escherichia coli against fluoride toxicity when heterologously expressed.

“When heterologously expressed, CLCF representatives from both gram-negative and gram-positive bacteria protect Escherichia coli against fluoride toxicity (1).”

Representatives with and without riboswitches share basic transport properties, and the proteins in this clade were renamed CLCFs.

“Not all proteins in this clade are associated with a riboswitch, but representatives with and without riboswitches share basic transport properties, and the proteins in this clade were thus renamed CLCFs(1).”

Proteins in the CLCF clade lack many of the essential chloride-coordinating residues that are well-conserved in other chloride-transporting family members.

“Sequence alignments show that proteins in the CLCF clade lack many of the essential chloride-coordinating residues that are well-conserved in other chloride-transporting family members(1).”

One type of fluoride transport is passive, gradient-controlled passage via Fluc-F− channels.

“One such type of transport is a passive, gradient‐controlled passage of fluoride via what is known as Fluc‐F− channels.”

The cell can be rescued from fluoride by producing one or both of two non-homologous classes of transport proteins.

“Ultimately, the cell can be rescued from fluoride by producing one or both of two non‐homologous classes of transport proteins (Stockbridge et al., 2012; Ji et al., 2014; Last et al., 2018).”

The other class are F−/H+ antiporters that act similarly to multi-drug resistance transporters and use a proton gradient or ATP hydrolysis to expel fluoride against a gradient.

“The other class are F−/H+ antiporters that act similarly to multi‐drug resistance transporters and use a proton gradient or ATP hydrolysis to expel fluoride against a gradient (Fig. 5).”