Fluoride resistance and transport by riboswitch-controlled CLC antiporters

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• A novel subclass of bacterial CLC anion-transporting proteins, termed CLC(F), has been identified.
• These CLC(F) proteins are phylogenetically distinct from previously studied CLC proteins.
• The CLC(F) subclass is specifically upregulated by fluoride (F⁻).

Purpose of the Study:

• To investigate the protective role of CLC(F) proteins against fluoride toxicity in Escherichia coli.
• To characterize the mechanism of fluoride transport catalyzed by purified CLC(F) proteins.
• To elucidate the unique mechanistic features distinguishing CLC(F) proteins from other CLCs.

Main Methods:

• Functional assays using Escherichia coli expressing CLC(F) proteins.
• In vitro transport assays with purified CLC(F) proteins reconstituted into liposomes.
• Biophysical techniques including (19)F NMR, osmotic response assays, and planar lipid bilayer recordings.
• Sequence alignments and site-directed mutagenesis.

Main Results:

• Randomly selected CLC(F) representatives conferred protection against fluoride toxicity in E. coli.
• Purified CLC(F) proteins were shown to catalyze fluoride transport across liposomal membranes.
• Four unique mechanistic traits were identified: absence of canonical anion binding site residues, high selectivity for F⁻ over Cl⁻, a channel-like valine residue mediating F⁻/H⁺ antiport, and a 1:1 stoichiometry for F⁻/H⁺ exchange.

Conclusions:

• CLC(F) proteins represent a distinct class of anion transporters with unique functional properties.
• These proteins play a crucial role in bacterial defense against fluoride toxicity.
• The identified mechanistic features offer new insights into the diversity of CLC protein function and ion transport.