Fluoride resistance and transport by riboswitch-controlled CLC antiporters

Stockbridge, Randy B.; Lim, Hyun–Ho; Otten, Renee; Williams, Carole; Shane, Tania; Weinberg, Zasha; Miller, Christopher

doi:10.1073/pnas.1210896109

Cited by 129 publications

(180 citation statements)

References 34 publications

(39 reference statements)

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“…concentration of fluoride than WT, and the growth of this strain can be rescued on high fluoride media by the addition of the eriC F gene (22), a variant of eriC that encodes a F − /H + antiporter (31). Because eriC F can substitute for crcB, it is likely that these proteins have some level of functional equivalence, namely the transportation of fluoride ions.…”

Section: Significancementioning

confidence: 99%

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

Smith²,

Davis

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

132

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Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, 18 F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions. toxicity resistance | environmental toxin | ion transport | dual membrane topology

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Section: Significancementioning

confidence: 99%

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

Smith²,

Davis

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

132

View full text Add to dashboard Cite

show abstract

“…We hypothesize that the increased sensitivity of UF35 to low pH is due to an energy-spilling "futile proton cycle" (19). The fluoride antiporter not only acts as an F Ϫ /H ϩ antiporter but also facilitates proton influx (20). The protons extruded by proton pumps can reenter the cells through the antiporter, causing a futile cycle.…”

mentioning

confidence: 99%

A Single Nucleotide Change in the Promoter mutp Enhances Fluoride Resistance of Streptococcus mutans

Liao

Brandt

Zhang

et al. 2016

Antimicrob Agents Chemother

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dPreviously, we identified a single nucleotide mutation in the promoter (mutp) of the fluoride antiporter-coding genes in a naturally fluoride-resistant Streptococcus mutans strain. Here, we studied the role of this mutation in a defined genetic background. The results confirmed that this mutation alone confers fluoride resistance on S. mutans, as shown by growth and lactic acid production assays. This resistance was explained by constitutively higher mutp promoter activity and upregulation of the fluoride antiporter-coding genes. Fluoride is highly abundant in nature and is extensively used in oral hygiene products (1, 2). At high concentrations, it can be bactericidal. Due to the extensive use of fluoride, many microorganisms have developed resistance to fluoride (3). Recent studies revealed that a conserved fluoride antiporter protein (CrcB) mediates microbial fluoride resistance by exporting fluoride ions (F Ϫ ) to maintain a low F Ϫ level in the cell (4, 5). The expression of the fluoride antiporter genes can be regulated by fluoride-sensing riboswitches (4). However, these riboswitches are absent in microorganisms such as Streptococcus mutans and staphylococci (4). Little is known about the regulation of the fluoride antiporter genes in these microorganisms.In a previous study, we compared the genome sequence of a naturally selected fluoride-resistant S. mutans strain to that of a fluoride-sensitive strain using whole-genome shotgun sequencing and discovered a single nucleotide mutation (Ϫ44A¡C) located in the promoter of two tandem genes coding for the fluoride antiporters (6). The expression of these genes was 10-fold higher in the fluoride-resistant strain than in the fluoride-sensitive strain. Although this evidence indicated that this mutation is involved in the expression regulation of the fluoride antiporters, it is uncertain whether this mutation alone causes fluoride resistance, since an additional seven mutations were found in the genome comparison. Thus, we aimed to investigate the role of this single nucleotide mutation in the fluoride resistance of S. mutans in a defined genomic environment. The promoter containing this mutation is referred to as mutp.We constructed a clean mutp mutant from S. mutans strain UA159 using site-directed mutagenesis and an unlabeled gene replacement system (7). The successful introduction of the base change, Ϫ44A¡C, in mutp was confirmed by Sanger sequencing. The corresponding mutant strain was designated UF35 and char-

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“…The first, called CLC F , is a variant in the CLC superfamily, known for the transport or channeling of chloride ions. CLC F genes encode F Ϫ /H ϩ antiporters in eubacteria and are generally under the control of F Ϫ -sensitive riboswitches (8,14). The second, much more broadly distributed family is called Fluc in bacteria (formerly crcB) (15) and FEX 2 in eukaryotes (16).…”

mentioning

confidence: 99%

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

Smith

Gordon

Rivetta³

et al. 2015

Journal of Biological Chemistry

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Background: Fluoride is broadly toxic, and organisms use fluoride export (FEX) proteins to expel it. Results: FEX is a constitutively expressed fluoride channel, and mutations to the C-and N-terminal domains have asymmetric effects. Conclusion: Protection from fluoride is constantly needed, and a positive residue in the membrane is required. Significance: Understanding FEX furthers our knowledge of fluoride resistance mechanisms.

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Fluoride resistance and transport by riboswitch-controlled CLC antiporters

Cited by 129 publications

References 34 publications

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

A Single Nucleotide Change in the Promoter mutp Enhances Fluoride Resistance of Streptococcus mutans

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

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