Widespread genetic switches and toxicity resistance proteins for fluoride

Abstract

Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

Fig. 1

Fluoride binding by crcB motif RNAs. (A) Consensus sequence and structural model for crcB RNAs based on 2188 representatives from bacterial and archaeal species. P1, P2, P3 and pseudoknot labels identify extended base-paired substructures. (B) Sequence and secondary structure model for the WT 78 Psy RNA from P. syringae. Colored circles summarize the in-line probing results presented in (C). The two G residues preceding nucleotide 1 were added to facilitate RNA production. (C) Polyacrylamide gel electrophoresis analysis of an in-line probing assay with 78 Psy RNA and various amounts of fluoride. NR, T1, and ⁻OH designate no reaction, partial digestion with RNase T1 (cleaves after guanosines), or partial digestion with hydroxide ions (cleaves after any nucleotide), respectively. Precursor RNA (Pre) band and some RNase T1 product bands are labeled (left). Locations of fluoride-mediated spontaneous RNA cleavage suppression (regions 1, 3, 5, 6) and enhancement (regions 2, 4) are identified by vertical bars. (D) Plot of the normalized fraction of RNA cleavage versus fluoride ion concentration from the data in (C). Curves represent those expected for one-to-one binding with a K_D of 60 μM.

Fig. 2

Fluoride riboswitch-mediated gene control. (A) Solid media cultures of WT E. coli cells or crcB KO E. coli cells transformed with a riboswitch reporter fusion construct carrying the P. syringae eriC fluoride riboswitch. (B) Plot of the β-galactosidase reporter activity versus fluoride concentration (c) in liquid media supporting growth of transformed E. coli cells [see (A)] as quantified using Miller assays. WT and crcB KO E. coli cells grown in media supplemented with 50 mM NaCl (no added fluoride) yielded 0.06 and 15.5 Miller units, respectively.

Fig. 3

Evaluation of putative fluoride transport proteins. (A) Liquid cultures supplemented with specific amounts of NaF were inoculated with identical amounts of WT E. coli cells and the optical density (O.D.) at 600 nm was periodically recorded over a 16-hour period. (B) Growth curve plots for the E. coli crcB KO strain. (C) Anion efflux of an EriC^F protein associated with the P. syringae fluoride riboswitch. Gray and black lines depict ion-transport measurements from liposomes in the absence or presence of protein, respectively. The high fluoride baseline is due to the relative membrane permeability of HF (pK_a of 3.4) compared with chloride (pK_a of −7). Asterisks in (C) and (D) identify the times at which protein-mediated anion transport is initiated. (D) Anion efflux by an EriC protein from E. coli that is known to serve as a chloride transporter.