We transformed this ΔcrcB strain with an arabinose-inducible rescue vector bearing CLC F genes to be tested, monitoring growth in LB supplemented with NaF. coli does not carry any CLC F genes, but instead uses an exporter from the unrelated crcB family, deletion of which renders the bacterium highly sensitive to F - ( 12). We first examined these for their ability to protect E. Eight homologues produce protein of sufficient quality for studying F - transport in reconstituted proteoliposomes. We have so far screened 29 members of the CLC F clade ( 12) for overexpression in E. coli and catalyzes robust F - efflux in reconstituted liposomes. Accordingly, these CLCs were suggested ( 12) to act as F - exporters that protect bacteria from environmental F - toxicity, an idea supported by the demonstration that CLC-psy rescues F -dependent growth-arrest in E. Riboswitch-controlled CLCs differ substantially from the canonical members of the superfamily for instance, CLC-psy, a homologue from the plant pathogen Pseudomonas syringae, shares only 22% identity with CLC-ec1 and lacks the serine and tyrosine residues that directly coordinate the central Cl - ion in all CLCs of known structure. The riboswitch-associated CLCs cluster within a single clade in the phylogenetic tree, although not all CLCs in this clade are regulated by riboswitches.
#CLC SEQUENCE VIEWER NCBI CODE#
The genes controlled by these riboswitches code for F -sensitive enzymes, including enolase and pyrophosphatase, as well as membrane proteins, including members of the CLC superfamily. Baker and colleagues ( 12) described a conserved regulatory RNA motif that induces transcription of downstream genes upon binding F - ion an X-ray crystal structure of a F -riboswitch was recently solved ( 13). What biological roles might these strange CLCs play?Ī first hint at an unusual CLC physiology in bacteria recently emerged from a small clade evolutionarily distant from the well studied members of the superfamily ( Fig. 1). Many prokaryotic genomes encode CLCs, in some cases more than one, and some lack certain key residues found in every currently described CLC ( 10). But the CLC family is vast, with highly divergent sequences among its bacterial members. All of this known functional diversity resides in a remarkably narrow region of the family’s phylogeny ( Fig. 1) that includes all of the eukaryotic CLCs and their closest bacterial counterparts CLC-ec1, a Cl -/H + antiporter from Escherichia coli that has been subject to extensive structural and mechanistic study ( 9– 11) also resides in this region of the tree. Most CLCs thus far studied use Cl - for their physiological purposes, but has been identified as the substrate anion in a plant vacuolar CLC ( 8). CLCs participate in diverse biological tasks requiring transmembrane anion conductance, such as acidification of lysosomes, control of skeletal muscle excitability, renal regulation of blood pressure, and extreme acid resistance in enteric bacteria ( 7). This family turned out to be split into proteins of two mechanistically disparate subtypes: anion channels and Cl -/H + antiporters ( 3– 6). The CLC family of membrane proteins derives its name from its charter member, a double-barreled Cl - channel used by electric rays to generate high-power pulses to stun prey ( 1, 2). Finally, F -/H + exchange occurs with 1∶1 stoichiometry, in contrast to the usual value of 2∶1. Third, at a residue thought to distinguish CLC channels and transporters, CLC Fs bear a channel-like valine rather than a transporter-like glutamate, and yet are F -/H + antiporters.
Second, CLC Fs exhibit high anion selectivity for F - over Cl.
First, CLC Fs lack conserved residues that form the anion binding site in canonical CLCs. Sequence alignments and membrane transport experiments using 19F NMR, osmotic response assays, and planar lipid bilayer recordings reveal four mechanistic traits that set CLC F proteins apart from all other known CLCs. We establish here that a set of randomly selected representatives from this “CLC F” clade protect Escherichia coli from F - toxicity, and that the purified proteins catalyze transport of F - in liposomes. A subclass of bacterial CLC anion-transporting proteins, phylogenetically distant from long-studied CLCs, was recently shown to be specifically up-regulated by F.
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