Fluoride Transport in a Strange Subclass of Bacterial CLC Proteins

Abstract

Among prokaryotes, CLC-type Cl- channels and transporters are phylogenetically abundant but functionally obscure. Enteric bacteria such as E. coli are known to use CLCs in extreme acid resistance, but many bacteria have several CLC genes from far-flung branches of the phylogenetic tree. We have been studying an evolutionarily distant CLC subclass that in sequence alignments lacks the canonical, mechanistically crucial central serine, which coordinates the central Cl- ion in E. coli CLC-ec1 and other well-studied homologues. The protein-level function of these “strange” CLCs, whose genes have recently been shown to be specifically upregulated by F- ion (J.L. Baker et al., in press), is unknown. Here we describe the overexpression, purification, and functional reconstitution of several CLC homologues that appear to protect diverse bacterial species from F- toxicity. These “fluoride-CLCs” catalyze robust F- transport in liposomes, as assayed by osmotic responses, F-/Cl- exchange, planar bilayer recording, and 19F-NMR. We are currently carrying out a detailed characterization of one of these homologues, CLC-ps from the plant pathogen Pseudomonas syringae, including quaternary architecture, anion selectivity, transport rate and mechanism, and the presence or absence of H+ coupling.