Functional characterization and Me2+ ion specificity of a Ca2+-citrate transporter from Enterococcus faecalis

Abstract

Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg2+- citrate, Ca2+-citrate or Fe3+-citrate. The Fe 3+-citrate transporter of Streptococcus mutans clusters on the phylogenetic tree on a separate branch with a group of transporters found in the phylum Firmicutes which are believed to be involved in anaerobic citrate degradation. We have cloned and characterized the transporter from Enterococcus faecalis EfCitH in this cluster. The gene was functionally expressed in Escherichia coli and studied using right-side-out membrane vesicles. The transporter catalyzes proton-motive-force-driven uptake of the Ca 2+-citrate complex with an affinity constant of 3.5 μm. Homologous exchange is catalyzed with a higher efficiency than efflux down a concentration gradient. Analysis of the metal ion specificity of EfCitH activity in right-side-out membrane vesicles revealed a specificity that was highly similar to that of the Bacillus subtilis Ca2+-citrate transporter in the same family. In spite of the high sequence identity with the S. mutans Fe 3+-citrate transporter, no transport activity with Fe3+ (or Fe2+) could be detected. The transporter of E. faecalis catalyzes translocation of citrate in complex with Ca2+, Sr2+, Mn2+, Cd2+ and Pb2+ and not with Mg 2+, Zn2+, Ni2+ and Co2+. The specificity appears to correlate with the size of the metal ion in the complex. © 2006 The Authors.