Phylogenomic analysis of Cation Diffusion Facilitator proteins uncovers Ni2+/Co2+ transporters

The ubiquitous Cation Diffusion Facilitator proteins (CDF) play a key role in maintaining the cellular homeostasis of essential metal ions. Previous neighbor-joining phylogenetic analysis classified CDF proteins into three substrate-defined groups: Zn2+, Fe2+/Zn2+ and Mn2+. These studies were unable to discern substrate-defined clades for Ni2+, Co2+, Cd2+ and Cu2+ transporters, despite their existence in this family. In this study we improved the accuracy of this previous functional classification using a phylogenomic approach based on a thorough maximum-likelihood phylogeny and the inclusion of recently characterized CDF transporters. The inference of CDF protein function predicted novel clades for Zn2+, Fe2+, Cd2+ and Mn2+. The Ni2+/Co2+ and Co2+ substrate specificities of two clades containing uncharacterized proteins were defined through the functional characterization of nepA and cepA metal inducible genes which independently conferred Ni2+ and Co2+ resistances to Rhizobium etli CFN42 and increased, respectively, N-i2+/Co2+ and Co2+ resistances to Escherichia coli. Neither NepA nor CepA confer Zn2+, Fe2+ and Mn2+ resistances. The ability of NepA to confer Ni2+/Co2+ resistance is dependent on clade-specific residues Asn(88) and Arg(197) whose mutations produce a non-functional protein.