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: Zn(2+), Fe(2+)/Zn(2+) and Mn(2+). These studies were unable to discern substrate-defined clades for Ni(2+), Co(2+), Cd(2+) and Cu(2+) 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 Zn(2+), Fe(2+), Cd(2+) and Mn(2+). The Ni(2+)/Co(2+) and Co(2+) substrate specificities of two clades containing uncharacterized proteins were defined through the functional characterization of nepA and cepA metal inducible genes which independently conferred Ni(2+) and Co(2+) resistances to Rhizobium etli CFN42 and increased, respectively, Ni(2+)/Co(2+) and Co(2+) resistances to Escherichia coli. Neither NepA nor CepA confer Zn(2+), Fe(2+) and Mn(2+) resistances. The ability of NepA to confer Ni(2+)/Co(2+) resistance is dependent on clade-specific residues Asn(88) and Arg(197) whose mutations produce a non-functional protein.