Phylogenomic analysis of Cation Diffusion Facilitator proteins uncovers Ni 2+/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 ofnepAandcepAmetal inducible genes which independently conferred Ni 2+ and Co 2+ resistances to Rhizobium etliCFN42 and increased, respectively, Ni 2+ /Co 2+ and Co 2+ resistances 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 roduce a non-functional protein.