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

Abstract

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, Ni²⁺/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 Asn⁸⁸ and Arg¹⁹⁷ whose mutations produce a non-functional protein.