The cation diffusion facilitator protein EmfA of Rhizobium etli belongs to a novel subfamily of Mn2+/Fe2+ transporters conserved in α-proteobacteriaElectronic supplementary information (ESI) available. See DOI: 10.1039/c4mt00135d

Manganese (Mn 2+ ) plays a key role in important cellular functions such as oxidative stress response and bacterial virulence. The mechanisms of Mn 2+ homeostasis are not fully understood, there are few data regarding the functional and taxonomic diversity of Mn 2+ exporters. Our recent phylogeny of the cation diffusion facilitator (CDF) family of transporters classified the bacterial Mn 2+ -CDF transporters characterized to date, Streptococcus pneumoniae MntE and Deinococcus radiodurans DR1236, into two monophyletic groups. DR1236 was shown to belong to the highly-diverse metal specificity clade VI, together with TtCzrB, a Zn 2+ /Cd 2+ transporter from Thermus thermophilus , the Fe 2+ transporter Sll1263 from Synechocystis sp and eight uncharacterized homologs whose potential Mn 2+ /Zn 2+ /Cd 2+ /Fe 2+ specificities could not be accurately inferred because only eleven proteins were grouped in this clade. A new phylogeny inferred from the alignment of 197 clade VI homologs revealed three novel subfamilies of uncharacterized proteins. Remarkably, one of them contained 91 uncharacterized α-proteobacteria transporters (46% of the protein data set) grouped into a single subfamily. The Mn 2+ /Fe 2+ specificity of this subfamily was proposed through the functional characterization of the Rhizobium etli RHE_CH03072 gene. This gene was upregulated by Mn 2+ , Zn 2+ , Cd 2+ and Fe 2+ but conferred only Mn 2+ resistance to R. etli. The expression of the RHE_CH03072 gene in an E. coli mntP/zitB/zntA mutant did not relieve either Zn 2+ or Mn 2+ stress but slightly increased its Fe 2+ resistance. These results indicate that the RHE_CH03072 gene, now designated as emfA , encodes for a bacterial Mn 2+ /Fe 2+ resistance CDF protein, having orthologs in more than 60 α-proteobacterial species. Phylogenetic and functional analysis of a novel Mn 2+ /Fe 2+ -CDF transporter.