Solute carriers 11 (Slc11) evolved from bacterial permease (MntH) to eukaryotic antibacterial defense (Nramp) while continuously mediating proton (H⁺)-dependent manganese (Mn²⁺) import. Also, Nramp horizontal gene transfer (HGT) toward bacteria led to mntH polyphyly. Prior demonstration that evolutionary rate-shifts distinguishing Slc11 from outgroup carriers dictate catalytic specificity suggested that resolving Slc11 family tree may provide a function-aware phylogenetic framework. Hence, MntH C (MC) subgroups resulted from HGTs of prototype Nramp (pNs) parologs while archetype Nramp (aNs) correlated with phagocytosis. PHI-Blast based taxonomic profiling confirmed MntH B phylogroup is confined to anaerobic bacteria vs. MntH A (MA)’s broad distribution; suggested niche-related spread of MC subgroups; established that MA-variant MH, which carries ‘eukaryotic signature’ marks, predominates in archaea. Slc11 phylogeny shows MH is sister to Nramp. Site-specific analysis of Slc11 charge network known to interact with the protonmotive force demonstrates sequential rate-shifts that recapitulate Slc11 evolution. 3D mapping of similarly coevolved sites across Slc11 hydrophobic core revealed successive targeting of discrete areas. The data imply that pN HGT could advantage recipient bacteria for H⁺-dependent Mn²⁺ acquisition and Alphafold 3D models suggest conformational divergence among MC subgroups. It is proposed that Slc11 originated as a bacterial stress resistance function allowing Mn²⁺-dependent persistence in conditions adverse for growth, and that archaeal MH could contribute to eukaryogenesis as a Mn²⁺ sequestering defense perhaps favoring intracellular growth-competent bacteria.

Transmembrane carriers of the Slc11 family catalyze proton (H⁺)-dependent uptake of divalent metal ions (Me²⁺) such as manganese and iron—vital elements coveted during infection. The Slc11 mechanism of high-affinity Me²⁺ cell import is selective and conserved between prokaryotic (MntH) and eukaryotic (Nramp) homologs, though processes coupling the use of the proton motive force to Me²⁺ uptake evolved repeatedly. Adding bacterial piracy of Nramp genes spread in distinct environmental niches suggests selective gain of function that may benefit opportunistic pathogens. To better understand Slc11 evolution, Alphafold (AF2)/Colabfold (CF) 3D predictions for bacterial sequences from sister clades of eukaryotic descent (MCb and MCg) were compared using both native and mutant templates. AF2/CF model an array of native MCb intermediates spanning the transition from outwardly open (OO) to inwardly open (IO) carriers. In silico mutagenesis targeting (i) a set of (evolutionarily coupled) sites that may define Slc11 function (putative synapomorphy) and (ii) residues from networked communities evolving during MCb transition indicates that Slc11 synapomorphy primarily instructs a Me²⁺-selective conformation switch which unlocks carrier inner gate and contributes to Me²⁺ binding site occlusion and outer gate locking. Inner gate opening apparently proceeds from interaction between transmembrane helix (h) h5, h8 and h1a. MCg1 xenologs revealed marked differences in carrier shape and plasticity, owing partly to an altered intramolecular H⁺ network. Yet, targeting Slc11 synapomorphy also converted MCg1 IO models to an OO state, apparently mobilizing the same residues to control gates. But MCg1 response to mutagenesis differed, with extensive divergence within this clade correlating with MCb-like modeling properties. Notably, MCg1 divergent epistasis marks the emergence of the genus Bordetella-Achromobacter. Slc11 synapomorphy localizes to the 3D areas that deviate least among MCb and MCg1 models (either IO or OO) implying that it constitutes a 3D network of residues articulating a Me²⁺-selective carrier conformation switch which is maintained in fast-evolving clades at the cost of divergent epistatic interactions impacting carrier shape and dynamics.

Transmembrane carriers of the Slc11 family catalyze proton (H+)-dependent uptake of divalent metal-ions (Me2+) such as manganese and iron - vital elements coveted during infection. Slc11 mechanism of high affinity Me2+ cell import is selective and conserved between prokaryotic (MntH) and eukaryotic (Nramp) homologs, though processes coupling the use of the proton motive force to Me2+ uptake evolved repeatedly. Adding bacterial piracy of Nramp genes spread in distinct environmental niches suggests selective gain of function that may benefit opportunistic pathogens. To better understand Slc11 evolution Alphafold (AF2)/Colabfold (CF) 3D predictions for bacterial sequences from sister clades of eukaryotic descent (MCb and MCg) were compared using both native and mutant templates. AF2/CF model an array of native MCb intermediates spanning the transition from outward open (OO) to inward open (IO) carrier. In silico mutagenesis targeting i) a set of (evolutionary coupled) sites that may define Slc11 function (putative synapomorphy), and ii) residues from networked communities evolving during MCb transition, indicate Slc11 synapomorphy primarily instructs Me2+-selective conformation switch which unlocks carrier inner gate, and contributes to Me2+ binding site occlusion and outer gate locking. Inner gate opening apparently proceeds from interaction between transmembrane helix (h) h5, h8 and h1a. MCg1 xenologs revealed marked differences in carrier shape and plasticity, owing partly to altered intramolecular H+-network. Yet, targeting Slc11 synapomorphy also converted MCg1 IO models to OO state, apparently mobilizing the same residues to control gates. But MCg1 response to mutagenesis differed, extensive divergence within this clade correlating with MCb-like modeling properties. Notably, MCg1 divergent epistasis marks emergence of the genus Bordetella-Achromobacter. Slc11 synapomorphy localizes to the 3D areas that deviate least among MCb and MCg1 models (either IO or OO) implying it constitutes a 3D network of residues articulating Me2+-selective carrier conformation switch which is maintained in fast evolving clades at the cost of divergent epistatic interactions impacting carrier shape and dynamics.

The Lactobacillales (LB) stand apart among bacterial orders, using manganese (Mn) instead of iron to support their growth and swiftly ferment complex foods while acidifying their environment. The present work investigates whether a shift in the use of Mn could mark the origin of LB. Transmembrane carriers of the ubiquitous Slc11 family play key roles in LB physiology by catalyzing proton-dependent Mn import. In prior studies, the Slc11 clade found in LB (MntH Cb, MCb) showed both remarkable structural plasticity and highly efficient Mn uptake, and another Slc11 clade, MCg1, demonstrated divergent evolution coinciding with emergence of bacterial genera (e.g., Bordetella, Achromobacter). Herein, the Slc11 clade MCb is subdivided in sister groups: MCb_ie and MCb_gut. MCb_ie derives directly from the Slc11 clade MCa, pointing an intermediate stage in the evolution of MCb_gut. MCb_ie predominates in marine Bacillaceae, is more conserved than MCb_gut, lacks the structural plasticity that typify MCb_gut carriers, and responds differently to identical mutagenesis. Exchanging MCb_ie/MCb_gut amino acid residues at sites that distinguish these clades showed conformation-dependent effects with both MCb_ie and MCb_gut templates, and the 3D location of the targeted sites in the carrier structure together suggests that the mechanism to open the inner gate, and release Mn into the cytoplasm, differs between MCb_ie and MCb_gut. Building on the established phylogeny for Enterococcus revealed that a pair of genes encoding MCb_gut was present in the common ancestor of LB, as MCb_gu1 and MCb_gu2 templates exhibited distinct structural dynamics properties. These data are discussed when examining whether MCb_gut⁺ LB could emerge in the upper gut of early vertebrates (ca. 540 mya), through genome contraction and evolution toward Mn-centrism, as they specialized as gastric aids favoring stomach establishment in jawed vertebrates through bi-directional communication with host nervous, endocrine and immune systems.

The Lactobacillales (LB) stand apart among bacterial orders, using manganese (Mn) instead of iron to support their growth and swiftly ferment complex foods while acidifying their environment. The present work investigates whether a shift in the use of Mn could mark the origin of LB. Transmembrane carriers of the ubiquitous Slc11 family play key roles in LB physiology by catalyzing proton-dependent Mn import. In prior studies, the Slc11 clade found in LB (MntH Cb, MCb) showed both remarkable structural plasticity and highly efficient Mn uptake, and another Slc11 clade, MCg1, demonstrated divergent evolution coinciding with emergence of bacterial genera (e.g., Bordetella , Achromobacter ). Herein, Slc11 clade MCb is subdivided in sister groups: MCb _ie and MCb _gut . MCb _ie derives directly from Slc11 clade MCa, pointing an intermediate stage in the evolution of MCb _gut . MCb _ie predominates in marine Bacillaceae, is more conserved than MCb _gut , lacks the structural plasticity that typify MCb _gut carriers, and responds differently to identical mutagenesis. Exchanging MCb _ie /MCb _gut amino acid residues at sites that distinguish these clades showed conformation-dependent effects with both MCb _ie and MCb _gut templates and the 3D location of the targeted sites in the carrier structure together suggest the mechanism to open the inner gate, and release Mn into the cytoplasm, differs between MCb _ie and MCb _gut . Building on the established phylogeny for Enterococcus revealed that a pair of genes encoding MCb _gut was present in the common ancestor of LB, as MCb _gu1 and MCb _gu2 templates exhibit distinct structural dynamics properties. These data are discussed examining whether MCb ⁺ LB could emerge in the upper gut of early vertebrates (ca. 540 mya), through genome contraction and evolution toward Mn-centrism, as they specialized as gastric aids favoring stomach establishment in jawed vertebrates through bi-directional communication with host nervous, endocrine and immune systems.