The bacterial schizorhodopsins: novel light-driven inward proton pumps from Antarctic Minisyncoccota (Patescibacteria) and cyanobacteria, with implications for the proton-pumping mechanism

Abstract

Microbial rhodopsins represent a diverse superfamily of light-sensitive seven-transmembrane proteins with expanding phylogenetic diversity driven by advances in metagenomics. Among these, schizorhodopsins constitute a divergent family originally identified as inward proton pumps from Promethearchaeota (Asgard archaea). Here, we report that in addition to archaeal schizorhodopsins, many members of the family originate from bacteria and detail a comprehensive biophysical characterization of novel schizorhodopsins from Antarctic Minisyncoccota (Patescibacteria) and cyanobacteria, designated as paSzR and psSzR, respectively. Both proteins function as light-driven inward proton pumps, as confirmed through pH measurements in Escherichia coli cells. Laser-flash photolysis experiments identified multiple photointermediates (K, L, and M) characteristic of microbial rhodopsin photocycles, though with slower turnover rates compared to archaeal schizorhodopsins. Site-directed mutagenesis of conserved residues in the third and sixth transmembrane helices demonstrates differential structural requirements between paSzR and psSzR. Our phylogenetic reconstruction reveals that most bacterial schizorhodopsins cluster in a single lineage distinct from archaeal variants. These findings significantly expand our understanding of microbial rhodopsin diversity and provide crucial insights into alternative molecular mechanisms for light-driven proton translocation, with implications for microbial ecology in extreme environments. Statement of significanceSchizorhodopsins represent an unusual family of light-driven proteins that pump protons inwardly--opposite to the classical microbial rhodopsin pumps. Originally discovered in archaea, we revealed their unprecedented prevalence in bacteria. We characterized two novel bacterial schizorhodopsins from the Antarctic: paSzR from Minisyncoccota and psSzR from Cyanobacteriota, demonstrating their inward proton-pumping activity. Our phylogenetic reconstruction showed that most bacterial schizorhodopsins form a distinct evolutionary lineage and two exchange events between Minisyncoccota and cyanobacteria. Through targeted mutagenesis, we identified critical residues that govern spectral tuning and transport efficiency, revealing mechanistic differences between members of the family. These findings expand the known diversity of light-energy conversion mechanisms across microbial life and provide insights into alternative proton translocation strategies, with implications for understanding microbial adaptation to extreme environments.