A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage

Abstract

Microbial rhodopsin is a photoreceptor protein found in various bacteria and archaea, and it is considered to be a light-utilization device unique to heterotrophs. Recent studies have shown that several cyanobacterial genomes also include genes that encode rhodopsins, indicating that these auxiliary light-utilizing proteins may have evolved within photoautotroph lineages. To explore this possibility, we performed a large-scale genomic survey to clarify the distribution of rhodopsin and its phylogeny. Our surveys revealed a novel rhodopsin clade, cyanorhodopsin (CyR), that is unique to cyanobacteria. Genomic analysis revealed that rhodopsin genes show a habitat-biased distribution in cyanobacterial taxa, and that the CyR clade is composed exclusively of non-marine cyanobacterial strains. Functional analysis using a heterologous expression system revealed that CyRs function as light-driven outward H+ pumps. Examination of the photochemical properties and crystal structure (2.65 Å resolution) of a representative CyR protein, N2098R from Calothrix sp. NIES-2098, revealed that the structure of the protein is very similar to that of other rhodopsins such as bacteriorhodopsin, but that its retinal configuration and spectroscopic characteristics (absorption maximum and photocycle) are distinct from those of bacteriorhodopsin. These results suggest that the CyR clade proteins evolved together with chlorophyll-based photosynthesis systems and may have been optimized for the cyanobacterial environment.

Patterns of Gene Content and Co-occurrence Constrain the Evolutionary Path toward Animal Association in Candidate Phyla Radiation Bacteria

“…Alignment columns with 95% or more gaps were trimmed manually in Geneious for the purposes of visualization. Transmembrane domains were identified by BLASTp searches ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and conserved residues were defined by manual comparison with an annotated alignment of previously published reference sequences ( 112 ).…”

Earliest Photic Zone Niches Probed by Ancestral Microbial Rhodopsins

“…Given the comparatively high statistical support and functional significance of ancestral binding pocket residues, we interrogated these specific sequence features to provide complementary insights into plausible functionality of ancient rhodopsins. For extant, uncharacterized rhodopsins, functional annotation is often guided by a characteristic, three-residue motif that includes sites homologous to the D85, T89, and D96 residues (a.k.a., “DTD” motif) in bacteriorhodopsin ( H. salinarum ) ( Kandori 2015 ; Hasegawa et al 2020 ; Inoue 2020 ; Rozenberg et al 2021 ). These sites are located within a section of the third transmembrane helix (“Helix C”; figs.…”

Earliest photic zone niches probed by ancestral microbial rhodopsins

“…Given the comparatively high statistical support and functional significance of ancestral binding pocket residues, we interrogated these specific sequence features to provide complementary insights into plausible functionality of ancient rhodopsins. For extant, uncharacterized rhodopsins, functional annotation is often guided by a characteristic, three-residue motif that includes sites homologous to the D85, T89, and D96 residues (a.k.a., “DTD” motif) in bacteriorhodopsin ( H. salinarum ) (Kandori 2015; Hasegawa et al 2020; Inoue 2020; Rozenberg et al 2021). These sites are located within a section of the third transmembrane helix (“Helix C”; Fig.…”