Demonstration of a sensory rhodopsin in eubacteria

Abstract

Summary: We report the first sensory rhodopsin observed in the eubacterial domain, a green light‐activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all‐trans retinal to form a pink pigment (λmax 543 nm) with a photochemical reaction cycle of 110 ms half‐life (pH 6.8, 18°C). Co‐expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane‐embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS‐PAGE and autofluorography of 3 H‐labelled all‐trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix–helix interactions with membrane‐embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.