Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

Abstract

Rhodopsin-encoding microorganisms are common in many environments. However, knowing that rhodopsin genes are present provides little insight into how the host cells utilize light. The genome of the freshwater actinobacterium Rhodoluna lacicola encodes a rhodopsin of the uncharacterized actinorhodopsin family. We hypothesized that actinorhodopsin was a light-activated proton pump and confirmed this by heterologously expressing R. lacicola actinorhodopsin in retinal-producing Escherichia coli. However, cultures of R. lacicola did not pump protons, even though actinorhodopsin mRNA and protein were both detected. Proton pumping in R. lacicola was induced by providing exogenous retinal, suggesting that the cells lacked the retinal cofactor. We used high-performance liquid chromatography (HPLC) and oxidation of accessory pigments to confirm that R. lacicola does not synthesize retinal. These results suggest that in some organisms, the actinorhodopsin gene is constitutively expressed, but rhodopsin-based light capture may require cofactors obtained from the environment. IMPORTANCEUp to 70% of microbial genomes in some environments are predicted to encode rhodopsins. Because most microbial rhodopsins are light-activated proton pumps, the prevalence of this gene suggests that in some environments, most microorganisms respond to or utilize light energy. Actinorhodopsins were discovered in an analysis of freshwater metagenomic data and subsequently identified in freshwater actinobacterial cultures. We hypothesized that actinorhodopsin from the freshwater actinobacterium Rhodoluna lacicola was a light-activated proton pump and confirmed this by expressing actinorhodopsin in retinalproducing Escherichia coli. Proton pumping in R. lacicola was induced only after both light and retinal were provided, suggesting that the cells lacked the retinal cofactor. These results indicate that photoheterotrophy in this organism and others may require cofactors obtained from the environment. Rhodopsin-containing photoheterotrophic microbes are common inhabitants of marine, terrestrial, and freshwater environments, where they have been identified by cultivation (1-3), metagenomic sequencing (4-6), targeted amplicon sequencing (7-9), and quantitative PCR (9, 10). The cosmopolitan distribution of rhodopsin-containing microbes in diverse habitats is reflected in the variety of effects the rhodopsins have on their hosts. For instance, certain rhodopsins transport protons or other ions, while others affect gene expression through signaling networks (11). Within a single organism, multiple rhodopsins can be present and perform different roles (12, 13). Closely related rhodopsin-containing organisms have been shown to react to light differently: in Dokdonia spp., light exposure has been shown to provide a growth advantage for one species while offering no measurable benefit to another (14-16). In addition, rhodopsins may differ in their maximum absorption peaks (480 to 560 nm [17]) or their abilities to bind additional carotenoids (18,19) ...

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively-synthesized Retinal

“…Many species of cyanobacteria encode functional β-carotene oxygenases (33, 45, 46), and retinoid concentrations in eutrophic lakes during cyanobacterial blooms are measurable (47). This proposal is consistent with actinorhodopsin studies in the culturable freshwater organisms Rhodoluna lacicola and Candidatus Rhodoluna planktonica (12, 13). While both encode actR , only the latter contains blh and thus exhibits self-sufficient rhodopsin activity in the laboratory.…”

“…Additionally, the relevant genes must be shown to be expressed in the native freshwater environments where acI is dominant. That these requirements are satisfied by acI is not guaranteed; Rhodoluna lacicola holo-ActR was shown to be active if, and only if, exogenous retinal was added (12). Without an encoded, retinal-producing enzyme or a solution for obtaining dilute environmental chromophores, it is unclear what the physiological role and ecosystem implications of ActR in such organisms are.…”

Photochemical characterization of actinorhodopsin and its functional existence in the natural host

“…As described above, the actinobacterium R. lacicola also expressed ActR, but it did not exhibit light-induced activity due to the lack of retinal binding [25]. Thus, MWH-Dar1 is the first actinobacterium confirmed to perform ActR phototrophy.…”

“…Thus, RpActR probably conserves this binding ability. Similarly to R. lacicola [25], however, the host strain MWH-Dar1 lacks carotenoid ketolases, indicating that this strain does not contain keto-carotenoids, such as salinixanthin for XR and echinenone for GR.…”

“…In actinobacterium Rhodoluna lacicola strain MWH-Ta8, the ActR opsin was constitutively expressed but did not contain the retinal [25]. This immature ActR required exogenous retinal to perform the H + -pumping function.…”