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Assembly of Natively Synthesized Dual Chromophores Into Functional Actinorhodopsin.

ABSTRACT: Microbial rhodopsin is a simple solar energy-capturing molecule compared to the complex photosynthesis apparatus. Light-driven proton pumping across the cell membrane is a crucial mechanism underlying microbial energy production. Actinobacteria is one of the highly abundant bacterial phyla in freshwater habitats, and members of this lineage are considered to boost heterotrophic growth via phototrophy, as indicated by the presence of actino-opsin (ActR) genes in their genome. However, it is difficult to validate their function under laboratory settings because Actinobacteria are not consistently cultivable. Based on the published genome sequence of Candidatus aquiluna sp. strain IMCC13023, actinorhodopsin from the strain (ActR-13023) was isolated and characterized in this study. Notably, ActR-13023 assembled with natively synthesized carotenoid/retinal (used as a dual chromophore) and functioned as a light-driven outward proton pump. The ActR-13023 gene and putative genes involved in the chromophore (retinal/carotenoid) biosynthetic pathway were detected in the genome, indicating the functional expression ActR-13023 under natural conditions for the utilization of solar energy for proton translocation. Heterologous expressed ActR-13023 exhibited maximum absorption at 565 nm with practical proton pumping ability. Purified ActR-13023 could be reconstituted with actinobacterial carotenoids for additional light-harvesting. The existence of actinorhodopsin and its chromophore synthesis machinery in Actinobacteria indicates the inherent photo-energy conversion function of this microorganism. The assembly of ActR-13023 to its synthesized chromophores validated the microbial community's importance in the energy cycle.

SUBMITTER: Chuon K

PROVIDER: S-EPMC8113403 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI Actinobacteria Members of this cosmopolitan lineage are proposed to bolster heterotrophic growth using phototrophy because their genomes encode actino-opsins (actR). This model has been difficult to validate experimentally because acI Actinobacteria are not consistently culturable. Based primarily on genomes from single cells and metagenomes, we provide a detailed biosynthetic route for members of acI clades A and B to synthesize retinal and its carotenoid precursors. Consequently, acI cells should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, unlike many bacteria that encode opsins but may need to exogenously obtain retinal because they lack retinal machinery. Moreover, we show that all acI clades contain genes for a secondary branch of the carotenoid pathway, implying synthesis of a complex carotenoid. Transcription analysis of acI Actinobacteria in a eutrophic lake shows that all retinal and carotenoid pathway operons are transcribed and that actR is among the most highly transcribed of all acI genes. Furthermore, heterologous expression of acI retinal pathway genes showed that lycopene, retinal, and ActR can be made using the genes encoded in these organisms. Model cells producing ActR and the key acI retinal-producing β-carotene oxygenase formed holo-ActR and acidified solution during illumination. Taken together, our results prove that acI Actinobacteria containing both ActR and acI retinal production machinery have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin.IMPORTANCE Microbes play critical roles in determining the quality of freshwater ecosystems, which are vital to human civilization. Because acI Actinobacteria are ubiquitous and abundant in freshwater lakes, clarifying their ecophysiology is a major step in determining the contributions that they make to nitrogen and carbon cycling. Without accurate knowledge of these cycles, freshwater systems cannot be incorporated into climate change models, ecosystem imbalances cannot be predicted, and policy for service disruption cannot be planned. Our work fills major gaps in microbial light utilization, secondary metabolite production, and energy cycling in freshwater habitats.

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Assembly of Natively Synthesized Dual Chromophores Into Functional Actinorhodopsin.

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