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bioRxiv · 10.1101/367102

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively-synthesized Retinal

Dwulit-Smith, J. R.·Hamilton, J. J.·Stevenson, D. M.·He, S.·Oyserman, B. O.·Moya-Flores, F.·Amador-Noguez, D.·McMahon, K.·Forest, K. T.
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Abstract

Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI Actinobacteria from three clades (acI-A, acI-B, acI-C). 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 experimentally validate because acI are not consistently culturable. In this study, using genomes from single cells and metagenomes, we provide a detailed biosynthetic route for many acI-A and -B members to synthesize retinal and its carotenoid precursors. Accordingly, these acI should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, in contrast to acI-C members and other bacteria that encode opsins but lack retinal-production machinery. Moreover, we show that all acI clades contain genes for a complex carotenoid pathway that starts with retinal precursors. Transcription analysis of acI 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 retinal pathway genes shows that lycopene, retinal, and ActR can be made. Model cells producing ActR and the key acI retinal-producing {beta}-carotene oxygenase formed acI-holo-ActR and acidified solution during illumination. Our results prove that acI containing both ActR and retinal-production enzymes have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin.\n\nIMPORTANCEMicrobes play critical roles in determining the quality of freshwater ecosystems that 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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Dwulit-Smith, J. R., Hamilton, J. J., Stevenson, D. M., He, S., Oyserman, B. O., Moya-Flores, F., Amador-Noguez, D., McMahon, K., Forest, K. T.. 2018-07-11. acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively-synthesized Retinal. https://doi.org/10.1101/367102

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