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

Nakamura, Shintaro; Kikukawa, Takashi; Tamogami, Jun; Kamiya, Masakatsu; Aizawa, Tomoyasu; Hahn, Martin W.; Ihara, Kenji; Kamo, Naoki; Demura, Makoto

doi:10.1016/j.bbabio.2016.09.006

Cited by 20 publications

(27 citation statements)

References 60 publications

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“…Indeed, many species of cyanobacteria encode functional ␤-carotene oxygenases (38,53,54), and retinoid concentrations in eutrophic lakes during cyanobacterial blooms are measurable (55). This proposal is consistent with actinorhodopsin studies in the culturable freshwater organisms Rhodoluna lacicola and "Candidatus Rhodoluna planktonica" (14,15). While both carry actR, only the latter contains blh and thus exhibits self-sufficient rhodopsin activity in the laboratory.…”

Section: Discussionsupporting

confidence: 67%

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively Synthesized Retinal

Dwulit-Smith

Hamilton

Stevenson

et al. 2018

Appl Environ Microbiol

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Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acIActinobacteria. 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 acIActinobacteriaare 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 acIActinobacteriain a eutrophic lake shows that all retinal and carotenoid pathway operons are transcribed and thatactRis 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 acIActinobacteriacontaining both ActR and acI retinal production machinery have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin.IMPORTANCEMicrobes play critical roles in determining the quality of freshwater ecosystems, which are vital to human civilization. Because acIActinobacteriaare 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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Section: Discussionsupporting

confidence: 67%

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively Synthesized Retinal

Dwulit-Smith

Hamilton

Stevenson

et al. 2018

Appl Environ Microbiol

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“…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.…”

Section: Discussionsupporting

confidence: 90%

“…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. On the other hand, the Luna1 bacterium Candidatus Rhodoluna planktonica encodes a putative retinal-synthesizing enzyme (13). This family of Actinobacteria is radically different from acI and much less abundant and prevalent in freshwater lakes (2), but this organism provides motivation to determine if acI truly use rhodopsins for photoheterotrophy.…”

Section: Introductionmentioning

confidence: 99%

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively-synthesized Retinal

Dwulit-Smith

Hamilton

Stevenson

et al. 2018

Preprint

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18Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI 19Actinobacteria from three clades (acI-A, acI-B, acI-C). Members of this cosmopolitan lineage are proposed 20 to bolster heterotrophic growth using phototrophy because their genomes encode actino-opsins (actR). 21This model has been difficult to experimentally validate because acI are not consistently culturable. In this 22 study, using genomes from single cells and metagenomes, we provide a detailed biosynthetic route for 23 many acI-A and -B members to synthesize retinal and its carotenoid precursors. Accordingly, these acI 24 should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, in contrast 25 to acI-C members and other bacteria that encode opsins but lack retinal-production machinery. Moreover, 26we show that all acI clades contain genes for a complex carotenoid pathway that starts with retinal 27 precursors. Transcription analysis of acI in a eutrophic lake shows that all retinal and carotenoid pathway 28 operons are transcribed and that actR is among the most highly-transcribed of all acI genes. Furthermore, 29 heterologous expression of retinal pathway genes shows that lycopene, retinal, and ActR can be made. 30Model cells producing ActR and the key acI retinal-producing β-carotene oxygenase formed acI-holo-ActR 31 and acidified solution during illumination. Our results prove that acI containing both ActR and retinal-32 production enzymes have the capacity to natively synthesize a green light-dependent outward proton-33 pumping rhodopsin. 35 IMPORTANCE 36Microbes play critical roles in determining the quality of freshwater ecosystems that are vital to human 37 civilization. Because acI Actinobacteria are ubiquitous and abundant in freshwater lakes, clarifying their 38 ecophysiology is a major step in determining the contributions that they make to nitrogen and carbon 39 cycling. Without accurate knowledge of these cycles, freshwater systems cannot be incorporated into 40 climate change models, ecosystem imbalances cannot be predicted, and policy for service disruption 41 cannot be planned. Our work fills major gaps in microbial light utilization, secondary metabolite production, 42and energy cycling in freshwater habitats.

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“…Since NSY medium does not include retinal, the actinorhodopsins cannot be responsible for the enhanced growth. Although rhodopsins might contribute to enhanced growth in the light in some actinobacterial species and under some conditions (32), they are not responsible for the observed growth rate phenotype here. We also grew Microbacterium sp.…”

Section: Resultsmentioning

confidence: 63%

“…Genome comparisons. The small genome sizes of the two Aurantimicrobium species are characteristic of freshwater Actinobacteria (24,28,32,35). The genome sequences suggest that both are heterotrophic aerobes with the ability to utilize amino acids and other small organic molecules.…”

Section: Resultsmentioning

confidence: 99%

Light Modulates the Physiology of NonphototrophicActinobacteria

et al. 2019

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Light is a source of energy and an environmental cue that is available in excess in most surface environments. In prokaryotic systems, conversion of light to energy by photoautotrophs and photoheterotrophs is well understood, but the conversion of light to information and the cellular response to that information have been characterized in only a few species. Our goal was to explore the response of freshwaterActinobacteria, which are ubiquitous in illuminated aquatic environments, to light. We found thatActinobacteriawithout functional photosystems grow faster in the light, likely because sugar transport and metabolism are upregulated in the light. Based on the action spectrum of the growth effect and comparisons of the genomes of threeActinobacteriawith this growth rate phenotype, we propose that the photosensor in these strains is a putative CryB-type cryptochrome. The ability to sense light and upregulate carbohydrate transport during the day could allow these cells to coordinate their time of maximum organic carbon uptake with the time of maximum organic carbon release by primary producers.IMPORTANCESunlight provides information about both place and time. In sunlit aquatic environments, primary producers release organic carbon and nitrogen along with other growth factors during the day. The ability ofActinobacteriato coordinate organic carbon uptake and utilization with production of photosynthate enables them to grow more efficiently in the daytime, and it potentially gives them a competitive advantage over heterotrophs that constitutively produce carbohydrate transporters, which is energetically costly, or produce transporters only after detection of the substrate(s), which delays their response. Understanding how light cues the transport of organic carbon and its conversion to biomass is key to understanding biochemical mechanisms within the carbon cycle, the fluxes through it, and the variety of mechanisms by which light enhances growth.

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

Cited by 20 publications

References 60 publications

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively Synthesized Retinal

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively Synthesized Retinal

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively-synthesized Retinal

Light Modulates the Physiology of NonphototrophicActinobacteria

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