acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively-synthesized Retinal

Abstract

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.

Aquiluna borgnonia gen. nov., sp. nov., a member of a Microbacteriaceae lineage of freshwater bacteria with small genome sizes

“…Many representatives are yellow-, orange- or red-pigmented due to various carotenoids; light-induced biosynthesis of these pigments was found in some strains [4]. Actinorhodopsins from bacteria of the phylum Actinobacteria with a putative proton pumping function were found predominantly in non-marine aquatic environments [5] and also among members of the family Microbacteriaceae [6, 7]. Genome-based phylogenies reconstructed from whole proteomes suggested that the family Microbacteriaceae is monophyletic [8].…”

“…Nevertheless, available genomes belonging to the same phylogenetic cluster showed a similar pattern (). The so-called acl Actinobacteria , a group of bacteria with similar small genome sizes, showed a comparable pattern concerning the positions of these genes [7]. In contrast, genes predicted to encode complete rhodopsin/retinal systems in genomes of other members of the Microbacteriaceae shown in (i.e.…”

Bacterium Lacking a Known Gene for Retinal Biosynthesis Constructs Functional Rhodopsins

“…Although we showed that A. minutum KNC T produced retinal, the genes responsible for its biosynthesis have not yet been identified. Among rhodopsin-possessing Actinobacteria, a lycopene cyclase encoded by the crtYc/Yd genes catalyzes the conversion of lycopene to β-carotene, which is then supplied for endogenous retinal (Dwulit-Smith et al, 2018). However, A. minutum has putative crtYe/Yf cluster genes, which encode C50 carotenoid epsilon cyclase (Krubasik et al, 2001;Netzer et al, 2010), not crtYc/Yd genes.…”

Omega Rhodopsins: A Versatile Class of Microbial Rhodopsins

“…ActR in Rhodoluna lacicola belonging to the acI lineage was active only if the exogenous retinal was supplied, suggesting that the cells lacked the retinal synthesis machinery [38]; in contrast, the Luna bacterium Candidatus Rhodoluna planktonica encodes a putative retinal-synthesizing system [39]. The biosynthetic route for retinal and carotenoids, which are necessary for the ActR function is elucidated in the acI lineage [40]. ActRs also contain the glycine residue for binding ketolated antenna carotenoids.…”

“…The ecological roles of ActR in actinobacteria have been characterized mainly in a ubiquitous freshwater actinobacterial lineage, acI. All retinal biosynthesis pathway genes were transcribed, and ActR was most highly transcribed in a eutrophic lake [40]. A metatranscriptomic study of acI also reported the high expression of ActR and some transport proteins [75].…”