Characterization of heliorhodopsins detected via functional metagenomics in freshwater Actinobacteria, Chloroflexi and Archaea

Abstract

Rhodopsins are widespread in microbes residing in diverse aquatic environments across the globe. Recently, a new unusual rhodopsin family, the heliorhodopsins (HeRs), was discovered, distributed among diverse bacteria, archaea, eukarya and even viruses. Here, using functional metagenomics on samples from Lake Ha'Hula and Ein Afek reserve, we found and characterized ten HeRs representing divergent members of the family. The expressed HeRs absorb light in the green and yellow wavelengths and originate from Actinobacteria, Chloroflexi and Archaea. The photocycle of the HeR from Chloroflexi revealed a low accumulation of the M-intermediate that we connect to the lack of two conserved histidine residues in transmembrane helices 1 and 2 in this protein. Another of HeR, from Actinobacteria, exhibited an unusually fast photocycle (166 ms, 5 times faster than HeR-48C12). To further explore the still unresolved question of the HeR function, we performed an analysis of protein families among genes neighboring HeRs, in our clones and thousands of other microbes. This analysis revealed a putative connection between HeRs and genes involved in oxidative stress. At the same time, very few protein families were found to distinguish genes surrounding prokaryotic HeRs from those surrounding rhodopsin pumps. The strongest association was found with the DegV family involved in activation of fatty acids and uncharacterized family DUF2177, which allowed us to hypothesize that HeRs are involved in membrane lipid remodeling. This work further establishes functional metagenomics as a simple and fruitful method of screening for new rhodopsins.

Energy transfer in ubiquitous rhodopsin pumps with xanthophyll antennas

“…Next, we tested whether members of the PR family would be able to bind carotenoids as well. We selected the Gly156-containing PRs EinA29G6 [GenBank UJI09384] from an uncultured freshwater flavobacterium 19 and KR1 [GenBank BAN14807] from the marine flavobacterium Dokdonia eikasta 20 , and GPR 31A08 [GenBank AAG10475] from a marine gammaproteobacterium 7 , which has Phe at XR position 156 instead (Fig. 2h, Extended Data Fig.…”

“…Inspired by the known XR/keto-carotenoid light-harvesting systems, we designed a strategy to search for rhodopsin/carotenoid complexes originating from aquatic microorganisms. For the first exploratory series of experiments, we picked a rhodopsin with fenestration discovered using functional metagenomics [17][18][19] in a freshwater lake (Lake Kinneret, Israel) and exposed it to a concentrated chromophore extract from the same lake (Fig. 1a, see METHODS).…”

Earliest photic zone niches probed by ancestral microbial rhodopsins

“…However, it has been demonstrated that rhodopsin-based phototrophy can also be carried out in hypoxic and anoxic conditions (DasSarma et al 2012). In addition, an alternative, uncharacterized biosynthetic pathway for retinal synthesis has been reported (Nakajima et al 2020;Chazan et al 2022). Thus, the necessity of oxygen for retinal synthesis remains an open question.…”

Diversity, distribution, and expression of opsin genes in freshwater lakes

“…More recently, a novel family of microbial rhodopsins distinct from Type I rhodopsins was discovered through functional metagenomics and named heliorhodopsin (HeR) (Pushkarev et al 2018). As HeRs are only distantly related to Type I rhodopsins and their functions are largely unknown, we solely focused on Type I rhodopsins in our study, although we note that HeRs have been found in diverse monoderm bacteria in freshwater environments (Chazan et al 2022;Flores-Uribe et al 2019).…”

“…2018). As HeRs are only distantly related to Type I rhodopsins and their functions are largely unknown, we solely focused on Type I rhodopsins in our study, although we note that HeRs have been found in diverse monoderm bacteria in freshwater environments (Chazan et al . 2022; Flores-Uribe et al .…”