AbstractRhodopsins 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.SignificanceThe recently discovered divergent rhodopsin family of heliorhodopsins is abundant in freshwater environments. In this study, we sampled a habitat rich in dissolved organic matter to increase our chances of finding spectrally shifted rhodopsins. Using functional metagenomics, diverse heliorhodopsins absorbing green and yellow light were discovered. The metagenomic clones originated from diverse prokaryotic groups: Actinobacteria, Chloroflexi and even Archaea, emphasizing the versatility of the E. coli expression system used. Photocycles of representative heliorhodopsins were measured and exhibited diverse kinetic characteristics. Analysis of genes neighboring heliorhodopsins in diverse prokaryotes revealed their putative connection to membrane lipid re-modeling and oxidative stress. Our findings suggest that functional metagenomics is a productive method for the discovery of new and diverse rhodopsins.
Discovery of new protein families and functions: new challenges in functional metagenomics for biotechnologies and microbial ecology
