Rhodopsins are widely distributed across all domains of life where they perform a plethora of functions through the conversion of electromagnetic radiation into physicochemical signals. As a result of an extensive survey of available genomic and metagenomic sequencing data, we reported the existence of novel clades and exotic sequence motifs scattered throughout the evolutionary radiations of both Type-1 and Type-3 rhodopsins that will likely enlarge the optogenetics toolbox. We expanded the typical rhodopsin blueprint by showing that a highly conserved and functionally important arginine residue (i.e., Arg82) was substituted multiple times during evolution by an extensive amino acid spectrum. We proposed the umbrella term Alt-rhodopsins (AltRs) for all such proteins that departed Arg82 orthodoxy. Some AltRs formed novel clades in the rhodopsin phylogeny and were found in giant viruses. Some newly uncovered AltRs were phylogenetically close to heliorhodopsins, which allowed a closer examination of the phylogenetic border between Type-1 rhodopsins and heliorhodopsins. Comprehensive phylogenetic trees and ancestral sequence reconstructions allowed us to advance the hypothesis that proto-heliorhodopsins were a eukaryotic innovation before their subsequent diversification into the extant Type-3 rhodopsins. IMPORTANCE The rhodopsin scaffold is remarkably versatile and widespread, coupling light availability to energy production and other light-dependent cellular responses with minor alterations to critical residues. We described an unprecedented spectrum of substitutions at one of the most conserved amino acids in the rhodopsin fold, Arg82. We denoted such phylogenetically diverse rhodopsins with the umbrella name Alt-rhodopsins (AltR) and described a distinct branch of AltRs in giant viruses. Intriguingly, some AltRs were the closest phylogenetic neighbors to Heliorhodopsins (HeRs) whose origins have remained enigmatic. Our analyses of HeR origins in the light of AltRs led us to posit a most unusual evolutionary trajectory that suggested a eukaryotic origin for HeRs before their diversification in prokaryotes.

• Keywords
• Alt-rhodopsins, AltRs, heliorhodopsins, metagenomics, optogenetics, rhodopsins,
• MeSH
• Phylogeny MeSH
• Rhodopsins, Microbial * genetics   MeSH
• Rhodopsin * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Rhodopsins, Microbial * MeSH
• Rhodopsin * MeSH

BACKGROUND: The planetary sulfur cycle is a complex web of chemical reactions that can be microbial-mediated or can occur spontaneously in the environment, depending on the temperature and pH. Inorganic sulfur compounds can serve as energy sources for specialized prokaryotes and are important substrates for microbial growth in general. Here, we investigate dissimilatory sulfur cycling in the brine and sediments of a southwestern Siberian soda lake characterized by an extremely high pH and salinity, combining meta-omics analyses of its uniquely adapted highly diverse prokaryote communities with biogeochemical profiling to identify key microbial players and expand our understanding of sulfur cycling under haloalkaline conditions. RESULTS: Peak microbial activity was found in the top 4 cm of the sediments, a layer with a steep drop in oxygen concentration and redox potential. The majority of sulfur was present as sulfate or iron sulfide. Thiosulfate was readily oxidized by microbes in the presence of oxygen, but oxidation was partially inhibited by light. We obtained 1032 metagenome-assembled genomes, including novel population genomes of characterized colorless sulfur-oxidizing bacteria (SOB), anoxygenic purple sulfur bacteria, heterotrophic SOB, and highly active lithoautotrophic sulfate reducers. Surprisingly, we discovered the potential for nitrogen fixation in a new genus of colorless SOB, carbon fixation in a new species of phototrophic Gemmatimonadetes, and elemental sulfur/sulfite reduction in the "Candidatus Woesearchaeota." Polysulfide/thiosulfate and tetrathionate reductases were actively transcribed by various (facultative) anaerobes. CONCLUSIONS: The recovery of over 200 genomes that encoded enzymes capable of catalyzing key reactions in the inorganic sulfur cycle indicates complete cycling between sulfate and sulfide at moderately hypersaline and extreme alkaline conditions. Our results suggest that more taxonomic groups are involved in sulfur dissimilation than previously assumed.

• Keywords
• Gemmatimonadetes, Haloalkaliphiles, Metagenomics, Metatranscriptomics, Nitrogen fixation, Polysulfide, Soda lake, Tetrathionate, Thiosulfate, Woesearchaeota,
• MeSH
• Archaea classification   genetics   metabolism   MeSH
• Bacteria classification   genetics   metabolism   MeSH
• Phylogeny MeSH
• Lakes chemistry   microbiology   MeSH
• Hydrogen-Ion Concentration MeSH
• Metagenome MeSH
• Oxidation-Reduction MeSH
• Salinity MeSH
• Sulfur analysis   metabolism   MeSH
• Salts chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Siberia MeSH
• Names of Substances
• brine MeSH Browser
• Sulfur MeSH
• Salts MeSH

BACKGROUND: Hypersaline soda lakes are characterized by extreme high soluble carbonate alkalinity. Despite the high pH and salt content, highly diverse microbial communities are known to be present in soda lake brines but the microbiome of soda lake sediments received much less attention of microbiologists. Here, we performed metagenomic sequencing on soda lake sediments to give the first extensive overview of the taxonomic diversity found in these complex, extreme environments and to gain novel physiological insights into the most abundant, uncultured prokaryote lineages. RESULTS: We sequenced five metagenomes obtained from four surface sediments of Siberian soda lakes with a pH 10 and a salt content between 70 and 400 g L-1. The recovered 16S rRNA gene sequences were mostly from Bacteria, even in the salt-saturated lakes. Most OTUs were assigned to uncultured families. We reconstructed 871 metagenome-assembled genomes (MAGs) spanning more than 45 phyla and discovered the first extremophilic members of the Candidate Phyla Radiation (CPR). Five new species of CPR were among the most dominant community members. Novel dominant lineages were found within previously well-characterized functional groups involved in carbon, sulfur, and nitrogen cycling. Moreover, key enzymes of the Wood-Ljungdahl pathway were encoded within at least four bacterial phyla never previously associated with this ancient anaerobic pathway for carbon fixation and dissimilation, including the Actinobacteria. CONCLUSIONS: Our first sequencing effort of hypersaline soda lake sediment metagenomes led to two important advances. First, we showed the existence and obtained the first genomes of haloalkaliphilic members of the CPR and several hundred other novel prokaryote lineages. The soda lake CPR is a functionally diverse group, but the most abundant organisms in this study are likely fermenters with a possible role in primary carbon degradation. Second, we found evidence for the presence of the Wood-Ljungdahl pathway in many more taxonomic groups than those encompassing known homo-acetogens, sulfate-reducers, and methanogens. Since only few environmental metagenomics studies have targeted sediment microbial communities and never to this extent, we expect that our findings are relevant not only for the understanding of haloalkaline environments but can also be used to set targets for future studies on marine and freshwater sediments.

• Keywords
• Candidate Phyla Radiation, Haloalkaliphilic extremophiles, Metagenomics, Soda lake sediments, Wood-Ljungdahl pathway,
• MeSH
• Bacteria classification   genetics   isolation & purification   metabolism   MeSH
• Sodium Chloride analysis   MeSH
• Phylogeny MeSH
• Genetic Variation MeSH
• Genome, Bacterial MeSH
• Geologic Sediments analysis   microbiology   MeSH
• Lakes analysis   microbiology   MeSH
• Nitrogen Cycle MeSH
• Carbon Cycle MeSH
• Metagenome MeSH
• Metagenomics MeSH
• Microbiota MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Sodium Chloride MeSH

Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.

• MeSH
• Archaea metabolism   MeSH
• Cell Line MeSH
• Escherichia coli metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Protein Conformation MeSH
• Humans MeSH
• Liposomes MeSH
• Models, Molecular MeSH
• Optogenetics * methods   MeSH
• Proton Pumps metabolism   MeSH
• Protons MeSH
• Retina metabolism   MeSH
• Rhodopsin chemistry   metabolism   MeSH
• Spectrum Analysis MeSH
• Light MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Chromatography, High Pressure Liquid MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Liposomes MeSH
• Proton Pumps MeSH
• Protons MeSH
• Rhodopsin MeSH