bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Heliorhodopsin evolution is driven by photosensory promiscuity in monoderms 2 Paul-Adrian Bulzu1, Vinicius Silva Kavagutti1,2, Maria-Cecilia Chiriac1, Charlotte 3 D. Vavourakis3, Keiichi Inoue4, Hideki Kandori5,6, Adrian-Stefan Andrei7, Rohit 4 Ghai1*

5 1Department of Aquatic Microbial Ecology, Institute of Hydrobiology, Biology Centre of 6 the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic.

7 2Department of Ecosystem Biology, Faculty of Science, University of South Bohemia, 8 Branišovská 1760, České Budějovice, Czech Republic.

9 3EUTOPS, Research Institute for Biomedical Aging Research, University of Innsbruck, 10 Austria.

11 4The Institute for Solid State Physics, The University of Tokyo, Kashiwa, Japan.

12 5Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, 13 Showa, Nagoya 466-8555, Japan.

14 6OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa, Nagoya 15 466-8555, Japan

16 7Limnological Station, Department of Plant and Microbial Biology, University of Zurich, 17 Kilchberg, Switzerland.

18 *Corresponding author: Rohit Ghai 19 Department of Aquatic Microbial Ecology, Institute of Hydrobiology, Biology Centre of 20 the Academy 21 of Sciences of the Czech Republic, Na Sádkách 7, 370 05, České Budějovice, Czech 22 Republic. 23 Phone: +420 387 775 881 24 Fax: +420 385 310 248 25 E-mail: [email protected] 26 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

27 The ability to harness Sun’s electromagnetic radiation by channeling it into high-energy 28 phosphate bonds empowered microorganisms to tap into a cheap and inexhaustible 29 source of energy. Life`s billion-years history of metabolic innovations led to the 30 emergence of only two biological complexes capable of harvesting light: one based on 31 rhodopsins and the other on (bacterio)chlorophyll. Rhodopsins encompass the most 32 diverse and abundant photoactive proteins on Earth and were until recently canonically 33 split between type-1 (microbial rhodopsins) and type-2 (animal rhodopsins) families. 34 Unexpectedly, the long-lived type-1/type-2 dichotomy was recently amended through the 35 discovery of heliorhodopsins (HeRs) (Pushkarev et al. 2018), a novel and exotic family of 36 rhodopsins (i.e. type-3) that evaded recognition in our current homology-driven scrutiny 37 of life’s genomic milieu. Here, we bring to resolution the debated monoderm/diderm 38 occurrence patterns by conclusively showing that HeR distribution is restricted to 39 monoderms. Furthermore, through investigating protein domain fusions, contextual 40 genomic information, and gene co-expression data we show that HeRs likely function as 41 generalised light-dependent switches involved in the mitigation of light-induced oxidative 42 stress and metabolic circuitry regulation. We reason that HeR’s ability to function as 43 sensory rhodopsins is corroborated by their photocycle dynamics (Pushkarev et al. 2018) 44 and that their presence and function in monoderms is likely connected to the increased 45 sensitivity to light-induced damage of these organisms (Maclean et al. 2009). 46 Type-1 and -2 rhodopsins families share a similar topological conformation and little or no 47 sequence similarity among each other. Despite dissimilarities in function, structure and 48 phylogeny, type-1 and -2 rhodopsins have a similar membrane orientation with their N- 49 terminus being situated in the extracellular space. Identified during a functional 50 metagenomics screen and characterised by low sequence similarity when compared to 51 type-1 rhodopsins, HeRs attracted increasing research interest due to their peculiar 52 membrane orientation (i.e. N-terminus in the cytoplasm and the C-terminus in the 53 extracellular space)(Pushkarev et al. 2018), unusual protein structure (Kovalev et al. 2020) 54 and controversial taxonomic distribution (Flores-Uribe, Hevroni, and Ghai 2019). While 55 electrophysiological (Pushkarev et al. 2018), physicochemical (Tanaka et al. 2020) and 56 structural (Shihoya et al. 2019; Kovalev et al. 2020) studies achieved great progress in 57 elucidating a series of characteristics ranging from photocycle length (indicating no 58 pumping activity) to detailed protein organization, they provide no data regarding the 59 biological function of HeRs. Moreover, polarized opinions regarding the putative 60 ecological role and taxonomic distribution of HeR-encoding organisms (Flores-Uribe, 61 Hevroni, and Ghai 2019; Kovalev et al. 2020) call for the use of novel approaches in 62 establishing HeR functionality. This work draws its essence from the tenet that functionally 63 linked genes within prokaryotes are co-regulated, and thus occur close to each other 64 (Aravind 2000; Huynen et al. 2000). Within this framework, the functions of 65 uncharacterised genes (i.e. HeRs) can be inferred from their genomic surroundings. Here 66 we couple HeR’s distributional patterns with contextual genomic information involving 67 protein domain fusions and operon organization, and gene expression data to shed light 68 on HeRs functionality. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

69 70 Figure 1. Modelled three-dimensional (3D) structures of MORN-HeR and Znf-HeR protein 71 domain fusions. (A) 3D model of a heliorhdodopsin (HeR) containing three N-terminal 72 MORN domain repeats. (B) 3D model of a HeR containing an N-terminal Zn ribbon motif. 73 Both models are oriented with the extracellular side up and intracellular side down. 74 Retinal is coloured green and cysteine residues are depicted with yellow-topped orange 75 sticks. 76 Previous assessments of taxonomic distribution of HeRs reported conflicting data 77 regarding their presence in monoderm (Flores-Uribe, Hevroni, and Ghai 2019) and 78 diderm (Kovalev et al. 2020) prokaryotes. In order to accurately map HeR taxonomic 79 distribution we used the GTDB database (release 89), since it contains a wide-range of 80 high-quality genomes derived from isolated strains and environmental metagenome- 81 assembled genomes, classified within a robust phylogenomic framework (Parks et al. 82 2020). By scanning 24,706 genomes, we identified 450 bona fide HeR sequences 83 (topology: C-terminal inside and N-terminal outside, seven transmembrane helices and a 84 SxxxK motif in helix 7; Supplementary Table S1) spanned across 17 phyla (out of 151; 85 Supplementary Table S2). In order to assign HeR-containing genomes to either 86 monoderm or diderm categories, we employed a set of 27 manually curated protein 87 domain markers that are expected to be restricted to organisms possessing double- 88 membrane cellular envelopes (i.e. diderms) (Taib et al. 2020). While most analyses were 89 expected to be influenced by varying levels of genome completeness , we found that a 90 conservative criterion of presence of at least ten marker domains singled out all diderm 91 lineages (i.e. Negativicutes, Halanaerobiales and Limnochondria) (Taib et al. 2020; 92 Megrian et al. 2020) within the larger monoderm phylum Firmicutes, apart from correctly 93 identifying other well-known diderms. Except for three genomes (one each belonging to bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

94 Myxococcota, Spirochaetota and Dictyoglomota phyla), all other HeR occurrences were 95 restricted to monoderms (Supplementary Table S2). Examination of the HeR-encoding 96 Myxoccoccota contig by querying its predicted proteins against the RefSeq and GTDB 97 databases revealed it to be an actinobacterial contaminant. The Spirochaeta genome was 98 incomplete (60% estimated completeness) and only encoded for two outer membrane 99 marker genes, making any inferences regarding its affiliation to monoderm or diderm 100 bacteria impossible. However, we could not rule out that this genome could belong to a 101 member lacking lipopolysaccharides (LPS) (Taib et al. 2020). The Dictyoglomota genome 102 belongs to an isolate, and despite its high completeness, it encodes only five markers. 103 Combined with the notion that Dictyoglomota are known to have atypical membrane 104 architectures (Saiki et al. 1985), the presence of only five markers points towards the 105 absence of a classical diderm cell envelope. Apart from these exceptions, all other HeR- 106 encoding genomes are monoderm and, at least within this collection, we find no strong 107 evidence of HeRs being present in any organism that is conclusively diderm. We also 108 identified HeRs in several assembled metagenomes and metatranscriptomes (see 109 Methods). For improved resolution of taxonomic origin, we considered only contigs of at 110 least 5 Kb in length (n = 1,340 from metagenomes and n = 4 from metatranscriptomes). 111 Following a strict approach to taxonomy assignment (i.e. at least 60 % genes giving best- 112 hits to the same phylum and not just majority-rule), we could designate a phylum for most 113 HeRs. Without any exception, we found that all the contigs that received robust taxonomic 114 classification (n = 1,319) belonged to known monoderm phyla (Supplementary Table S3).

115 116 Figure 2. Genomic context of HeR-protein domain fusion genes. A) Representative 117 MORN-HeR encoding contigs identified in strictly anaerobic Firmicutes. B) Contigs 118 encoding Znf-HeR domain fusions. Neighbouring genes were depicted within an interval bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

119 spanning ~ 7 kb, centered on HeR. Genes occurring only once within the considered 120 intervals are coloured grey; genes encoding HisKA, PAS, regulatory domains, as well as 121 other discussed HeR neighbours are depicted bright yellow. Homologous genes 122 occurring multiple times found within each category of HeR-protein fusion contigs are 123 depicted using matching colours. Hypothetical genes are white. 124 Domain fusions with rhodopsins are recently providing novel insights into the diverse 125 functional couplings that enhance the utility of a light sensor, e.g. the case of a 126 phosphodiesterase domain fused with a type-1 rhodopsin (Ikuta et al. 2020). As far as we 127 are aware, no domain fusions have been described for HeRs yet. In our search for such 128 domain fusions that may shed light on HeR functionality, the MORN repeat (Membrane 129 Occupation and Recognition Nexus, PF02493) was found in multiple copies (typically 3) at 130 the cytoplasmic N-terminus of some HeRs (n = 36). A tentative 3D model for a 131 representative MORN-HeR could be generated and is shown in Figure 1A. These MORN- 132 HeR sequences were phylogenetically restricted to two environmental branches of MAGs 133 recovered from haloalkaline sediments that affiliate to the family Syntrophomonadaceae 134 (phylum Firmicutes) (Timmers et al. 2018; Vavourakis et al. 2018, 2019) (Supplementary 135 Figure 1). The prototypic MORN repeat, consisting of 14 amino acids with the consensus 136 sequence YEGEWxNGKxHGYG, was first described in 2000 (Takeshima et al. 2000) from 137 junctophilins present in skeletal muscle and later recognized to be ubiquitous in both 138 eukaryotes and prokaryotes (El-Gebali et al. 2019). This conserved signature can be seen 139 in the alignment of MORN-repeats fused to HeRs (Supplementary Figure 2). MORN- 140 repeats have been shown to bind to phospholipids (Im et al. 2007; Ma et al. 2006), 141 promoting stable interactions with plasma membranes (Takeshima et al. 2000) and also 142 function as protein-protein interaction modules involved in di- and oligomerization (Sajko 143 et al. 2020). They are expected to be intracellular and provide a large putative interaction 144 surface (either with other MORN-HeRs or other proteins). A widespread adaptation of 145 bacteria to alkaline environmental conditions is the increased fluidity of their plasma 146 membranes achieved by the incorporation of branched-chain and unsaturated fatty acids 147 which ultimately influences the configuration and activity of membrane integral proteins 148 such as ATP synthases and various transporters (Kanno et al. 2015). Microbial rhodopsins 149 typically associate as oligomers in vivo, which is also the case with heliorhodopsins that 150 are known to form dimers (Shibata et al. 2018; Shihoya et al. 2019). The presence of 151 MORN-repeats in HeRs exclusively within extreme haloalkaliphilic bacteria (class 152 Dethiobacteria) may be accounted for via their potential role in stabilizing HeR dimers in 153 conditions of increased membrane fluidity (Supplementary Figure 4). Another possibility 154 would be the interaction of MORN-repeats with other MORN-repeat containing proteins 155 encoded in these MAGs. We could indeed identify multiple MORN-protein domain 156 fusions co-occurring in genomes of analysed Dethiobacteria (Supplementary Figures 1 157 and 3; Supplementary Table S15). Even though the nature of interactions amongst these 158 proteins with intracellular MORN-repeats is unclear, they raise the possibility that MORN- 159 repeats act as downstream transducers of conformational changes occuring in HeRs. Such 160 tandem repeat structures may function as versatile target recognition sites capable of 161 binding not only small molecules like nucleotides but also peptides and larger proteins 162 (Kajava 2012). If true, this would render HeRs as sensory rhodopsins. In support of this, we 163 found several genes in close proximity to MORN-HeRs encoding signature protein bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

164 domains (e.g. PAS, HisKA, HATPase_c) that are known to be involved in histidine kinase 165 signalling (Aravind, Iyer, and Anantharaman 2010) (Figure 2A).

166 167 Figure 3. Schematic representation of genes that may be transcriptionally linked to HeRs. 168 Taxonomic categories and number of occurrences are shown at the top of each putative 169 operon. Intergenic distances (in bp) are indicated at gene junctions. Negative distance 170 values indicate overlapping genes. Pfam or COG identifiers are used to represent domain 171 architectures. A star (*) indicates a fused gene (two domains: Glutaredoxin and COG4270) 172 found in at least 473 genomes from GTDB and 231 unique sequences in UniProt 173 suggesting a functional linkage of COG4270 with Glutaredoxin. 174 As no other obvious domains were found to be fused with HeRs using standard profile 175 searches, we examined all N and C-terminal extensions as well as loops longer than 50 aa 176 by performing more sensitive profile-profile searches using HHPred (Zimmermann et al. 177 2018). We found at least ten N-terminal extensions of HeRs (ntv1-ntv10), 22 variants of 178 ECL1 (extracellular loop 1), a single type of loop extension for ICL2 (intracellular loop 2) 179 and three variants of ICL3 (intracellular loop 3). A complete listing of all alignments and 180 summary results of HHpred can be found in Supplementary Table S8. Remarkably, we 181 found significant matches in a set of six sequences (all originating from 182 Thermoplasmatales archaea) to zinc ribbon proteins (Pfam domain zinc_ribbon_4) at the 183 N-terminus of some heliorhodopsins (these extensions are termed N-terminal variant 1 or 184 ntv1, Supplementary Table S8). Zinc ribbons belong to the larger family of Zinc-finger 185 domains (Krishna et al., 2003). A CxxC-17x-CxxC was found in this region that likely 186 coordinates a metal (e.g., zinc or iron). These CxxC_CxC type motifs are common to a 187 wider family of Zinc finger-like proteins that were initially found to bind to DNA and later 188 shown to be capable of binding to RNAs, proteins and small molecules (Krishna, 189 Majumdar, and Grishin 2003). Similar motifs are also seen in Rubredoxins and 190 Cys_rich_KTR domains. We term these fused ntv1 protein variants as Znf-HeRs (Zinc finger 191 Heliorhodopins). A modelled structure for a representative Znf-HeR is shown in Figure 1. 192 In one contig encoding a Znf-HeR we identified a histidine kinase that could be 193 functionally linked (Figure 2B). Notably, most identified Znf-HeRs are flanked by genes 194 known to be triggered by light exposure and play key roles in photoprotection (i.e. 195 carotenoid biosynthesis genes Lycopene cyclase, phytoene desaturase – Amino-oxidase, 196 squalene/phytoene synthase – SQS-PSY) and UV-induced DNA damage repair (DNA 197 photolyases, UV-DNA damage endonucleases – UvdE) (Rastogi et al. 2010; Yatsunami et 198 al. 2014). Recent research showed that HeRs from Thermoplasmatales archaea (TaHeR) bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

199 and uncultured freshwater Actinobacteria (48C12) (for which the structure is resolved and 200 lacks the ntv1 extension) might bind zinc (Hashimoto et al. 2020). As the zinc binding site 201 could not be precisely identified it was suggested that it could be located in the 202 cytoplasmic part, and responsible for modifying the function of HeR. Our discovery of Znf- 203 HeRs offers additional, more direct indications of the role of zinc in the possible 204 downstream signalling by HeRs.

205 206 Figure 4. A) Genes encoding HisKA domain signalling proteins identified in the proximity 207 of HeR genes from diverse phyla. All genes containing HisKA domains are coloured bright 208 yellow, HeRs are shown in red, and all other genes in grey. B) Transcripts obtained by 209 strand-specific metatranscriptomics from freshwater encoding genes co-expressed with 210 HeR. 211 Given the large number of long contigs encoding HeRs (from genomes and 212 metagenomes), we sought to identify candidate genes that could be transcribed together 213 with HeRs (in the same operon). We used the following strict criteria for obtaining such 214 genes 1) the intergenic distance between such a gene and the HeR must be less than 10 215 bp, and 2) the gene must be located on the same strand. A number of interesting 216 candidates emerged in this analysis with the most frequent ones being summarized in 217 Figure 3 (a complete table can be found in Supplementary Table S9). We identified 218 multiple instances in which genes with Glutaredoxin and GSHPx PFAM domains were 219 found adjacent to HeRs (n = 31). Glutaredoxins are small redox proteins with active 220 disulphide bonds that utilize reduced glutathione as an electron donor to catalyze thiol- 221 disulphide exchange reactions. They are involved in a wide variety of critical cellular 222 processes like the maintenance of cellular redox state, iron and redox-sensing, and 223 biosynthesis of iron-sulphur clusters (Lillig, Berndt, and Holmgren 2008; Rouhier et al. 224 2010). Glutathione is also used by glutathione peroxidase (GSHPx) to reduce hydrogen 225 peroxide and peroxide radicals i.e. as an anti-oxidative stress protection system (Bhabak 226 and Mugesh 2010). Additionally, there are also instances where Glutaredoxin and genes 227 containing Glyoxalase_2 domains may be co-transcribed with HeRs. Glyoxalases, in bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

228 concert with glutaredoxins, are critical for detoxification of methylglyoxal, a toxic 229 byproduct of glycolysis (Ferguson et al. 1998). Moreover, adjacent to HeRs we find at least 230 three instances where a catalase gene is also present (in Actinobacteria; see 231 Supplementary Figures S10-S11). Collectively, these observations suggest a role for HeRs 232 in oxidative stress mitigation. In one case, we found a gene encoding the DICT domain 233 (Figure 3) which is frequently associated to GGDEF, EAL, HD-GYP, STAS, and two- 234 component system histidine kinases. Notably, it has been predicted to have a role in light 235 response (Aravind, Iyer, and Anantharaman 2010). 236 Although we assembled contigs encoding HeRs from previously published 237 metatranscriptomes, the lack of strand-specific transcriptomes hampered any clear 238 conclusions on whether or not genes adjacent to HeRs are indeed co-transcribed, leaving 239 open the possibility that they might simply be artefacts of assembly (Zhao et al. 2015). In 240 order to gather more definitive evidence for co-transcription we performed strand- 241 specific metatranscriptome sequencing for a freshwater sample (see Methods). We 242 recovered six HeR-encoding transcripts that were > 1 kb in length. All these transcripts are 243 predicted to originate from highly abundant freshwater Actinobacteria with streamlined 244 genomes (four transcripts from “Ca. Planktophila” and two from “Ca. Nanopelagicus”) 245 (Supplementary Table S12) (Neuenschwander et al. 2018). Overall, there are three types 246 of transcripts based upon gene content: class1 - encoding Glutamine synthetase catalytic 247 subunit and NAD+ synthetase; class2 - encoding a hydrolase, a peptidase and a DUF393 248 domain containing protein, and class3 - encoding glucose/sorbosone dehydrogenase 249 (GSDH) (Figure 4B and Supplementary Table S12). A common theme for glutamine 250 synthetase and NAD+ synthetase is that both utilize ammonia and ATP to produce 251 glutamine and NAD+ respectively. Moreover, some NAD+ synthetases may be glutamine 252 dependent (Resto, Yaffe, and Gerratana 2009). Glutamine synthetase in particular is a key 253 enzyme for nitrogen metabolism in prokaryotes at large (García-Domínguez, Reyes, and 254 Florencio 1999). For hydrolases and peptidases, the function prediction is somewhat 255 broad. Glucose/sorbosone dehydrogenase catalyses the production of gluconolactone 256 from glucose (Oubrie et al. 1999). Therefore, it appears that all six HeRs are generally co- 257 transcribed with genes involved in nitrogen assimilation and degradation/assimilation of 258 sugars and peptides. This would suggest that these processes are also influenced by light, 259 with such a link between light-dependent increase in sugar uptake and metabolic activity 260 being recently proposed in non-phototrophic Actinobacteria (Maresca et al. 2019). Light 261 also triggers photosynthetic activity, increasing availability of sugars and other nutrients 262 (e.g. glutamine and ammonia) for heterotrophs. In this vein, a link between a light sensing 263 mechanism, e.g. via heliorhodopsins, may lead to elevated metabolic activity. 264 In a previous study, histidine kinases were deemed absent in the vicinity of HeRs (Kovalev 265 et al. 2020). Given that our initial analyses predicted a sensory function, we examined 266 genomic regions spanning 10 kb up- and downstream of HeRs. Already in the case of 267 MORN-HeRs and Znf-HeRs we observed histidine kinase signalling components in close 268 proximity to them (Figure 2). In our search we detected multiple instances of histidine 269 kinases (HisKA) fused with PAS, GAF, MCP_Signal, HAMP or HATPase_c domains in the 270 gene neighbourhoods of HeRs in distinct phyla (e.g. Actinobacteria, Chloroflexi, 271 Patescibacteria, Firmicutes, Dictyoglomota, Thermoplasmatota) (Figure 4B; more details 272 in Supplementary Figures S5-S14). Moreover, in many cases multiple response regulator 273 genes were present in the same regions (Pfam domains Response_reg, Trans_reg_C). bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

274 Less frequently, GGDEF and EAL domains, usually associated with bacterial signalling 275 proteins, were also present. Using overrepresentation analysis (Shmakov et al. 2018), we 276 found that the occurrence of two-component system protein domains in the vicinity of 277 HeRs is statistically significant (see Methods and Supplementary Table S11). In addition to 278 these two-component system proteins, the same regions also appear enriched in redox 279 proteins (e.g. thioredoxin, peroxidase, catalase). The close association of two-component 280 systems, genes involved in oxidative stress mitigation and HeRs points towards a 281 functional interaction. 282 Conclusions 283 In conclusion, contextual genomic information shows that monoderm prokaryotes use 284 HeRs in multiple mechanisms for the activation of downstream metabolic pathways post 285 light sensing. Furthermore, we offer tantalizing clues regarding the involvement of HeRs 286 in multiple cellular processes and add new lines of inquiry for the primary role of HeRs in 287 light signal transduction. Additional support for the role of HeRs in light sensing is 288 inferred from the frequent association of HeRs with classical histidine kinases and 289 associated protein domains in multiple phyla. Furthermore, multiple types of N-terminal 290 domain fusions found in specific subfamilies of HeRs (i.e. MORN domains in 291 haloalkaliphilic Firmicutes and Zinc ribbon type domains in Thermoplasmatales archaea) 292 point to possible downstream signalling which may be effected by recruitment of 293 additional, as yet unknown, partner proteins. 294 We further propose a critical role for HeRs in protecting monoderm cells from light- 295 induced oxidative damage. In this sense, we observed a close association and probable 296 transcriptional linkage of HeRs to glyoxylases and glutaredoxins (sometimes seen as 297 overlapping genes). Given that light can induce the uptake and metabolism of sugars, as 298 previously discussed for certain Actinobacteria (Maresca et al. 2019), it is expected that 299 increased sugar availability resulting from photosynthesis leads to increased glycolytic 300 activity in heterotrophic bacteria. Glycolysis also produces small amounts of toxic 301 methylglyoxal that can be neutralized by the combined action of glyoxylases and 302 glutaredoxins. In this sense, it appears that at least in some Actinobacteria, glyoxylases 303 and glutaredoxins may be transcribed together with HeRs, but how the transcription is 304 controlled remains unclear. Additional evidence of transcriptional linkages of HeRs to 305 proteins like peroxiredoxin and catalase also imply a light-dependent activation, boosting 306 the cellular response to light induced oxidative damage which may be critical for both 307 aerobes and anaerobes. Evidence from strand-specific HeR transcripts originating from 308 freshwater Actinobacteria suggests the further involvement of HeRs in nitrogen and sugar 309 metabolism via glutamate synthase, NAD+ synthases and glucose/sorbosone 310 dehydrogenases in these organisms. 311 Overall, the picture that emerges (at least for some organisms) is one of HeR’s role in 312 responding to light and transmitting the signal via histidine kinases. Downstream 313 processes that are ultimately regulated are diverse, including possible roles for HeRs in 314 the mitigation of light-induced oxidative damage and in the regulation of nitrogen 315 assimilation and carbohydrate metabolism, processes that may benefit from a light- 316 dependent activation through more efficient utilization of available resources. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

317 Recent work has shown more support for the diderm-first ancestor (Coleman et al. 2020) 318 and given the far broader distribution of type-1 rhodopsins in both mono- and di-derm 319 organisms it appears likely that type-1 rhodopsins emerged prior to HeRs. The very 320 restricted distribution of HeRs to monoderms would support this view as well. Even so, 321 HeRs are not universally present in monoderms and when present, appear to be 322 associated with diverse genes involved in signal transduction, oxidative stress mitigation, 323 nitrogen and glucose metabolism. This would suggest they have been exapted as 324 generalized sensory switches that may allow light-dependent control of metabolic activity 325 in multiple lineages, somewhat similar to type-1 rhodopsins where minor modifications 326 have led to emergence of a wide variety of ion-pumps (Kandori 2020). The frequent 327 distribution of HeRs in aquatic environments (habitats characterised by increased light 328 penetration), where they commonly occur within phylum Actinobacteriota, helps us to 329 explain their monoderm-restricted presence. Abundant freshwater actinobacterial 330 lineages are generally typified by lower GC content (Ghai, McMahon, and Rodriguez- 331 Valera 2012) and increased vulnerability to oxidative stress damage (Kim et al. 2019). This 332 susceptibility is also illustrated by actinobacterial phages that exhibit positive selection 333 towards reactive oxygen species defense mechanisms (Kavagutti et al. 2019). Given the 334 fact that monoderms are generally more sensitive to light-induced damage and 335 corroborated with up-mentioned metabolic implications, we consider that HeRs evolved 336 as sensory switches capable of triggering a fast response against photo-oxidative stress in 337 prokaryotic lineages more sensitive to light. 338 Methods 339 Metagenomes and metatranscriptomes. We used previously published metagenomics 340 and metatranscriptomics data from freshwaters (Andrei et al. 2019; Kavagutti et al. 2019; 341 Mehrshad et al. 2018), haloalkaline brine and sediment (Vavourakis et al. 2018, 2019), 342 brackish sediments (Bulzu et al. 2019), GEOTRACES cruise (Biller et al. 2018) and TARA 343 expeditions (Salazar et al. 2019). In addition, we downloaded multiple environmental 344 metagenomes (sludge, marine, pond, estuary, etc.) from EBI MGnify 345 (https://www.ebi.ac.uk/metagenomics/) (Mitchell et al. 2020) and assembled them using 346 Megahit v1.2.9 (D. Li et al. 2016). All contigs in this work are named or retain existing 347 names that allow tracing them to their original datasets.

348 Sequence search for bona fide rhodopsins. Genes were predicted in metagenomics 349 contigs using Prodigal (Hyatt et al. 2010). Candidate rhodopsin sequences were scanned 350 with hmmsearch (Eddy 2011) using PFAM models (PF18761: heliorhodopsin, PF01036: 351 bac_rhodopsin) and only hits with significant e-values ( < 1e-3) were retained. Homologs 352 for these sequences were identified by comparison to a known set of rhodopsin 353 sequences (Bulzu et al. 2019) using MMSeqs2 (Hauser, Steinegger, and Söding 2016) and 354 alignments were made using MAFFT-linsi(Katoh and Standley 2013). These alignments 355 were used as input to Polyphobius (Käll, Krogh, and Sonnhammer 2005) for 356 transmembrane helix prediction. Only those sequences that had seven transmembrane 357 helices and either a SxxxK motif (for heliorhodopsins) or DxxxK motif (for 358 proteorhodopsins) in TM7 were retained. In addition, we also screened the entire 359 UniProtKB for HeRs. In total, we accumulated at least 4,108 (3,606+502) bona fide HeR 360 sequences. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

361 Taxonomic classification of assembled contigs. Contigs were dereplicated using cd-hit 362 (W. Li and Godzik 2006) (95% sequence identity and 95% coverage). Only contigs ≥ 10 kb 363 were retained for this analysis. A custom protein database was created by predicting and 364 translating genes in all GTDB genomes (release 89) (Parks et al. 2020) using Prodigal 365 (Hyatt et al. 2010). These sequences were supplemented with viral and eukaryotic 366 proteins from UniProtKB (UniProt Consortium 2019). Best-hits against predicted proteins 367 in contigs were obtained using MMSeqs2 (Hauser, Steinegger, and Söding 2016). 368 Taxonomy was assigned to a contig (minimum length 5 kb) only if ≥ 60% of genes in the 369 contig gave best-hits to the same phylum. All contigs that appeared to originate from 370 diderms were cross-checked against NCBI RefSeq (accessed online on 15th December 371 2020).

372 Outer-envelope detection. A set of protein domains found in genes encoding for the 373 outer-envelope (Taib et al. 2020) was further reduced to include only those domains that 374 were found mostly in known diderms. These domains were searched against the 375 predicted proteins in all genomes in GTDB using hmmsearch (e-value < 1e-3). The results 376 are shown in Supplementary Table S13.

377 Protein function/structure predictions. Predicted proteins were annotated using 378 TIGRFAMs (Haft, Selengut, and White 2003) and COGs (Galperin et al. 2015). Domain 379 predictions were carried out using the pfam_scan.pl script against the PFAM database 380 (release 32) (El-Gebali et al. 2019). Profile-profile searches were carried out online using 381 the HHPred server (Zimmermann et al. 2018). Additional annotations were added using 382 Interproscan (Mitchell et al. 2019). Protein structure predictions were carried out using the 383 Phyre2 server (Kelley et al. 2015) and structures were visualized with CueMol 384 (http://www.cuemol.org/en/).

385 Domains overrepresentation near heliorhodopsin. A subset of high-quality MAGs (n = 386 240) containing HeR-encoding genes flanked both up- and downstream by a minimum of 387 10 genes were selected from GTDB (release 89) (Parks et al. 2020). For each genome, the 388 probability of finding any particular domain by chance in a random subset of 20 genes 389 was calculated using the hypergeometric distribution (without replacement) in R with the 390 function phyper (stats package) (Johnson, Kemp, and Kotz 2005). In order to account for 391 type I errors arising from multiple comparisons, hypergeometric test P-values were 392 adjusted using the Benjamini-Hochberg procedure (Benjamini and Hochberg 1995). 393 Further, we selected domains located in the proximity of HeR in at least 10% of genomes 394 with low probability (FDR corrected P-value < 0.05). This procedure that was initially 395 employed for the whole GTDB genome collection was repeated for individual phyla 396 containing HeR-encoding genes within at least five genomes. 397 Strand-specific freshwater transcriptome sequencing and assembly. Sampling was 398 performed on the 16th of August 2020 at 9:00 in Řimov reservoir, Czech Republic, 399 (48°50'54.4"N, 14°29'16.7"E) using a hand-held vertical Friedinger (2 L) sampler. A total 400 of 20 L of water were collected from a depth of 0.5 m and immediately transported to the 401 laboratory. Serial filtration was carried out by passing water sample through a 20 µm pore 402 size pre-filter mesh followed by a 5 µm pore size PES filter (Sterlitech) and a 0.22 µm pore 403 size PES filter (Sterlitech, USA) using a Masterflex peristaltic pump (Cole-Palmer, USA). bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

404 Filtration was done at maximum speed for 15 minutes to limit cell lysis and RNA damage 405 as much as possible. A total volume of 3.7 L was filtered during this time. PES filters (5 µm 406 and 0.22 µm pore sizes) were loaded into cryo-vials pre-filled with 500 µl of DNA/RNA 407 Shield (Zymo Research, USA) and stored at -80˚C. RNA was extracted from filters using the 408 Direct-zol RNA MicroPrep (Zymo Research, USA) after they had been previously thawed, 409 partitioned, and subjected to mechanical lysis by bead-beating in ZR BashingBead™ Lysis 410 tubes (with 0.1 and 0.5 mm spheres). DNase treatment was performed to remove 411 genomic DNA during RNA extraction as an “in-column” step described in the Direct-zol 412 protocol and was repeated after RNA elution, by using the Ambion Turbo DNA-freeTM Kit 413 (Life Technologies, USA). RNA was quantified using a NanoDrop® ND-1000 UV-Vis 414 spectrophotometer (Thermo Fisher Scientific, USA) and integrity was verified by agarose 415 gel (1%) electrophoresis. A total of 4.6 µg of RNA from the 0.22 µm pore size filter and 2.6 416 µg from the 5 µm pore size filter were sent for dUTP-marking based strand-specific 417 metatranscriptomic sequencing at Novogene (www.novogene.com). Following quality 418 control at Novogene, samples were mixed into one single reaction, then subjected to 419 rRNA depletion and used for stranded library preparation. Strand-specificity was achieved 420 by incorporation of dUTPs instead of dTTPs in the second-strand cDNA followed by 421 digestion of dUTPs by uracil-DNA glycosylase to prevent PCR amplification of this strand. 422 Paired-end (PE 150 bp) sequencing was carried out using a Novaseq 6000 platform. A 423 total of 166,213,184 raw sequencing reads, amounting to 24.9 Gb, were produced. De 424 novo assembly of metatranscriptomic data was performed using rnaSPAdes v.3.14.1 425 (Bushmanova et al. 2019) in reverse-forward strand-specific mode (--ss rf) with a custom k- 426 mers list 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 127. A total of 156,235 hard-filtered 427 transcripts of a minimum length of 1 kb were assembled. Protein coding sequences were 428 predicted de novo using Prodigal (Hyatt et al. 2010) in metagenomic mode (-p meta). 429 Protein domains were annotated by scanning with InterProScan(Mitchell et al. 2019) while 430 PFAM (Protein Families)(El-Gebali et al. 2019) domains were identified using the publicly 431 available perl script pfam_scan.pl (ftp://ftp.ebi.ac.uk/pub/databases/Pfam/Tools/). 432 Proteins were scanned locally using HMMER3 (Eddy 2011) against the COGs (Clusters of 433 Orthologous Groups) (Galperin et al. 2015) HMM database (e-value < 1e-5) and the 434 TIGRFAMs (TIGR Families) (Haft, Selengut, and White 2003) HMM collection with trusted 435 score cutoffs. BlastKOALA (Kanehisa, Sato, and Morishima 2016) was used to assign KO 436 identifiers (KO numbers). Annotations for representative transcripts encoding HeR are 437 summarised in Supplementary Table S12. 438 Data availability 439 Sequence data generated in this study have been deposited in the European Nucleotide 440 Archive (ENA) at EMBL-EBI under project accession number PRJEB35770 (run 441 ERR5100021). The derived data that support the findings of this paper, including R code 442 used for statistical analyses, are available in FigShare 443 (https://figshare.com/s/7bb42426f2ad5e891fec). All other relevant data supporting the 444 findings of this study are available within the paper and its supplementary information 445 files. 446 447 448 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

449 Acknowledgements

450 P.-A.B., V.S.K. and R.G. were supported by the research grant 20-12496X (Grant Agency 451 of the Czech Republic). V.S.K. was additionally supported by the research grant 452 116/2019/P (Grant Agency of the University of South Bohemia in České Budějovice, 2019- 453 2021). A.-Ş.A. was supported by Ambizione grant PZ00P3_193240 (Swiss National 454 Science Foundation). M.-C. C. was supported by the Program for the Support of 455 Perspective Human Resources (PPLZ), Czech Academy of Sciences (Grant No. 456 L200961953). K. I. was supported by Grants-in-Aid from the Japan Society for the 457 Promotion of Science (JSPS) for Scientific Research (KAKENHI grant Nos. 20K21383 and 458 20H05758). H. K. was supported by a research grant from the Japanese Ministry of 459 Education, Culture, Sports, Science and Technology (18H03986) and a grant from CREST, 460 Japan Science and Technology Agency (JPMJCR1753).

461 Author contributions

462 R.G. and P.-A.B. designed the study. P.-A.B., A.-Ş.A. and R.G. wrote the manuscript. P.- 463 A.B., R.G., V.S.K, M.-C.C., C.D.V and A.-Ş.A. analysed and interpreted the data. K.I. and 464 H.K. performed rhodopsin structural analyses. All authors commented on and approved 465 the manuscript.

466 Competing interests

467 The authors declare no competing interests.

468 References

469 Andrei, Adrian-Ştefan, Michaela M. Salcher, Maliheh Mehrshad, Pavel Rychtecký, Petr 470 Znachor, and Rohit Ghai. 2019. “Niche-Directed Evolution Modulates Genome 471 Architecture in Freshwater Planctomycetes.” The ISME Journal 13 (4): 1056–71. 472 Aravind, L. 2000. “Guilt by Association: Contextual Information in Genome Analysis.” 473 Genome Research 10 (8): 1074–77. 474 Aravind, L., L. M. Iyer, and V. Anantharaman. 2010. “Natural History of Sensor Domains in 475 Bacterial Signaling Systems.” In Sensory Mechanisms in Bacteria: Molecular Aspects of 476 Signal Recognition, edited by Ray Dixon Stephen Spiro, 1–38. Caister Academic Press 477 Norfolk, UK. 478 Benjamini, Y., and Y. Hochberg. 1995. “Controlling the False Discovery Rate: A Practical 479 and Powerful Approach to Multiple Testing.” Journal of the Royal Statistical Society. 480 https://rss.onlinelibrary.wiley.com/doi/abs/10.1111/j.2517-6161.1995.tb02031.x. 481 Bhabak, Krishna P., and Govindasamy Mugesh. 2010. “Functional Mimics of Glutathione 482 Peroxidase: Bioinspired Synthetic Antioxidants.” Accounts of Chemical Research 43 (11): 483 1408–19. 484 Biller, Steven J., Paul M. Berube, Keven Dooley, Madeline Williams, Brandon M. Satinsky, 485 Thomas Hackl, Shane L. Hogle, et al. 2018. “Marine Microbial Metagenomes Sampled 486 across Space and Time.” Scientific Data 5 (September): 180176. 487 Bulzu, Paul-Adrian, Adrian-Ştefan Andrei, Michaela M. Salcher, Maliheh Mehrshad, Keiichi 488 Inoue, Hideki Kandori, Oded Beja, Rohit Ghai, and Horia L. Banciu. 2019. “Casting Light bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

489 on Asgardarchaeota Metabolism in a Sunlit Microoxic Niche.” Nature Microbiology 4 (7): 490 1129–37. 491 Bushmanova, Elena, Dmitry Antipov, Alla Lapidus, and Andrey D. Prjibelski. 2019. 492 “RnaSPAdes: A de Novo Transcriptome Assembler and Its Application to RNA-Seq Data.” 493 GigaScience 8 (9). https://doi.org/10.1093/gigascience/giz100. 494 Coleman, Gareth A., Adrián A. Davín, Tara Mahendrarajah, Anja Spang, Philip Hugenholtz, 495 Gergely J. Szöllősi, and Tom A. Williams. 2020. “A Rooted Phylogeny Resolves Early 496 Bacterial Evolution.” Cold Spring Harbor Laboratory. 497 https://doi.org/10.1101/2020.07.15.205187. 498 Eddy, Sean R. 2011. “Accelerated Profile HMM Searches.” PLoS Computational Biology 7 499 (10): e1002195. 500 El-Gebali, Sara, Jaina Mistry, Alex Bateman, Sean R. Eddy, Aurélien Luciani, Simon C. 501 Potter, Matloob Qureshi, et al. 2019. “The Pfam Protein Families Database in 2019.” 502 Nucleic Acids Research 47 (D1): D427–32. 503 Ferguson, G. P., S. Tötemeyer, M. J. MacLean, and I. R. Booth. 1998. “Methylglyoxal 504 Production in Bacteria: Suicide or Survival?” Archives of Microbiology 170 (4): 209–18. 505 Flores-Uribe, J., G. Hevroni, and R. Ghai. 2019. “Heliorhodopsins Are Absent in Diderm 506 (Gram-negative) Bacteria: Some Thoughts and Possible Implications for Activity.” 507 Environmental Microbiology Reports. 508 https://onlinelibrary.wiley.com/doi/abs/10.1111/1758-2229.12730. 509 Galperin, Michael Y., Kira S. Makarova, Yuri I. Wolf, and Eugene V. Koonin. 2015. 510 “Expanded Microbial Genome Coverage and Improved Protein Family Annotation in the 511 COG Database.” Nucleic Acids Research 43 (Database issue): D261-9. 512 García-Domínguez, M., J. C. Reyes, and F. J. Florencio. 1999. “Glutamine Synthetase 513 Inactivation by Protein-Protein Interaction.” Proceedings of the National Academy of 514 Sciences of the United States of America 96 (13): 7161–66. 515 Ghai, Rohit, Katherine D. McMahon, and Francisco Rodriguez-Valera. 2012. “Breaking a 516 Paradigm: Cosmopolitan and Abundant Freshwater Actinobacteria Are Low GC.” 517 Environmental Microbiology Reports 4 (1): 29–35. 518 Haft, Daniel H., Jeremy D. Selengut, and Owen White. 2003. “The TIGRFAMs Database of 519 Protein Families.” Nucleic Acids Research 31 (1): 371–73. 520 Hashimoto, Masanori, Kota Katayama, Yuji Furutani, and Hideki Kandori. 2020. “Zinc 521 Binding to Heliorhodopsin.” Journal of Physical Chemistry Letters 11 (20): 8604–9. 522 Hauser, Maria, Martin Steinegger, and Johannes Söding. 2016. “MMseqs Software Suite 523 for Fast and Deep Clustering and Searching of Large Protein Sequence Sets.” 524 Bioinformatics 32 (9): 1323–30. 525 Huynen, M., B. Snel, W. Lathe 3rd, and P. Bork. 2000. “Predicting Protein Function by 526 Genomic Context: Quantitative Evaluation and Qualitative Inferences.” Genome Research 527 10 (8): 1204–10. 528 Hyatt, Doug, Gwo-Liang Chen, Philip F. Locascio, Miriam L. Land, Frank W. Larimer, and 529 Loren J. Hauser. 2010. “Prodigal: Prokaryotic Gene Recognition and Translation Initiation 530 Site Identification.” BMC Bioinformatics 11 (March): 119. 531 Ikuta, Tatsuya, Wataru Shihoya, Masahiro Sugiura, Kazuho Yoshida, Masahito Watari, 532 Takaya Tokano, Keitaro Yamashita, et al. 2020. “Structural Insights into the Mechanism of 533 Rhodopsin Phosphodiesterase.” Nature Communications 11 (1): 5605. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

534 Im, Yang Ju, Amanda J. Davis, Imara Y. Perera, Eva Johannes, Nina S. Allen, and Wendy F. 535 Boss. 2007. “The N-Terminal Membrane Occupation and Recognition Nexus Domain of 536 Arabidopsis Phosphatidylinositol Phosphate Kinase 1 Regulates Enzyme Activity.” The 537 Journal of Biological Chemistry 282 (8): 5443–52. 538 Johnson, Norman L., Adrienne W. Kemp, and Samuel Kotz. 2005. Univariate Discrete 539 Distributions. John Wiley & Sons. 540 Kajava, Andrey V. 2012. “Tandem Repeats in Proteins: From Sequence to Structure.” 541 Journal of Structural Biology 179 (3): 279–88. 542 Käll, Lukas, Anders Krogh, and Erik L. L. Sonnhammer. 2005. “An HMM Posterior Decoder 543 for Sequence Feature Prediction That Includes Homology Information.” Bioinformatics 21 544 Suppl 1 (June): i251-7. 545 Kandori, Hideki. 2020. “Biophysics of Rhodopsins and Optogenetics.” Biophysical Reviews 546 12 (2): 355–61. 547 Kanehisa, Minoru, Yoko Sato, and Kanae Morishima. 2016. “BlastKOALA and 548 GhostKOALA: KEGG Tools for Functional Characterization of Genome and Metagenome 549 Sequences.” Journal of Molecular Biology 428 (4): 726–31. 550 Kanno, Manabu, Hideyuki Tamaki, Yasuo Mitani, Nobutada Kimura, Satoshi Hanada, and 551 Yoichi Kamagata. 2015. “PH-Induced Change in Cell Susceptibility to Butanol in a High 552 Butanol-Tolerant Bacterium, Enterococcus Faecalis Strain CM4A.” Biotechnology for 553 Biofuels 8 (April): 69. 554 Katoh, Kazutaka, and Daron M. Standley. 2013. “MAFFT Multiple Sequence Alignment 555 Software Version 7: Improvements in Performance and Usability.” Molecular Biology and 556 Evolution 30 (4): 772–80. 557 Kavagutti, Vinicius S., Adrian-Ştefan Andrei, Maliheh Mehrshad, Michaela M. Salcher, and 558 Rohit Ghai. 2019. “Phage-Centric Ecological Interactions in Aquatic Ecosystems Revealed 559 through Ultra-Deep Metagenomics.” Microbiome 7 (1): 135. 560 Kelley, Lawrence A., Stefans Mezulis, Christopher M. Yates, Mark N. Wass, and Michael J. 561 E. Sternberg. 2015. “The Phyre2 Web Portal for Protein Modeling, Prediction and 562 Analysis.” Nature Protocols 10 (6): 845–58. 563 Kim, Suhyun, Ilnam Kang, Ji-Hui Seo, and Jang-Cheon Cho. 2019. “Culturing the 564 Ubiquitous Freshwater Actinobacterial AcI Lineage by Supplying a Biochemical ‘helper’ 565 Catalase.” The ISME Journal 13 (9): 2252–63. 566 Kovalev, K., D. Volkov, R. Astashkin, A. Alekseev, I. Gushchin, J. M. Haro-Moreno, I. 567 Chizhov, et al. 2020. “High-Resolution Structural Insights into the Heliorhodopsin Family.” 568 Proceedings of the National Academy of Sciences of the United States of America 117 (8): 569 4131–41. 570 Krishna, S. Sri, Indraneel Majumdar, and Nick V. Grishin. 2003. “Structural Classification of 571 Zinc Fingers: Survey and Summary.” Nucleic Acids Research 31 (2): 532–50. 572 Li, Dinghua, Ruibang Luo, Chi-Man Liu, Chi-Ming Leung, Hing-Fung Ting, Kunihiko 573 Sadakane, Hiroshi Yamashita, and Tak-Wah Lam. 2016. “MEGAHIT v1.0: A Fast and 574 Scalable Metagenome Assembler Driven by Advanced Methodologies and Community 575 Practices.” Methods 102 (June): 3–11. 576 Li, Weizhong, and Adam Godzik. 2006. “Cd-Hit: A Fast Program for Clustering and 577 Comparing Large Sets of Protein or Nucleotide Sequences.” Bioinformatics 22 (13): 1658– 578 59. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

579 Lillig, Christopher Horst, Carsten Berndt, and Arne Holmgren. 2008. “Glutaredoxin 580 Systems.” Biochimica et Biophysica Acta 1780 (11): 1304–17. 581 Ma, Hui, Ying Lou, Wen Hui Lin, and Hong Wei Xue. 2006. “MORN Motifs in Plant PIPKs 582 Are Involved in the Regulation of Subcellular Localization and Phospholipid Binding.” Cell 583 Research 16 (5): 466–78. 584 Maclean, Michelle, Scott J. MacGregor, John G. Anderson, and Gerry Woolsey. 2009. 585 “Inactivation of Bacterial Pathogens Following Exposure to Light from a 405-Nanometer 586 Light-Emitting Diode Array.” Applied and Environmental Microbiology 75 (7): 1932–37. 587 Maresca, Julia A., Jessica L. Keffer, Priscilla P. Hempel, Shawn W. Polson, Olga 588 Shevchenko, Jaysheel Bhavsar, Deborah Powell, Kelsey J. Miller, Archana Singh, and 589 Martin W. Hahn. 2019. “Light Modulates the Physiology of Nonphototrophic 590 Actinobacteria.” Journal of Bacteriology 201 (10). https://doi.org/10.1128/JB.00740-18. 591 Megrian, Daniela, Najwa Taib, Jerzy Witwinowski, Christophe Beloin, and Simonetta 592 Gribaldo. 2020. “One or Two Membranes? Diderm Firmicutes Challenge the Gram- 593 Positive/Gram-Negative Divide.” Molecular Microbiology 113 (3): 659–71. 594 Mehrshad, Maliheh, Michaela M. Salcher, Yusuke Okazaki, Shin-Ichi Nakano, Karel Šimek, 595 Adrian-Stefan Andrei, and Rohit Ghai. 2018. “Hidden in Plain Sight-Highly Abundant and 596 Diverse Planktonic Freshwater Chloroflexi.” Microbiome 6 (1): 176. 597 Mitchell, Alex L., Alexandre Almeida, Martin Beracochea, Miguel Boland, Josephine 598 Burgin, Guy Cochrane, Michael R. Crusoe, et al. 2020. “MGnify: The Microbiome Analysis 599 Resource in 2020.” Nucleic Acids Research 48 (D1): D570–78. 600 Mitchell, Alex L., Teresa K. Attwood, Patricia C. Babbitt, Matthias Blum, Peer Bork, Alan 601 Bridge, Shoshana D. Brown, et al. 2019. “InterPro in 2019: Improving Coverage, 602 Classification and Access to Protein Sequence Annotations.” Nucleic Acids Research 47 603 (D1): D351–60. 604 Neuenschwander, Stefan M., Rohit Ghai, Jakob Pernthaler, and Michaela M. Salcher. 605 2018. “Microdiversification in Genome-Streamlined Ubiquitous Freshwater 606 Actinobacteria.” The ISME Journal 12 (1): 185–98. 607 Oubrie, A., H. J. Rozeboom, K. H. Kalk, A. J. Olsthoorn, J. A. Duine, and B. W. Dijkstra. 608 1999. “Structure and Mechanism of Soluble Quinoprotein Glucose Dehydrogenase.” The 609 EMBO Journal 18 (19): 5187–94. 610 Parks, Donovan H., Maria Chuvochina, Pierre-Alain Chaumeil, Christian Rinke, Aaron J. 611 Mussig, and Philip Hugenholtz. 2020. “A Complete Domain-to-Species Taxonomy for 612 Bacteria and Archaea.” Nature Biotechnology 38 (9): 1079–86. 613 Pushkarev, Alina, Keiichi Inoue, Shirley Larom, José Flores-Uribe, Manish Singh, Masae 614 Konno, Sahoko Tomida, et al. 2018. “A Distinct Abundant Group of Microbial Rhodopsins 615 Discovered Using Functional Metagenomics.” Nature 558 (7711): 595–99. 616 Rastogi, Rajesh P., Richa, Ashok Kumar, Madhu B. Tyagi, and Rajeshwar P. Sinha. 2010. 617 “Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair.” 618 Journal of Nucleic Acids 2010 (December): 592980. 619 Resto, Melissa, Jason Yaffe, and Barbara Gerratana. 2009. “An Ancestral Glutamine- 620 Dependent NAD(+) Synthetase Revealed by Poor Kinetic Synergism.” Biochimica et 621 Biophysica Acta 1794 (11): 1648–53. 622 Rouhier, Nicolas, Jérémy Couturier, Michael K. Johnson, and Jean-Pierre Jacquot. 2010. 623 “Glutaredoxins: Roles in Iron Homeostasis.” Trends in Biochemical Sciences 35 (1): 43–52. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

624 Saiki, Takashi, Yasuhiko Kobayashi, Kiyotaka Kawagoe, and Teruhiko Beppu. 1985. 625 “Dictyoglomus Thermophilum Gen. Nov., Sp. Nov., a Chemoorganotrophic, Anaerobic, 626 Thermophilic Bacterium.” International Journal of Systematic and Evolutionary 627 Microbiology 35 (3): 253–59. 628 Sajko, S., I. Grishkovskaya, J. Kostan, and M. Graewert. 2020. “Structures of Three MORN 629 Repeat Proteins and a Re-Evaluation of the Proposed Lipid-Binding Properties of MORN 630 Repeats.” BioRxiv. https://www.biorxiv.org/content/10.1101/826180v2.abstract. 631 Salazar, Guillem, Lucas Paoli, Adriana Alberti, Jaime Huerta-Cepas, Hans-Joachim 632 Ruscheweyh, Miguelangel Cuenca, Christopher M. Field, et al. 2019. “Gene Expression 633 Changes and Community Turnover Differentially Shape the Global Ocean 634 Metatranscriptome.” Cell 179 (5): 1068-1083.e21. 635 Shibata, Mikihiro, Keiichi Inoue, Kento Ikeda, Masae Konno, Manish Singh, Chihiro 636 Kataoka, Rei Abe-Yoshizumi, Hideki Kandori, and Takayuki Uchihashi. 2018. “Oligomeric 637 States of Microbial Rhodopsins Determined by High-Speed Atomic Force Microscopy and 638 Circular Dichroic Spectroscopy.” Scientific Reports 8 (1): 8262. 639 Shihoya, Wataru, Keiichi Inoue, Manish Singh, Masae Konno, Shoko Hososhima, Keitaro 640 Yamashita, Kento Ikeda, et al. 2019. “Crystal Structure of Heliorhodopsin.” Nature 574 641 (7776): 132–36. 642 Shmakov, Sergey A., Kira S. Makarova, Yuri I. Wolf, Konstantin V. Severinov, and Eugene V. 643 Koonin. 2018. “Systematic Prediction of Genes Functionally Linked to CRISPR-Cas Systems 644 by Gene Neighborhood Analysis.” Proceedings of the National Academy of Sciences of 645 the United States of America 115 (23): E5307–16. 646 Taib, Najwa, Daniela Megrian, Jerzy Witwinowski, Panagiotis Adam, Daniel Poppleton, 647 Guillaume Borrel, Christophe Beloin, and Simonetta Gribaldo. 2020. “Genome-Wide 648 Analysis of the Firmicutes Illuminates the Diderm/Monoderm Transition.” Nature Ecology 649 & Evolution, October. https://doi.org/10.1038/s41559-020-01299-7. 650 Takeshima, H., S. Komazaki, M. Nishi, M. Iino, and K. Kangawa. 2000. “Junctophilins: A 651 Novel Family of Junctional Membrane Complex Proteins.” Molecular Cell 6 (1): 11–22. 652 Tanaka, Tatsuki, Manish Singh, Wataru Shihoya, Keitaro Yamashita, Hideki Kandori, and 653 Osamu Nureki. 2020. “Structural Basis for Unique Color Tuning Mechanism in 654 Heliorhodopsin.” Biochemical and Biophysical Research Communications 533 (3): 262–67. 655 Timmers, Peer H. A., Charlotte D. Vavourakis, Robbert Kleerebezem, Jaap S. Sinninghe 656 Damsté, Gerard Muyzer, Alfons J. M. Stams, Dimity Y. Sorokin, and Caroline M. Plugge. 657 2018. “Metabolism and Occurrence of Methanogenic and Sulfate-Reducing Syntrophic 658 Acetate Oxidizing Communities in Haloalkaline Environments.” Frontiers in Microbiology 9 659 (December): 3039. 660 UniProt Consortium. 2019. “UniProt: A Worldwide Hub of Protein Knowledge.” Nucleic 661 Acids Research 47 (D1): D506–15. 662 Vavourakis, Charlotte D., Adrian-Stefan Andrei, Maliheh Mehrshad, Rohit Ghai, Dimitry Y. 663 Sorokin, and Gerard Muyzer. 2018. “A Metagenomics Roadmap to the Uncultured 664 Genome Diversity in Hypersaline Soda Lake Sediments.” Microbiome 6 (1): 168. 665 Vavourakis, Charlotte D., Maliheh Mehrshad, Cherel Balkema, Rutger van Hall, Adrian- 666 Ştefan Andrei, Rohit Ghai, Dimitry Y. Sorokin, and Gerard Muyzer. 2019. “Metagenomes 667 and Metatranscriptomes Shed New Light on the Microbial-Mediated Sulfur Cycle in a 668 Siberian Soda Lake.” BMC Biology 17 (1): 69. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

669 Yatsunami, Rie, Ai Ando, Ying Yang, Shinichi Takaichi, Masahiro Kohno, Yuriko 670 Matsumura, Hiroshi Ikeda, et al. 2014. “Identification of Carotenoids from the Extremely 671 Halophilic Archaeon Haloarcula Japonica.” Frontiers in Microbiology 5 (March): 100. 672 Zhao, Shanrong, Ying Zhang, William Gordon, Jie Quan, Hualin Xi, Sarah Du, David von 673 Schack, and Baohong Zhang. 2015. “Comparison of Stranded and Non-Stranded RNA- 674 Seq Transcriptome Profiling and Investigation of Gene Overlap.” BMC Genomics 16 675 (September): 675. 676 Zimmermann, Lukas, Andrew Stephens, Seung-Zin Nam, David Rau, Jonas Kübler, Marko 677 Lozajic, Felix Gabler, Johannes Söding, Andrei N. Lupas, and Vikram Alva. 2018. “A 678 Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at Its 679 Core.” Journal of Molecular Biology 430 (15): 2237–43. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Supplementary Information

2 Heliorhodopsin evolution is driven by photosensory promiscuity in monoderms

3 Paul-Adrian Bulzu1, Vinicius Silva Kavagutti1,2, Maria-Cecilia Chiriac1, Charlotte 4 D. Vavourakis3, Keiichi Inoue4, Hideki Kandori5,6, Adrian-Stefan Andrei7, Rohit 5 Ghai1*

6 1Department of Aquatic Microbial Ecology, Institute of Hydrobiology, Biology Centre of 7 the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic.

8 2Department of Ecosystem Biology, Faculty of Science, University of South Bohemia, 9 Branišovská 1760, České Budějovice, Czech Republic.

10 3EUTOPS, Research Institute for Biomedical Aging Research, University of Innsbruck, 11 Austria.

12 4The Institute for Solid State Physics, The University of Tokyo, Kashiwa, Japan.

13 5Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, 14 Showa, Nagoya 466-8555, Japan.

15 6OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa, Nagoya 16 466-8555, Japan

17 7Limnological Station, Department of Plant and Microbial Biology, University of Zurich, 18 Kilchberg, Switzerland.

19 *Corresponding author: Rohit Ghai 20 Department of Aquatic Microbial Ecology, Institute of Hydrobiology, Biology Centre of the 21 Academy 22 of Sciences of the Czech Republic, Na Sádkách 7, 370 05, České Budějovice, Czech 23 Republic. 24 Phone: +420 387 775 881 25 Fax: +420 385 310 248 26 E-mail: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

27 We examined the distribution and co-occurrence of HeRs and type-1 rhodopsins 28 (Supplementary Tables S4-S7) in the GTDB database (release 89), as it has been previously 29 suggested that these proteins tend to coexist within the same organisms (Kovalev et al. 30 2020). From all 24,706 scanned genomes we identified and retrieved 1,455 bona fide 31 type-1 rhodopsin-containing genomes from which 69 (4.74 %) proved to also harbour 32 HeRs. Since 15.33 % from all identified HeRs (n = 450) co-occur with type-1 rhodopsins we 33 consider it as being more an exception than a norm and find no data to sustain a 34 physiological dependency between these two rhodopsin families. 35 MORN-protein domain fusions 36 The bacteria encoding MORN-HeRs were previously predicted to be strict anaerobes 37 (Timmers et al. 2018). Although mostly recovered from sediments, these MAGs also 38 encode other proteins directly or indirectly associated with the presence of light (i.e. 39 bacteriophytochrome COG4251, DNA repair photolyase COG1533, 40 Deoxyribodipyrimidine photolyase COG0415). Therefore, the co-occurrence of strict 41 anaerobiosis and light-dependent components indicates the top sediment layer as their 42 likely habitat. The available 3D structure of MORN-repeats shows a single repeat to consist 43 of short beta-pleated regions folded back upon themselves, creating a flat surface area 44 that expands when the repeats are present in multiple tandem copies (Wilson et al., 2002). 45 The presence of periplasmic proteins with MORN-Big_2 or MORN-Big2-PASTA domains 46 may indicate the extracellular MORN repeats as adaptors between typical sensor domains 47 (i.e. PASTA/Big_2) and the transducer protein kinases acting in the cytoplasm 48 (Supplementary Figure 3). However, as the MORN-repeats that are fused to HeRs in these 49 organisms are intracellular, they could only interact with MORN-repeats on the 50 cytoplasmic side. In one such case, intracellular MORN-repeats were fused to ATPase 51 component of an ABC-type efflux pump (likely involved in drug resistance or Cu2+/Na2+ 52 ion efflux). The other candidate found was a PknB protein kinase-FHA-MORN repeat fusion 53 that was predicted to be in an atypical membrane orientation. In such proteins, 54 dimerization domains (e.g. PASTA) are extracellular (Supplementary Figure 3C) while 55 cytoplasmic protein kinase domains function as part of signal transduction pathways in a 56 wide range of gram-positive bacteria (Kang et al. 2005). The dimerization of PknB is 57 essential for autophosphorylation and activation of the kinases through an allosteric 58 mechanism (Lombana et al. 2010). The predicted reverse orientation (if correct) of this 59 protein renders MORN-mediated interactions with HeR unlikely in these organisms. 60 However, the presence of MORN-repeats in PknB type proteins for which dimerization is 61 essential for function (most likely via MORN-repeats) strengthens the possibility that the 62 MORN-repeats aid in MORN-HeR dimerization as well. 63 Various Cysteine-rich motif-containing heliorhodopsins 64 Another extension found in four sequences (N-terminal variant 2 or ntv2 in Supplementary 65 Table S8) with many more cysteines (n = 10) that did not give any significant hits to known 66 proteins was also identified. The presence of multiple cysteine residues and a conserved 67 tryptophan residue is reminiscent of RINGv finger domains that coordinate two metals. 68 However, RING finger domains also have a highly conserved histidine residue that was not 69 detected. Indeed, we also found at least two instances in freshwater Actinobacteria (Ca. 70 Planktophila) where a RINGv domain-containing protein is located right upstream of the 71 heliorhodopsin gene and in the same orientation (see Figure 3). bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

72 A third variant (ntv3) with seven cysteines was found (in Thermoplasmatales and in 73 Euryarchaeota) but without conserved histidines or tryptophan. However, it shows broad 74 similarity with ntv3 in presence of conserved prolines and arginines before the cysteine 75 motifs. Apart from the n-terminal extensions motifs, at least three sequences of the 76 intracellular loop (ICL3), were also found to be rich in cysteines (n = 8) which also 77 presented a conserved tryptophan similar to ntv2 described above (intracellular loop 3 78 variant 1, icl3v1). 79 Thus, at least 17 sequences presented cysteine-rich motifs either at the N-terminus (ntv1, 80 ntv2 and ntv3) or in the intracellular loop (icl3v1) at the cytoplasmic side of 81 heliorhodopsins suggesting the possibility that these might be transducers of the 82 conformational change in heliorhodopsins upon light excitation. The conserved cysteines 83 in these proteins could bind either iron or zinc and are likely redox-active. While we did 84 find many other types of N-terminal extensions, we were unable to find any significant hits 85 to these even by sensitive sequence searches (Supplementary Table S8). 86 Sources of retinal for HeR function 87 We also found several NAD-dependent short-chain dehydrogenases that might encode 88 for retinol dehydrogenases in the vicinity of HeRs (adh_short in Figure 3, Supplementary 89 Table S9). It has been mentioned before that as HeRs are able to efficiently capture retinal 90 from exogenous sources and that HeR-encoding microbes do not have a retinal 91 biosynthesis pathway (Shihoya et al. 2019). 92 De novo retinal biosynthesis requires five genes that if supplied in-trans to a non-retinal 93 producing microbe may result in functional rhodopsins (Sabehi et al. 2005). The final step 94 of the de novo pathway uses a beta-carotene monooxygenase that converts beta-carotene 95 to retinal. However, retinal may also be converted from retinol by the action of retinol 96 dehydrogenases. We used the curated GTDB database to further probe the co- 97 occurrence of HeR and type-1 rhodopsins along with genes for retinal biosynthesis. Of the 98 total of 381 genomes that encoded only HeR, we find that only a single genome encoded 99 all genes necessary for the de novo production of retinal, but 213 (55%) also encoded the 100 beta-carotene monooxygenase and 241 genomes (63%) encoded at least one retinol 101 dehydrogenase (Supplementary Table S10). Considering genomes that encoded only 102 type-1 rhodopsins (n = 1,386), 596 (43%) encoded the complete pathway for retinal 103 biosynthesis and additionally 995 (71%) also encoded at least one retinol dehydrogenase. 104 It appears that microbes encoding only HeR mostly lack the complete pathway for de novo 105 retinal biosynthesis and that apart from exogenous capture of retinal, conversion from 106 beta-carotene (via beta-carotene monooxygenase) or from retinol (via retinol 107 dehydrogenases) may be at work. 108 HeR genomic context 109 We performed gene context analysis of HeRs by combining the maximum-likelihood 110 phylogenetic tree generated for representative HeR sequences (n = 872) with HeR gene 111 neighbourhood information (Supp. Figure 4; iTOL: 112 https://itol.embl.de/tree/14723125092152021608050562). The resulting tree places most 113 HeR sequences (n = 835) within 19 conspicuous phylogenetic clusters which we further 114 denominate as C1-C19 (see Supp. Figure 15). Among them, Actinobacteriota-encoded 115 HeRs are by far the most numerous (n = 533) accounting for eight well-defined clusters bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

116 (i.e. C1-7 and C11) and a small sub-cluster within Patescibacteria (CPR)-dominated C17. 117 Regarding Actinobacteriota, C1 and C3 are represented by order Nanopelagicales, C2 118 includes chiefly members of Microtrichales (class Acidimicrobiia), C4 comprises 119 Actinomycetales HeRs and C5 includes classes Coriobacteriia and Thermoleophilia. 120 Notably, C5 brings together HeRs recovered mainly from lesser studied sediment habitats 121 including nine Chloroflexota (class Dehalococcoidia) sequences. Predominantly marine 122 C6 includes Acidimicrobiia HeRs from order Microtrichales and other poorly classified 123 representatives from within this class while C7 has both marine and freshwater 124 Microtrichales together with Propionibacteriales genus Nocardioides. 125 Cluster C11 stands out in this analysis due to the high level of evolutionary conservation of 126 both HeRs and their neighbouring genes, bringing together exclusively members of 127 marine Actinobacteria from order Ca. Actinomarinales (TMED189). Importantly, in C11 we 128 notice that synteny is only conserved among genes sharing the same orientation as HeR 129 while gene “gains” and/or “losses” occur only in the opposite orientation. Despite very 130 high phylogenetic relatedness within C11 and therefore the unsurprisingly similar gene 131 context amongst its members, the differences between (+) and (-) strand feature 132 conservation (relative to HeR orientation) indicate a potentially relevant transcriptional unit 133 comprised of genes: afuA - iron(III) transport system substrate-binding protein (K02012), 134 afuB - iron(III) transport system permease protein (K02011), afuC - iron(III) transport system 135 ATP-binding protein (K02010), HeR – Heliorhodopsin (PF18761), an 11-subunit respiratory 136 complex I operon (nuoA, B, C, D, H, I, J, K, L, M, N), NDUFAF7 - NADH dehydrogenase 137 [ubiquinone] 1 alpha subcomplex assembly factor 7 (K18164/PF02636), htpX - heat shock 138 protein (K03799), pspE - phage shock protein E (K03972) containing a Rhodanese 139 (PF00581) domain and adenosine_kinase (cd01168) (Note: full-length annotated contigs 140 deposited in Figshare). In summary, HeRs from C11 are always preceded by genes 141 encoding a complete ABC-type ferric iron uptake system and followed by an operon 142 encoding a 11-subunit, “ancestral”-type (Moparthi and Hägerhäll 2011) respiratory 143 complex I and by accessory components required for correct assembly and function of this 144 complex (NDUFAF7, htpX, pspE) (Zurita Rendón et al. 2014; Pagani and Galante 1983; 145 Alexander and Volini 1987; Sakoh, Ito, and Akiyama 2005). The last conserved gene 146 encodes a pfkB family adenosine kinase (cd01168), a key purine salvage enzyme that 147 phosphorylates adenosine to generate adenosine monophosphate (AMP) (Long, Escuyer, 148 and Parker 2003). 149 The presence of HeRs within the same transcriptional unit as above mentioned energy 150 metabolism components could indicate them as modulators or even light-induced sensory 151 “switches” of such processes, a mechanism perhaps similar to cryptochrome-driven 152 metabolic synchronization with substrate availability described in other Actinobacteria 153 (Maresca et al. 2019). Notably, beside the 11-subunit complex I, that lacks the NADH 154 dehydrogenase module (subunits nuoE, nuoF, nuoG) (Moparthi and Hägerhäll 2011), Ca. 155 Actinomarina (TMED189) genomes also encode the full-sized 14-subunit variant of 156 respiratory complex I in close proximity to the first (for example in GCA-902516125.1). 157 Curiously, the association of HeRs with complex I genes is reminiscent of that between the 158 transmembrane, sensory, EAL-domain containing protein seen in Bacillus cereus located 159 upstream of a similar 11-subunit complex I operon (Moparthi and Hägerhäll 2011). bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

160 The last cluster featuring a significant number of Actinobacteria HeRs (n = 12) is C17. In 161 this CPR-dominated cluster, Actinobacteria HeRs form a well-defined group sharing a 162 common ancestor with a small CPR sub-cluster. While gene context appears conserved 163 within these Actinobacteria, this does not apply to the putative “sister” CPR sub-cluster. 164 Clusters C8-C10 share a common ancestor with C11 and include HeRs encoded largely in 165 strict or facultative anaerobic prokaryotes recovered from sediments (including activated 166 sludge). Notably, the phylogenetic tree (iTOL link above) shows two Asgardarchaeota 167 (class Heimdallarchaeia) HeRs branching with very high support (SH-test/UFBoot = 168 97.2/98) as a sister clade to all C8-C10 members, after the split with the common ancestor 169 shared with C11. Despite the high support for the Asgardarchaeota HeR split, defining a 170 credible cluster will require including additional sequences once more genomes become 171 available. The basal, C10 cluster, is comprised of Archaea-derived HeRs from phyla 172 Crenarchaeota (class Bathyarchaeia) and Euryarchaeota (class Methanobacteria). Clusters 173 C8 and mainly C9 include the MORN-HeR encoding Firmicutes as well as a few 174 Chloroflexota (class Anaerolineae) HeRs. 175 C12-C16 form a separate super-cluster showing moderate-to-low support for internal 176 branching patterns and include mostly, although not exclusively, anaerobic Archaea (C12 177 and C14, with the notable exception of aerobic Halobacterota within C12) and anaerobic 178 Chloroflexota (classes Anaerolineae - C13, C15 and Dehalococcoidia – C16). Notably, C16 179 includes one Thermoplasmatota HeR (encoded in contig SRR5506739-C331) with a zinc- 180 finger extension (Znf-HeR) at the N-terminus. C18 is the most basal cluster with confidently 181 assigned taxonomy. It includes exclusively aerobic Chloroflexota members of the 182 Ellin6529 lineage. 183 Although no consensus taxonomy could be determined for members of C19 – the first 184 branching group after the split with proteorhodopsins, the abundance of “eukaryotic 185 signature proteins” (ESP) (e.g. Arf, Roc, Rab, etc.) points towards either a eukaryotic origin 186 or unidentified, ESP-rich archaea (Hartman and Fedorov 2002; Dong, Wen, and Tian 187 2007). 188 An extended phylogenetic tree of HeRs, including additional dereplicated sequences 189 identified and retrieved from UniProtKB and GTDB, is available in FigShare (see Supp. 190 Methods - Phylogenetic tree of HeRs).

191 Supplementary Methods

192 Re-assembling of HeR-encoding Spirochaeta. The unexpected detection of HeR in a 193 previously published Spirochaeta (diderm organism) genome prompted further 194 investigation. The original Illumina short-read dataset SRX2623364 was downloaded from 195 NCBI SRA (Sequence Read Archive) and preprocessed by using a combination of tools 196 provided by the BBMap project (https://sourceforge.net/projects/bbmap/). This involved 197 removing poor-quality reads with bbduk.sh (qtrim = rl, trimq = 18), identifying phiX and p- 198 Fosil2 control reads (k = 21) and removing Illumina sequencing adapters (k = 21). Further, 199 de novo assembly of preprocessed paired-end reads was done by Megahit v1.2.9 (D. Li et 200 al. 2016) with k-mer list: 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 127, and with default 201 parameters. A total of 886 contigs with an average length of 4.98 kbp were produced. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

202 Protein-coding genes were predicted by Prodigal (Hyatt et al. 2010) and taxonomically 203 classified by scanning with MMSeqs2 against the GTDB database. A Spirochaeta contig 204 (length = 304,032 bp) was identified and scanned for the presence of HeR against the 205 PFAM database. Both taxonomy (Spirochaeta) and HeR presence were consistent with 206 previously published results. 207 Phylogenetic tree of HeRs. An extensive collection of predicted HeR amino acid 208 sequences (n = 4,108) was generated from: 1) all HeR sequences available in UniProtKB (n 209 = 502), 2) HeR identified within dereplicated contigs (using cd-hit-est -c 0.95 -aS 0.95) 210 assembled from publicly available metagenomes and metatranscriptomes (n = 3,145), 3) 211 HeR identified within dereplicated high-quality MAGs included in the GTDB database (n = 212 455) and 4) HeR assembled from the strand-specific metatranscriptomic dataset 213 generated in this study (n = 6). This collection was simplified by keeping only 214 representative sequences (n = 1,669) chosen following clustering with MMSeqs2 215 (Steinegger and Söding 2017) at 90% sequence identity and 90% coverage (mode: easy- 216 cluster; -c 0.90; --min-seq-id 0.90). Representative HeR sequences were aligned together 217 with 30 selected proteorhodopsins serving as outgroup by using PASTA (Mirarab et al. 218 2015) (resulting alignment with 1,699 sequences, 2,486 columns, 2,264 distinct patterns, 219 1,091 parsimony-informative sites, 698 singleton sites, 697 constant sites). A Maximum 220 Likelihood (ML) phylogenetic tree was constructed with IQ-TREE2 (Minh et al. 2020) (1,000 221 iterations for ultrafast bootstrapping (Hoang et al. 2018) and SH testing, respectively; best 222 model chosen by ModelFinder (Kalyaanamoorthy et al. 2017): LG+G4; additional 223 parameters recommended for short sequence alignments -nstop 500 -pers 0.2). The 224 generated tree was annotated to include labels containing: HeR-encoding contig name, 225 habitat of origin and consensus GTDB taxonomic classification (if available). Data including 226 alignment and the annotated phylogenetic tree are deposited in FigShare 227 (https://figshare.com/s/7bb42426f2ad5e891fec). 228 Phylogenetic tree of HeRs with gene context. A simplified depiction of HeR genomic 229 context across representative taxonomic groups harbouring such genes was constructed 230 by merging HeR phylogenetic information with available HeR gene neighbourhood data 231 (Supplementary Figure 4). For this purpose, we established a collection of representative, 232 dereplicated HeR-encoding contigs (n = 872) of at least 5 kb and with clear consensus 233 taxonomy from two sources: 1) HeR-encoding contigs assembled from publicly available 234 metagenomes and metatranscriptomes (n = 3,145) and 2) HeR contigs assembled from 235 the strand-specific metatranscriptomic dataset generated in this study (n = 6). 236 Dereplication of contigs was previously achieved using cd-hit-est (W. Li and Godzik 2006) 237 with identity cutoffs of 95% and coverage of 95% (-c 0.95 -aS 0.95). Phylogenetic tree 238 building: Curated HeR sequences recovered from selected contigs were aligned together 239 with 30 proteorhodopsins serving as outgroup by using PASTA (Mirarab et al. 2015) 240 (resulting alignment with 902 sequences with 957 columns, 895 distinct patterns, 550 241 parsimony-informative sites, 198 singleton sites, 209 constant sites). A Maximum 242 Likelihood (ML) phylogenetic tree was constructed with IQ-TREE2 (Minh et al. 2020) (1,000 243 iterations for ultrafast bootstrapping (Hoang et al. 2018) and SH testing, respectively; best 244 model chosen by ModelFinder (Kalyaanamoorthy et al. 2017): LG+I+G4; additional 245 parameters recommended for short sequence alignments -nstop 500 -pers 0.2). bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

246 Reconstruction of HeR gene neighborhoods: Coding sequences were predicted de 247 novo using Prodigal (Hyatt et al. 2010) in metagenomic (-p meta) mode. Protein domains 248 were annotated by scanning predicted coding sequences against the PFAM (Protein 249 Families) database using the publicly available perl script pfam_scan.pl. Predicted protein 250 sequences from all contigs were clustered together using the MMSeqs2 easy-cluster 251 workflow with 50% identity and 80% coverage cutoffs (--min-seq-id 0.5 -c 0.8 -e 1e-3 -- 252 cluster-reassign) and a minimum of 2 sequences per cluster. Clusters were sorted 253 according to their size (i.e. number of sequences) and colour codes were assigned to the 254 top largest 63. All clusters containing HeRs were assigned matching colours. HeR gene 255 neighbourhood data was combined with the reconstructed HeR phylogenetic tree in iTOL 256 (Letunic and Bork 2016) (https://itol.embl.de/). To facilitate visual interpretation, a number 257 of adjustments and rules were applied: 1) all contigs were oriented according to the sense 258 (+) of encoded HeRs, 2) contigs were centered on HeR genes with a maximum of 10 259 neighbouring genes depicted up- and downstream, 3) information regarding gene 260 lengths was not included, all of them being shown as equally sized rectangles, 4) 261 homologous genes (i.e. members of the same MMSeqs2 defined cluster) share matching 262 colours within each phylogenetically defined cluster, 5) all HeRs are coloured the same 263 across all phylogenetic clusters, 6) grey rectangles indicate genes with few homologues 264 and/or singletons, 7) taxonomy, habitat information and phylogenetic clusters are colour 265 coded on independent strips.

266 MORN-HeR phylogenomic tree. A maximum-likelihood (ML) phylogenomic tree was 267 constructed for Firmicutes MAGs encoding MORN-HeR protein domain fusions along with 268 other representatives of this phylum that assumably lack such genes. The established 269 collection of (n = 68) MAGs and reference genomes (Supplementary Table S15) was 270 scanned by hmmsearch against a previously published list of (n = 120) conserved protein 271 marker HMMs (Parks et al. 2018). Four divergent markers (TIGR00442, TIGR00539, 272 TIGR00643, TIGR00717) were identified by scanning with CD-search (e-value < 1e-2) and 273 removed. Curated amino acid sequences for the selected 116 phylogenetic markers were 274 aligned with PRANK (Löytynoja 2014) and resulting alignments were trimmed by BMGE 275 (parameters: -t AA -g 0.5 -b 3 -m BLOSUM30) (Criscuolo and Gribaldo 2010). Individually 276 trimmed alignments were concatenated resulting in a block of 68 sequences with 40,714 277 columns, 36,088 distinct patterns, 28,978 parsimony informative sites, 3,366 singleton 278 sites and 8,370 constant sites. The concatenated alignment was used with IQ-TREE 279 (v.1.6.12) to construct the ML phylogenomic tree (parameters: 1,000 iterations of ultrafast 280 bootstrapping (Hoang et al. 2018) and SH testing (Minh et al. 2020), respectively; best 281 model (LG+F+R5) chosen by ModelFinder (Kalyaanamoorthy et al. 2017). 282 Multiple sequence alignment (MSA) of MORN-HeR proteins. Predicted amino acid 283 sequences containing full-length MORN-MORN-MORN-HeR protein domain fusions (n = 284 36) were retrieved from Firmicutes MAGs (n = 35) previously used to construct the 285 phylogenomic tree presented in Supplementary Figure 1. All sequences were aligned 286 together using the PSI-Coffee alignment method (Chang et al. 2012) provided by the T- 287 Coffee online server (http://tcoffee.crg.cat) with default parameters. The resulting MSA is 288 presented with annotations in Supplementary Figure 2 while the original alignment file 289 generated by PSI-Coffee is available in FigShare 290 (https://figshare.com/s/7bb42426f2ad5e891fec). bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

291 Supplementary References

292 Alexander, K., and M. Volini. 1987. “Properties of an Escherichia Coli Rhodanese.” The 293 Journal of Biological Chemistry 262 (14): 6595–6604. 294 Chang, Jia-Ming, Paolo Di Tommaso, Jean-François Taly, and Cedric Notredame. 2012. 295 “Accurate Multiple Sequence Alignment of Transmembrane Proteins with PSI- 296 Coffee.” BMC Bioinformatics 13 Suppl 4 (March): S1. 297 Criscuolo, Alexis, and Simonetta Gribaldo. 2010. “BMGE (Block Mapping and Gathering with 298 Entropy): A New Software for Selection of Phylogenetic Informative Regions from 299 Multiple Sequence Alignments.” BMC Evolutionary Biology 10 (July): 210. 300 Dong, Jiu-Hong, Jian-Fan Wen, and Hai-Feng Tian. 2007. “Homologs of Eukaryotic Ras 301 Superfamily Proteins in Prokaryotes and Their Novel Phylogenetic Correlation with 302 Their Eukaryotic Analogs.” Gene 396 (1): 116–24. 303 Hartman, Hyman, and Alexei Fedorov. 2002. “The Origin of the Eukaryotic Cell: A Genomic 304 Investigation.” Proceedings of the National Academy of Sciences of the United States 305 of America 99 (3): 1420–25. 306 Hoang, Diep Thi, Olga Chernomor, Arndt von Haeseler, Bui Quang Minh, and Le Sy Vinh. 307 2018. “UFBoot2: Improving the Ultrafast Bootstrap Approximation.” Molecular 308 Biology and Evolution 35 (2): 518–22. 309 Hyatt, Doug, Gwo-Liang Chen, Philip F. Locascio, Miriam L. Land, Frank W. Larimer, and Loren 310 J. Hauser. 2010. “Prodigal: Prokaryotic Gene Recognition and Translation Initiation 311 Site Identification.” BMC Bioinformatics 11 (March): 119. 312 Kalyaanamoorthy, Subha, Bui Quang Minh, Thomas K. F. Wong, Arndt von Haeseler, and Lars 313 S. Jermiin. 2017. “ModelFinder: Fast Model Selection for Accurate Phylogenetic 314 Estimates.” Nature Methods 14 (6): 587–89. 315 Kang, Choong-Min, Derek W. Abbott, Sang Tae Park, Christopher C. Dascher, Lewis C. 316 Cantley, and Robert N. Husson. 2005. “The Mycobacterium Tuberculosis 317 Serine/Threonine Kinases PknA and PknB: Substrate Identification and Regulation of 318 Cell Shape.” Genes & Development 19 (14): 1692–1704. 319 Kovalev, K., D. Volkov, R. Astashkin, A. Alekseev, I. Gushchin, J. M. Haro-Moreno, I. Chizhov, 320 et al. 2020. “High-Resolution Structural Insights into the Heliorhodopsin Family.” 321 Proceedings of the National Academy of Sciences of the United States of America 117 322 (8): 4131–41. 323 Letunic, Ivica, and Peer Bork. 2016. “Interactive Tree of Life (ITOL) v3: An Online Tool for the 324 Display and Annotation of Phylogenetic and Other Trees.” Nucleic Acids Research 44 325 (W1): W242-5. 326 Li, Dinghua, Ruibang Luo, Chi-Man Liu, Chi-Ming Leung, Hing-Fung Ting, Kunihiko Sadakane, 327 Hiroshi Yamashita, and Tak-Wah Lam. 2016. “MEGAHIT v1.0: A Fast and Scalable 328 Metagenome Assembler Driven by Advanced Methodologies and Community 329 Practices.” Methods 102 (June): 3–11. 330 Li, Weizhong, and Adam Godzik. 2006. “Cd-Hit: A Fast Program for Clustering and Comparing 331 Large Sets of Protein or Nucleotide Sequences.” Bioinformatics 22 (13): 1658–59. 332 Lombana, T. Noelle, Nathaniel Echols, Matthew C. Good, Nathan D. Thomsen, Ho-Leung Ng, 333 Andrew E. Greenstein, Arnold M. Falick, David S. King, and Tom Alber. 2010. 334 “Allosteric Activation Mechanism of the Mycobacterium Tuberculosis Receptor 335 Ser/Thr Protein Kinase, PknB.” Structure 18 (12): 1667–77. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

336 Long, Mary C., Vincent Escuyer, and William B. Parker. 2003. “Identification and 337 Characterization of a Unique Adenosine Kinase from Mycobacterium Tuberculosis.” 338 Journal of Bacteriology 185 (22): 6548–55. 339 Löytynoja, Ari. 2014. “Phylogeny-Aware Alignment with PRANK.” Methods in Molecular 340 Biology 1079: 155–70. 341 Maresca, Julia A., Jessica L. Keffer, Priscilla P. Hempel, Shawn W. Polson, Olga Shevchenko, 342 Jaysheel Bhavsar, Deborah Powell, Kelsey J. Miller, Archana Singh, and Martin W. 343 Hahn. 2019. “Light Modulates the Physiology of Nonphototrophic Actinobacteria.” 344 Journal of Bacteriology 201 (10). https://doi.org/10.1128/JB.00740-18. 345 Minh, Bui Quang, Heiko A. Schmidt, Olga Chernomor, Dominik Schrempf, Michael D. 346 Woodhams, Arndt von Haeseler, and Robert Lanfear. 2020. “IQ-TREE 2: New Models 347 and Efficient Methods for Phylogenetic Inference in the Genomic Era.” Molecular 348 Biology and Evolution 37 (5): 1530–34. 349 Mirarab, Siavash, Nam Nguyen, Sheng Guo, Li-San Wang, Junhyong Kim, and Tandy Warnow. 350 2015. “PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino- 351 Acid Sequences.” Journal of Computational Biology: A Journal of Computational 352 Molecular Cell Biology 22 (5): 377–86. 353 Moparthi, Vamsi K., and Cecilia Hägerhäll. 2011. “The Evolution of Respiratory Chain 354 Complex I from a Smaller Last Common Ancestor Consisting of 11 Protein Subunits.” 355 Journal of Molecular Evolution 72 (5–6): 484–97. 356 Pagani, S., and Y. M. Galante. 1983. “Interaction of Rhodanese with Mitochondrial NADH 357 Dehydrogenase.” Biochimica et Biophysica Acta 742 (2): 278–84. 358 Parks, Donovan H., Maria Chuvochina, David W. Waite, Christian Rinke, Adam Skarshewski, 359 Pierre-Alain Chaumeil, and Philip Hugenholtz. 2018. “A Standardized Bacterial 360 Taxonomy Based on Genome Phylogeny Substantially Revises the Tree of Life.” 361 Nature Biotechnology 36 (10): 996–1004. 362 Sabehi, Gazalah, Alexander Loy, Kwang-Hwan Jung, Ranga Partha, John L. Spudich, Tal 363 Isaacson, Joseph Hirschberg, Michael Wagner, and Oded Béjà. 2005. “New Insights 364 into Metabolic Properties of Marine Bacteria Encoding Proteorhodopsins.” PLoS 365 Biology. https://doi.org/10.1371/journal.pbio.0030273. 366 Sakoh, Machiko, Koreaki Ito, and Yoshinori Akiyama. 2005. “Proteolytic Activity of HtpX, a 367 Membrane-Bound and Stress-Controlled Protease from Escherichia Coli.” The Journal 368 of Biological Chemistry 280 (39): 33305–10. 369 Shihoya, Wataru, Keiichi Inoue, Manish Singh, Masae Konno, Shoko Hososhima, Keitaro 370 Yamashita, Kento Ikeda, et al. 2019. “Crystal Structure of Heliorhodopsin.” Nature 371 574 (7776): 132–36. 372 Steinegger, Martin, and Johannes Söding. 2017. “MMseqs2 Enables Sensitive Protein 373 Sequence Searching for the Analysis of Massive Data Sets.” Nature Biotechnology 35 374 (11): 1026–28. 375 Timmers, Peer H. A., Charlotte D. Vavourakis, Robbert Kleerebezem, Jaap S. Sinninghe 376 Damsté, Gerard Muyzer, Alfons J. M. Stams, Dimity Y. Sorokin, and Caroline M. 377 Plugge. 2018. “Metabolism and Occurrence of Methanogenic and Sulfate-Reducing 378 Syntrophic Acetate Oxidizing Communities in Haloalkaline Environments.” Frontiers 379 in Microbiology 9 (December): 3039. 380 Zurita Rendón, Olga, Lissiene Silva Neiva, Florin Sasarman, and Eric A. Shoubridge. 2014. 381 “The Arginine Methyltransferase NDUFAF7 Is Essential for Complex I Assembly and 382 Early Vertebrate Embryogenesis.” Human Molecular Genetics 23 (19): 5159–70. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Tree scale: 0.1

HeliorhodopsinMORN~HeRMORN~ABC_tranPkinse~X~MORNGYF_2~MORNMORN~DUF4282 Completeness (%) Natranaerobius thermophilus 98.31 CSSed10_444 65.33 Syntrophothermus lipocalidus 98.81 Thermosyntropha lipolytica 99.36 Ca. Syntrophocurvum alkaliphilum 97.22 Syntrophomonas wolfei G311 97.35 monadales

Syntrophomonas palmitatica o. Syntropho- 96.92 CSSed10_148R1 89.83 T1Sed10_110R1 83.3 Ca. Syntrophonatronum acetioxidans 90.18 T1Sed10-67 67.34 Dethiobacter alkaliphilus AHT 1 95.76 CSSed11_131 74.72 CSSed11_298R1 g. Dethiobacter 86.43 CSSed165cm_560 73.28 CSSed11_181 73.94 T3Sed10_167 81.36 CSSed11_109 83.23 T1Sed10_166 83.84

CSSed10_9 f. UBA8154 84.93 CSSed11_70 72.32 B1Sed10_75 94.25 T1Sed10_174R1 92.77

T3Sed10_337 o. DTU022 57.88 CSSed162cmA_215 84.96 CSSed165cm_49 87.92 CSSed11_206R1 51.31 CSSed162cmB_282 87.5 Firmicutes bacterium UBA993 81.81 B1Sed10_173 86.42 B1Sed10_21 81.57 T1Sed10_56 77.47 T1Sed10_137m 53.93 T1Sed10_93 71.88 B1Sed10_74m 74.24 T1Sed10_71R1 72.43 CSSed10_277R1 73.42 T3Sed10_148R1 87.08 CSSed10_145R1 86.34 CSSed11_141 85.75 T1Sed10_145 60.59 T1Sed10_2 84.53 B1Sed10_235 62.61 B1Sed10_50 76.03 B1Sed10_123 83.31 B1Sed10_67 76.92

B1Sed10_154 g. UBA993 73.25 T1Sed10_71R2 76.21 CSSed165cm_530 82.06 CSSed162cmB_589 91.24 Tree: UFBootstraps CSSed11_224R1 83.87 ≥ 95 CSSed11_224R2 58.75 TE-sed-oct17-56 72.51 B1Sed10_8 71.34 90-94 CSSed165cm_390 69.55 CSSed165cm_595 83.02 < 90 CSSed10_167 65.05 CSSed11_158R1m 51.69 Heatmap: gene counts CSSed162cmA_337 93.86 CSSed162cmB_213 93.43 0 CSSed165cm_124 92.16 CSSed165cm_165 82.49 1 CSSed11_134 76.49 T3Sed10_127 72.88 2 T1Sed10_80R1 61.52 B1Sed10_39R1 88.12 3 CSSed10_39 85.59 CSSed11_39 88.98 Supplementary Figure 1. Phylogenomic tree of MORN-Heliorhodopsin (MORN-HeR) encoding MAGs. Green circles indicate high confidence UFBootstrap values (≥ 95). Occurences of genes encoding heliorhodopsins, MORN-heliorhodopsin as well as other MORN-domain fusions are depicted in the adjacent matrix. Genome completeness values are depicted as a histogram (estimated by CheckM). All genomes are members of the Firmicutes phylum, with different taxonomic subdivisions highlighted on the tree (taxonomy by GTDBtk). Natranaerobius thermophilus was used to root the tree. The majority of genomes included here have been previously used for phylogenomic analyses by Timmers et. al, 2018. Among reference genomes, Firmicutes bacterium UBA993 and Ca. Syntrophocurvum alkaliphilum are new additions. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

MORN MORN MORN

Heliorhodopsin

437

247 sites

Supplementary Figure 2. Multiple sequence alignment of MORN-HeR protein domain fusions predicted in Firmicutes MAGs. Each sequence shows 3 consecutive MORN domains (indicated by green rectan- gles). Aligned HeR domains display a high level of conservation and are truncated for illustration purposes (indicated by yellow rectangle). The original full-length alignment was deposited in FigShare. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A N-term C-term N-term C-term IN OUT T MORN MORN MORN Heliorhodopsin PK MORN MORN MORN M 1-4 x 2-12 x

IN OUT T MORN MORN MORN ABC_tran PK Big_2 MORN MORN M intracellular cell membrane 3/7 x

OUT IN T MORN MORN MORN Big_2 Big_2 PK FHA MORN MORN M 6 x

IN OUT T MORN MORN MORN Big_2 Big_2 PASTA FHA MORN MORN MORN M 8 x extracellular TM = Transmembrane helix

B Heliorhodopsin monomers ECL1 Heliorhodopsin dimer ABC-2 type efflux pump Heliorhodopsin loops (active form)

OUT C C C C C dimerization

IN N N N N AA N ATP-ase component of ABC transporters A ? A N-terminal MORN repeat Possible MORN-mediated on heliorhodopsins heliorhodopsin monomer ? interaction Potential MORN-mediated interactions regulating extracellular transport

PA PA = PASTA domain C Signaling molecule Physical stimulus B2 B2 B2 = Big_2 domain MORN-MORN Potential MORN-mediated mediated dimerization interactions involved in Protein kinase - MORN Activated B2 B2 of serine/threonine kinase? monomers protein kinase extracellular signal transduction

? ?? ?

? B2 OUT

IN Auto- FHA FHA phosphorylation C C P C C C N N N N Activation N P Phosphorylation of target protein

Supplementary Figure 3. Summary of MORN-repeat proteins predicted from metagenome-assembled genomes (MAGs) of anaerobic Firmicutes. A). Schematics of frequently co-occuring MORN-repeat containing proteins in Firmicutes MAGs including MORN-HeR, MORN-ABC transporters - likely involved in drug resistance or Cu2+/Na2+ ion efflux, periplasmic proteins containing bacterial immunoglobulin-like folds (Big_2, PASTA), MORN-protein kinase fusions where MORN repeats and p-kinase domains are commonly separated by transmembrane α-helices on opposite sides of the cellular membrane and MORN-forkhead associated (FHA) domains. B) Potential interac- tions between MORN-HeR monomers, MORN-ATPase components of ABC transporters and MORN-HeR and ABC transporters mediated or stabilized by the presence of MORN-repeat fusions. C) Potential interactions of MORN-Protein-kinases associated with functions such as extracellular signal transduction. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Tree scale 10 Taxonomy Actinobacteriota Euryarchaeota Thermoplasmatota

Asgardarchaeota Firmicutes Thermotogota Chloroflexota Halobacterota Unclassified

Crenarchaeota Patescibacteria

Znf-HeR

16 15 14 17 13 HeR 18

19 12

1 11

10

9

MORN- HeR

2 8

3 7

4 6 5

Habitat

Activated Sludge Marine

Fermentation Wastewater Cluster assignment Freshwater Other Sediment

Supplementary Figure 4. Genomic context of heliorhodopsin (HeR) genes across representa- tive taxonomic groups. The phylogenetic tree was constructed using 872 HeR amino acid sequences and 30 proteorhodopsins used as an outgroup for rooting. Gene neighbourhoods (10 genes up- and downstream) for each HeR were depicted schematically. Abundant homologues are coloured within each defined phylogenetic cluster while less abundant genes and/or single- tons are depicted in gray. All contigs were centered and oriented according to encoded HeR (dark red circle). Information regarding relative gene lengths was not included. Particular HeR-protein domain fusions are indicated separately: Znf-HeR and MORN-HeR. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 5. Histidine It is made kinases available and under related aCC-BY-NC-ND genes 4.0 (blue International labels) license in the. genomic neighborhood (within 10 kb) of Heliorhodopsins (red labels) originating from the phylum Chloroflexi.

GTP-cyclohydroIHeliorhodopsin GAF-2-PAS-HisKA-HATPase-cResponse-reg CH-Jul18-C51775

DUF2007DUF1461Pro-CA HYPO LigT-PEase-LigT-PEaseAmidohydro-2 SBP-bac-3-HisKA-HATPase-cHYPO HeliorhodopsinMu-like-ComHYPOHYPOHYPONifU-N Aminotran-5PALP MetW Abhydrolase-1Cys-Met-Meta-PP GCA-001005265-C1 Response-reg

HYPOMethyltransf-25HYPO ABC2-membraneABC-tran HYPOFer4-Nitroreductase FGGY-N-FGGY-C-2-Hacid-dh- HeliorhodopsinDUF43454HBT VWA MerR-1 MgtE-N-CBS-CBS-MgtEDUF2284 PAS-4-GAF-2-PAS-4-PAS-9-PAS-3- GCA-002328075-C7 2-Hacid-dh-C-SCP2-Aldol-ase-II HisKA-HATPase-c

MR-MLE-N-MR-MLE-CCOG1541-H-PhenylacetateGlyco-hydro-42-Glyco-hydro-42MNitroreductaseHYPO AP-endonuc-2GFO-IDH-MocADUF368 HeliorhodopsinMethyltransf-25HYPO CHASE-PAS-4-HisKA-HATPase-c-DUF4145 Glyco-hydro-2-N-Glyco-hydro-2-Response-regHPPK Pterin-bind GCA-002329325-C45 coenzyme-A-ligase-PaaK Response-reg Glyco-hydro-2-C

Exo-endo-phosHAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPO ABC-tranCbiQ CbiM-PDGLEFUR DNA-photolyase-FAD-binding-7HYPO HeliorhodopsinHYPO GCA-002399085-C26

Ribosomal-S21FtsK-alpha-FtsK-SpoIIIE-FAD-binding-4-FAD-oxidase-CGidB HYPO HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-Cadh-short-C2Y1-Tnp Heliorhodopsin PMT-2 Peptidase-M23 DUF4032-UspHYPO GCA-002423325-C75 FtsK-gamma

HYPO EamA-EamA HeliorhodopsinHYPO Acetyltransf-3RadC DZR MF-00999-2-7-1-184-Sulfofructose-kinaseAAA-31 ParBc-HTH-ParBAmidohydro-3HisKA-HATPase-cGAF-3-HisKA-HATPase-c GCA-002702705-C10

HYPO IMS-IMS-HHH-IMS-CMetallophos ABC2-membrane-2ABC-tranRibosomal-L9-CDUF1232 PAS-HisKA-HATPase-c-HeliorhodopsinLexA-DNA-bind-Peptidase-S24DnaB-DnaB-CDodecin DUF3048-DUF3048-C BTAD HD GCA-003152995-C31 Response-reg

TIGR01830HeliorhodopsinPAS-3-HisKA-HATPase-c-PhdYeFM-antitoxPIN Acetyltransf-1HYPO MBG-2 TIGR03466-HpnA-hopanoid-HYPO GCA-003171035-C85 Response-reg associated-sugar-epimera-se

Aminotran-3 CN-hydrolaseHeliorhodopsinDHO-dh GAF-2-HisKA-HATPase-c- RH-9nov16-C31966 Response-reg

Exo_Endo_PhosHAMP~HisKA~HATPase_cResponse-reg-Trans-reg-CHYPO ABC-tranCbiQ CbiM-PDGLEFURHYPO FAD-binding-7HYPOHYPOHeliorhodopsin SRR1562003-C2229

HAMP-HisKA-HATPase-cCBS NAD-binding-10-DUF2867MFS-1 HeliorhodopsinStaygreen SRR4030057-C3145

PALP HYPO HYPO HYPO HYPO Indigoidine-A DHH-DHHA1-RecJ-OBAcyltransferase Heliorhodopsin PAS-4-PAS-4-PAS-9-HisKA-HATPase-cResponse-reg-Trans-reg-CCOG0745-TK-DNA-binding-response-regulator-OmpR-HYPO SRR4236661-C224 f-amily+wHTH

HYPOHYPO Robl-LC7Response-regResponse-reg-GAF-3Response-reg-GAF-2-PAS-RsgA-N-RsgA-GTPaseHYPO TPR-2-TPR-8 Methyltransf-11HeliorhodopsinCOG3881Response-reg-GerEResponse-reg-PAS-GAF-HisKACsbD Peptidase-M10-PPCCOG4907-S-Uncharacterized-membrane-proteinChaBPRCDUF1328 SRR5007706-C940 HisKA-HATPase-c

TIR-2-WD40(6x)COG1313HYPOHYPO Robl-LC7Response-regResponse-reg-GAF-2Response-reg-GAF-2-PAS-RsgA-N-RsgA-GTPaseHeliorhodopsin TIGR01451 Sigma70-r2-Sigma70-r4-2HYPO SRR5506727-C398 HisKA-HATPase-c

TIR-2-WD40(6x)COG1313HYPOHYPO Robl-LC7Response-regHYPO Response-reg-GAF-2Response-reg-GAF-2-PAS-RsgA-N-RsgA-GTPaseHeliorhodopsin TIGR01451 Sigma70-r2-Sigma70-r4-2HYPO COG1520 SRR6048342-C735 HisKA-HATPase-c

HYPOResponse-regResponse-regPAS-8-HisKA-HATPase-cDUF4397(4x) Heliorhodopsin SRR7067999-C4278 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 6. Histidine It is made kinases available and under related aCC-BY-NC-ND genes 4.0 (blue International labels) license in the. genomic neighborhood (within 10 kb) of Heliorhodopsins (red labels) originating from the phylum Firmicutes.

HSP70 FGE-sulfataseHYPO PAS-4-GAF-PAS-4-PAS-8-Response-reg-HDDUF483 MMR-HSR1-TGSHYPOHeliorhodopsinTIGR04076(3x)Aldo-ket-red Beta-lactamaseAminotran-5Zn-dep-PLPCHYPO HYPOSNARE-assocAcetyltransf-1ATC-hydrolase HisKA-HATPase-c GCA-002345475-C4

Pentapeptide-Pentapeptide-4HYPO HYPO MacB-PCD-FtsXABC-tran Biotin-lipoyl-2-HlyD-3HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CTIGR03599Fer4-7 HeliorhodopsinHYPO HYPOHTH-27 HYPO Abhydrolase-5NYN-OST-HTHDEAD-Helicase-CTetR-NFlavodoxin-5zf-trcl GCA-002841395-C12

HeliorhodopsinEpimerase HisKA-HATPase-cResponse-reg-Trans-reg-CDUF4342HYPO GCA-003520745-C39

HYPODisA-N Aminotran-1-2 UvrB-inter-CarD-CdnL-TRCF-Pept-tRNA-hydroPribosyltran-N-Pribosyl-synthMatE-MatE Heliorhodopsin DUF5011-SBP-bac-5SpoVGGHMP-kinases-NRrnaADDUF4093HD PTAC-PTACHIT HisKA-HATPase-cResponse-reg-Trans-reg-C DEAD-Helicase-C-TRCF GCF-002838185-C1

DisA-N Aminotran-1-2DDE-Tnp-IS1595HYPO UvrB-inter-CarD-CdnL-TRCF-Pept-tRNA-hydroPribosyltran-N-Pribosyl-synthMatE-MatE HeliorhodopsinSpoVGGHMP-kinases-NRrnaADDUF4093HD DEDD-Tnp-IS110-Transposase-20PTAC-PTACHIT HisKA-HATPase-cResponse-reg-Trans-reg-C DEAD-Helicase-C-TRCF GCF-002838205-C1

COG5523 HI0933-like FtsX ABC-tranTetR-N AcyltransferaseMFS-2 PgpA NitroreductaseHeliorhodopsinTocopherol-cyclHisKA-HATPase-cResponse-reg-Trans-reg-CPhoU-PhoUABC-tran BPD-transp-1BPD-transp-1TIGR02136-ptsS-2-phosphate-binding-proteinCOG1814-P-Predicted-Fe2-Mn2-transporterP-II SRR5690703-C37

HYPODisA-N Aminotran-1-2 UvrB-inter-CarD-CdnL-TRCF-Pept-tRNA-hydroPribosyltran-N-Pribosyl-synthMatE-MatE Heliorhodopsin DUF5011-SBP-bac-5SpoVGGHMP-kinases-NRrnaADDUF4093HD PTAC-PTACHIT HisKA-HATPase-cResponse-reg-Trans-reg-C DEAD-Helicase-C-TRCF SRR6201693-C16 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 7. Histidine It is made kinases available andunder related aCC-BY-NC-ND genes 4.0 (blue International labels) license in the. genomic neighborhood (within 10 kb) of Heliorhodopsins (red labels) originating from the phylum Patescibacteria.

HisKA-7TM-HisKA-HATPase-cTIGR03725-bact-YeaZ-universal-bacterial-protein-YeaZBacA Spermine-synthSeryl-tRNA-N-tRNA-synt-2bDUF2007tRNA-synt-1fHD-3 HD-3-HD-3GreA-GreB-N-GreA-GreBHeliorhodopsinHYPO HYPO HYPO PrenyltransfAAA Inositol-P PGM-PMM-I-PGM-PMM-II-PGM-HYPODUF4430 PMM-III-PGM-PMM-IV GCA-001816655-C3

Sortase PEGA EPSP-synthaseMreB-Mbl tRNA-anti-codon-HDHYPO His-Phos-1HYPONYN tRNA-synt-1cHeliorhodopsinGreA-GreBResponse-regCAP Pyr-redox-2HYPOHYPO Peptidase-S11 PAS-HisKA-HATPase-cResponse-regtRNA-anti-codon-tRNA-synt-2 GCA-002338125-C1

NusG-KOWSecE Glycos-transf-2PAS-9-HisKA-HATPase-cDUF45AAA-33HYPO HYPOIron-permeaseHYPOHeliorhodopsinHYPO NUDIXPQ-loop Pyr-redox-2 Glyco-transf-4-Glycos-transf-1Glyco-hydro-57 Glyco-hydro-15HYPORibosomal-L33HYPO GCA-002422915-C2

HYPOHYPO HYPO HeliorhodopsinHYPOHYPO HYPO HYPO HYPO HYPO HisKA-HATPase-cHAMP-HATPase-cHYPO GCA-002771355-C7

HYPO HeliorhodopsinMF-00163-Peptide-deformylaseHisKA-7TM-HisKA-HATPase-cHYPOABC-tran FtsX LytR-cpsA-psrTranscrip-regRuvCHYPORuvA-N-HHH-5-RuvA-C GCA-003164595-C1

HYPO HYPO NAD-binding-10Ser-hydrolaseDUF1905His-Phos-1Acetyltransf-1HYPOHYPOHYPO HeliorhodopsinMF-03036-DNPH1Pterin-bindHYPOTHF-DHG-CYH-THF-DHG-CYH-CResponse-regHYPO HisKA-7TM-HisKA-HATPase-cHYPO DUF11 SRR5821655-C26

HYPOHYPONuc-deoxyrib-trDUF2568HeliorhodopsinHYPO Aldo-ket-redHYPOTHF-DHG-CYH-THF-DHG-CYH-CResponse-regMethyltransf-31 HisKA-7TM-HisKA-HATPase-cHYPO DUF11-Gram-pos-anchor SRR5821668-C515

HYPO DUF2142 LPG-synthase-TMMF-00146-dCTPCupin-2HYPO deaminaseHeliorhodopsinHYPO HisKA-HATPase-cHYPO SRR6007444-C3654 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this Supplementarypreprint (which was Figure not certified 8. Histidine by peer review) kinases is the author/funder,and related who genes has granted (blue bioRxiv labels) a license in the to displaygenomic the preprint neighborhood in (within 10 kb) of Heliorhodopsinsperpetuity. It is (red made labels) available originating under aCC-BY-NC-ND from the 4.0 phylum International Actinobacteriota, license. class Acidimicro- biia.

AstE-AspAMS-channel Amidohydro-3 HisKA-HATPase-cAminotran-1-2SCO1-SenCPCuAC Abhydrolase-6SpoIIAA-likeHeliorhodopsinHYPOSigma70-r4-2HYPO Cupin-2RibD-CHYPOCOG4803-S-Uncharacterized-DUF998TetR-N ParE-toxin membrane-protein GCA-002418265-C2

COG0668HeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPOHYPO ApbE Ferric-reductSNARE-assocFer2-3-Fer4-7Succ-DH-flav-C RE-14apr17-C14497

COG2318PIG-LHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-Response-reg-Trans-reg-CHYPO HATPase-c RE-15aug16-C29899

HYPOThioredoxinHaem-bdDUF664PIG-LHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-c RE-16jun15-C17802

Glycos-trans-3N-Glycos-HYPOThioredoxinHaem-bdDUF664PIG-LHYPOMS-channelHeliorhodopsinHAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPOHYPO ApbE Ferric-reductSNARE-assocFer4-7 transf-3-PYNP-C RE-18aug17-C8302

PIG-LHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-C RE-20APR16-C40370

HeliorhodopsinHAMP-HisKA-HATPase-cResponse-reg-Trans-reg-C RE-20APR16-C93831

COG0668M-Small-conductance-mechanosensitive-cha-nnelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPOHYPO ApbE HYPO RE-23may17-C92038

ThioredoxinHaem-bdDUF664PIG-LHYPOMS-channelHeliorhodopsinHisKA-HATPase-c RE-4nov15-C44399

COG0668-M-Small-conductance-mechanosensitive-cha-nnelHeliorhodopsinHAMP-HisKA-HATPase-cTrans-reg-C RE-9nov16-C108044

HYPOHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPOHYPO ApbE Ferric-reductSNARE-assoc RE-9nov16-C29599

Haem-bdDUF664 MFS-1 PIG-LHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPO HYPO ApbE Ferric-reductSNARE-assocFer4-7 RH-26jul17-C14910

DUF664 TIGR00900-Antiporter-proteinHYPOPIG-LHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-cTrans-reg-C RH-26jul17-C33965

COG0668-M-Small-conductance-mechanosensitive-channelHeliorhodopsinHAMP-HisKA-HATPase-cResponse-reg-Trans-reg-C RH-9nov16-C86972

COG0668-M-Small-conductance-mechanosensitive-cha-nnelHeliorhodopsinHisKA-HATPase-c SRR5468271-C28914

HYPOHYPOThioredoxinRhodaneseHaem-bdDUF664 MFS-1 PIG-LHYPOMS-channelHeliorhodopsinHAMP-HisKA-HATPase-c SRR5468365-C2567

TerC HYPOHYPOThioredoxinRhodaneseHaem-bdDUF664PIG-LHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-c SRR5468380-C1964

HYPO Glycos-trans-3N-Glycos-HYPOThioredoxinHaem-bdDUF664 MFS-1 PIG-LHYPOMS-channelHeliorhodopsinsCache-3-2-HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPOHYPO transf-3-PYNP-C SRR5468412-C1391 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 9. Histidine It is made kinases available underand arelatedCC-BY-NC-ND genes 4.0 (blueInternational labels) license in. the genomic neighborhood (within 10 kb) of Heliorhodopsins (red labels) originating from the phylum Actinobacteriota, class Actinobacte- ria.

HYPOHYPO HeliorhodopsinGTP-EFTU-GTP-EFTU-D2-HYPO TIGR00218PAS-3-HisKA-HATPase-cHYPO GCA-002693615-C113 EFG-C

Glyco-transf-4- Peptidase-M4- COG3386-G-Sugar-GTP-EFTU-GTP- His-Phos-1PhoU-PhoUHisKA-HATPase-cResponse-reg-Trans-reg-CEamA-EamAYbjN HYPOadh-short Peptidase-M4-CHeliorhodopsin MFS-1 PAP2 TIGR02983HYPO Bac-luciferaseRhomboid GCA-002698575-C11 Glycos-transf-1 lactone-lactonase-YvrEEFTU-D2-EFG-C

PAS-4-PAS-4-HisKA-CN-hydrolaseDoxXPfkB DUF998HeliorhodopsinBPD-transp-2BPD-transp-2ABC-tran-ABC-tranPeripla-BP-4ABC-tran-ABC-tranLacI-Peripla-BP-3 HATPase-c GCA-002729155-C71

Glyco-transf-4- MF-01175-tRNA- PhoU-PhoUHis-Phos-1YbjN adh-shortHYPODUF2516Asparaginase-IIDUF1794DrsEHeliorhodopsinHYPO Amidase modifying-protein-YgfZFURDUF1416Rhodanese-RhodaneseDUF4395ThioredoxinCOG1977-H-Molybdopterin-converting-factor-small-subunitTrans-reg-C GCA-002737595-C1 Glycos-transf-1

PAS-4-HisKA-HATPase-c- HYPO DUF4360HYPO NAD-binding-10CPBP FasciclinDUF378MerR-1-B12-binding-HeliorhodopsinCOG3328HYPOSigma70-r2Asp23HYPODUF2273Asp23HYPOHYPO SpoIIE Response-reg GCF-000721705-C42 2-B12-binding

HYPO Peptidase-S66-Peptidase-ResolvaseHYPOHisKA-HATPase-cCOG1961Endonuclease-NSHYPOHYPO TerC HeliorhodopsinCOG3328Transposase-mutCOG3464-X-TransposaseHTH-Tnp-ISL3adh-short DPRP HYPO DUF4232 COG3408-G-Glycogen-debranching-enzyme-alpha-1-6-glucosidaseHYPO GCF-002929535-C20 S66C

AP-endonuc-2SIMPLABC-tranFtsX-ECDPeptidase-M23PDZ-6-Peptidase-S41SmpB GAF-2-GGDEF-EALHNH-3HeliorhodopsinResIII-Helicase-CYdjMHYPO 4HBT-3 HisKA-HATPase-cAIRS-AIRS-CProk-JABThiS PALP HYPO GCF-003073135-C6

HisKA-3Response-reg-GerEHYPOPG-binding-1-Peptidase-HYPOPutative-PNPOxPA-Peptidase-M28NAD-binding-10Beta-lactamasePentapeptide(2x)-4-HeliorhodopsinHYPOF420H2-quin-redHYPOCOG0316Sulfotransfer-1PepSY HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPOCupin-2 GCF-900091585-C1 M15-3 Pentapeptide

Response-reg-GerECpn60-TCP1SBP-bac-8DUF3263CPBP Spermine-synthHeliorhodopsinPhnA-Zn-Ribbon-PhnAMFS-1 Na-H-ExchangerDUF347(4x)Response-reg-Trans-reg-CHisKA-HATPase-cCyt-b5Cyt-b5 Jr-28aug17-C3048

SBP-bac-8DUF3263CPBP Spermine-synthHeliorhodopsinPhnA-Zn-Ribbon-PhnAMFS-1 Na-H-ExchangerDUF347(4x)Response-reg-Trans-reg-CHisKA-HATPase-cCyt-b5Cyt-b5 Jr-7aug17-C3953

Ferric-reduct-FAD-binding-8-HYPO HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CAldedhLumazine-bd-2DUF4870COG5646-Speroxidase-peroxidaseHeliorhodopsinMF-00548-zupT-Zinc- RE-23may17-C20662 NAD-binding-1 transporter-ZupT

PhoU-PhoUHis-Phos-1YbjN Glyco-transf-4-Glycos-adh-shortCOG4106DUF2516Asparaginase-IIDUF1794DrsEHeliorhodopsinHYPO Amidase MF-00259-T-AminomethyltransferaseFURDUF1416Rhodanese-RhodaneseDUF4395ThioredoxinTrans-reg-CAcetyltransf-1 transf-1 RE-4nov15-C377

PhoU-PhoUHis-Phos-1YbjN Glyco-transf-4-Glycos-adh-shortHYPODUF2516Asparaginase-IIDUF1794DrsEHeliorhodopsinHYPO AmidaseGCV-T-C transf-1 RH-14apr17-C12203

HisKA-HATPase-cResponse-reg-Trans-reg-CEamA-EamAYbjN Glyco-transf-4-Glycos-transf-1HYPOadh-shortHYPO Asp-Glu-racePutative-PNPOxHeliorhodopsinadh-short GTP-EFTU-GTP-EFTU-D2-EFG-CHYPO HYPO PPDK-N RH-14apr17-C277

HTH-5HeliorhodopsinAldo-ket-redHAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CCOG3462COG3544 RH-18aug17-C51518

HeliorhodopsinAldo-ket-redHAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CCOG3462DUF305YHYH RH-26jul17-C41144

Glyco-transf-4-Glycos- DiS-P-DiS-Peptidase-A24EAL YbjN HYPOadh-shortAsp-Glu-racePutative-PNPOxHYPO HeliorhodopsinGTP-EFTU(2x)-D2-EFG-CSCFA-trans AMP-binding-AMP-binding-CHYPOTIGR00218-manA-mannose-6-phosphate-isomerase-cla-ss-IPAS-3-HisKA-HATPase-c transf-1 SRR3961935-C67

Glyco-transf-4-Glycos- AMP-binding-AMP- DiS-P-DiS-Peptidase-A24EAL YbjN HYPOadh-shortAsp-Glu-racePutative-PNPOxHYPO HeliorhodopsinGTP-EFTU(2x)-D2-EFG-CSCFA-trans binding-CHYPOTIGR00218-manA-mannose-6-phosphate-isomerase-cla-ss-IPAS-3-HisKA-HATPase-c SRR3962293-C33 transf-1

DiS-P-DiS-Peptidase-A24EAL YbjN Glyco-transf-4-Glycos-HYPOadh-shortAsp-Glu-racePutative-PNPOxHYPO HeliorhodopsinGTP-EFTU(2x)-D2-EFG-CSCFA-trans AMP-binding-AMP-HYPOTIGR00218-manA-mannose-6-phosphate-isomerase-cla-ss-IPAS-3-HisKA-HATPase-c SRR3962508-C37 transf-1 binding-C

Glyco-transf-4-Glycos- HYPOYbjN HYPOadh-shortAsp-Glu-racePutative-PNPOxHYPO HeliorhodopsinGTP-EFTU(2x)-D2-EFG-CHYPOTIGR00218PAS-3-HisKA-HATPase-cAMP-binding-C SRR3963456-C335 transf-1

Sbt-1HYPO DEAD-Helicase-C-HA2- Aldedh COG5646 MMPL-MMPLHeliorhodopsinCOG4244HYPOCOG1477FMN-bindFerric-reduct-NAD-binding-1HYPO HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPO ABC-membrane-ABC-tran SRR5468211-C1 OB-NTP-bind-DUF3418

Sbt-1HYPO DEAD-Helicase-C-HA2- Aldedh COG5646 MMPL-MMPLHeliorhodopsinCOG4244HYPOCOG1477FMN-bindFerric-reduct-NAD-binding-1HYPO HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CHYPO ABC-membrane SRR5468365-C469 OB-NTP-bind-DUF3418

His-Phos-1PhoU-PhoUHisKA-HATPase-cResponse-reg-Trans-reg-CEamA-EamAYbjN Glyco-transf-4-Glycos-HYPOadh-shortPeptidase-M4-Peptidase-HeliorhodopsinCOG3386 GTP-EFTU(2x)-D2-EFG-CMFS-1 PAP2 HYPO Bac-luciferaseRhomboidBac-luciferase SRR5821668-C164 transf-1 M4-C

Peptidase-M4-Peptidase- His-Phos-1PhoU-PhoUHisKA-HATPase-cResponse-reg-Trans-reg-CEamA-EamAYbjN Glyco-transf-4-Glycos-HYPOadh-short HeliorhodopsinCOG3386 GTP-EFTU(2x)-D2-EFG-CMFS-1 PAP2 HYPO Bac-luciferaseRhomboidBac-luciferase M4-C SRR5821670-C88 transf-1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 10. Histidine It is made kinases available underand relatedaCC-BY-NC-ND genes 4.0 (blue International labels) license in the. genomic neighborhood (within 10 kb) of Heliorhodopsins (red labels) originating from the phylum Actinobacteriota, class Coriobac- teriia.

adh-shortLDcluster4 MafB19-deamHYPOHYPO 5TM-5TMR-LYT-HisKA-HATPase-cCitrate-synt TIGR03820-lys-2-3-AblA-lysine-2-3-aminomutaseDUF1295 DegV HeliorhodopsinCOG0477-GEPR-MFS-family-permeasePeptidase-M20-M20-dimerLacI-Peripla-BP-3Peripla-BP-4ABC-tranBPD-transp-2PTE GATase1-likeIU-nuc-hydro GCA-001950395-C1

Spermine-synth sCache-3-2-HAMP-HisKA-Phosphonate-bdMethyltransf-25B12-binding-Radical-SAMUPF0126-UPF0126HYPOTspO-MBRHeliorhodopsinUbiA Dus PPDK-N E1-E2-ATPase-HydrolaseAcetyltransf-1HYPO NAD-kinaseBand-7 HATPase-c GCA-002423585-C4

MBOAT HYPO Amidinotransf B12-binding-Radical-SAMPAS-3-HisKA-HATPase-cPeripla-BP-6HeliorhodopsinNitroreductaseCupredoxin-1CN-hydrolase CW-binding-2(3x)LysEVF530Aminotran-4Aminotran-1-2YqeY GCA-002841295-C2

HYPO Amidinotransf HYPO B12-binding-Radical-SAMHAMP-PAS-3-HisKA-HATPase-cPeripla-BP-6HeliorhodopsinNitroreductasePspA-IM30 TPM-phosphatase Aminotran-1-2YqeYHYPO GCA-002841355-C16

HeliorhodopsinUbiA ABC2-membrane-4ABC2-membrane-4ABC-tran HAMP-HisKA-HATPase-cCOG0745 SRR5468109-C2146

HeliorhodopsinUbiA ABC2-membrane-4ABC2-membrane-4ABC-tran HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CDus HYPO SRR5468111-C1943

Peptidase-M78 HEAT-2 HD HYPOHYPO HAMP-HisKA-HATPase-cResponse-reg-Trans-reg-CDUF1287Polyketide-cyc2HeliorhodopsinSBF HYPO B12-binding-Radical-SAMHemerythrinTPP-enzyme-CPOR-N-PFOR-IIFer4 POR AMP-binding-AMP-binding-CHTH-31-Cupin-2 SRR5506721-C517

TIGR00933HYPO4HBT Methyltransf-23cNMP-bindingHYPO HYPOAcetyltransf-3DUF188FasciclinHeliorhodopsinCatalase-Catalase-relCOG4564 HYPO SRR5506727-C4132

Nitrate-Specific HisKA

TIGR00933HYPO4HBT cNMP-bindingHYPOHYPOAcetyltransf-3DUF188MetallophosFasciclinHeliorhodopsinCatalase-Catalase-relCOG3850-T-HYPO SRR5506739-C570

DEAD-Helicase-CHYPOHYPO Response-reg-Trans-reg-CHAMP-HisKA-HATPase-cHYPO Glt-symporterInhibitor-I42adh-short NAD-binding-10-DUF2867Heliorhodopsin SRR7299214-C485 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 11. Histidine It is made kinases available andunder relatedaCC-BY-NC-ND genes 4.0 (blue International labels) license in the. genomic neighborhood (within 10 kb) of Heliorhodopsins (red labels) originating from the phylum Actinobacteriota (classes RBG-13-55-18 and Thermoleophilia), phylum Dictyoglomota (class Dictyoglomia) and phylum Thermoplas- matota (class E2).

phylum Actinobacteriota, class RBG-13-55-18

UPF0126-UPF0126Peptidase-M20-M20-dimerAA-permease-2HYPO AA-permease-2 AAT HYPO Bile-Hydr-Trans-BAAT-CHeliorhodopsinHYPO Sigma70-r1-2-Sigma70-r2-Sigma70-r3-Sigma70-r4SpoVS DUF1624 AHSA1 PAS-PAS-4-GAF-2-PAS-4-PAS-3-PAS-HisKA-HATPase-c GCA-001768645-C27

Response-regResponse-reg-HisKA-HATPase-cPeptidase-M48 PG-binding-4-YkuDMethyltransf-25 CoA-binding-2-Succ-CoA-ligGGDEF DUF2892HeliorhodopsinOxidored-q6-NiFe-hyd-SSU-CHYPO FAD-binding-6-NAD-binding-1-Fer2Amino-oxidase ABC-tran ABC2-membrane-2zinc-ribbon-2 GCA-003157385-C5

phylum Actinobacteriota, class Thermoleophilia

Catalase-relHeliorhodopsinCOG1225-O-PeroxiredoxinAhpC-TSAHYPO sCache-like-HAMP-PAS-8-HisKA-HATPase-cResponse-reg-Trans-reg-CPBP-like-2 BPD-transp-1BPD-transp-1ABC-tran GCA-003157225-C134

G6PD-N-G6PD-C PGI HYPO DUF559HYPO TIGR04075Response-reg-Trans-reg-CHAMP-HisKA-HATPase-cHYPO HeliorhodopsinMethyltransf-3Cpn10 Fe-ADH DUF4337DUF4337 Aminotran-3 FAD-binding-4-MurB-CPMSR Abhydrolase-6Usp RH-23may17-C2104

phylum Dictyoglomota, class Dictyoglomia

COG1452-M-LPS-assembly-outer-membrane-protein- LacI-Peripla-BP-3 SBP-bac-5 BPD-transp-1BPD-transp-1ABC-tran-oligo-HPYABC-tran-oligo-HPYGlyco-hydro-4-Glyco-hydro-4CGlycos-transf-2HeliorhodopsinResponse-reg-Trans-reg-CHisKA-HATPase-cHYPO CN-hydrolaseCOG5375-S-Uncharacterized-proteinLptC LptF-LptG RubredoxinPyr-redox-2-Rubredoxin-C Lp-tD-organic-solvent-tolerance-protein-OstA GCF-000020965-C1

phylum Thermoplasmatota, class E2

flux

UbiA Rhodanese TIGR03462-CarR-dom-SF-lycopene-cyclase-domainAmino-oxidase SQS-PSY DUF523-DUF1722UvdE Putative-PNPOxDNA-photolyaseHYPO HeliorhodopsinDUF3335 Radical-SAM Cation-ef PAS-PAS-9-HisKA-HATPase-cHYPO NosD HYPO Propeptide-C25-Peptidase-C25 SRR5506739-C331

COG4249-R-Uncharacterized- Thy1 RibD-CMut7-C Peptidase-C1DUF424 Aminotran-3 Beta-propel Epimerase Radical-SAM HeliorhodopsinPMSRSelR SBP-bac-8 dCache-1-HAMP-HisKA-HATPase-cHYPO protein-contains-caspa-se-domainPeptidase-M28 Aminotran-3 PD40-PD40 SRR6201696-C64 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 12. Histidine It is made kinases available underand arelatedCC-BY-NC-ND genes 4.0 (blue International labels) license in the. genomic neighborhood (within 10 kb) of Heliorhodopsins (red labels) originating from the phylum Halobacterota, class Halobacte- ria.

-2 BPD-transp-1-BPD-transp-1ABC-tran-TOBE-2 PMT Gluconate-2-dh3GMC-oxred-N-GMC-oxred-CResponse-reg-HisKA-HATPase-cResponse-regPAS-3-HisKA-HATPase-cHeliorhodopsinGlycos-transf-2 HTH-IclR HYPO CarboxypepD-regHYPO HYPO ATPaseHYPOResponse-reg-HalXHYPO GCF-000023945-C1

HYPO TrkA-C TrkH HYPO TrkA-N-TrkA-CTrkH MMR-HSR1-MMR-HSR1-C-TGSHYPOCOG1753Asp-Glu-raceHeliorhodopsinPutative-PNPOx COG2730- PAS-3-HisKA-HATPase-cHisKA-7TM-HisKA-HATPase-cResponse-reg-PAS-9-PAS-8Glyco-tranf-2-3 GCF-000337515-C2

Response-reg-PAS-4-PAS-4-GAF-2-HYPO NifU-NAcetyltransf-10Methyltransf-25HYPO CDC48-N-CDC48-2-AAA-AAA-COG2108 HeliorhodopsinPGA-cap TrmB-Regulator-TrmBHYPOHYPOTRAMSigma70-r4-TFX-Cadh-short-C2 PKD-PKD HisKA-HATPase-c lid-3-AAA-AAA-lid-3 GCF-000755225-C2

GMC-oxred-N-GMC-oxred-CResponse-reg-HisKA-HATPase-cResponse-regPAS-3-HisKA-HATPase-cHeliorhodopsinGlycos-transf-2 HTH-IclR HYPO M20-dimer SRR5621770-C1065

BPD-transp-1-BPD-transp-1ABC-tran-TOBE-2 PMT-2 Gluconate-2-dh3GMC-oxred-N-GMC-oxred-CResponse-reg-HisKA-HATPase-cResponse-regPAS-3-HisKA-HATPase-cHeliorhodopsinGlycos-transf-2 HTH-IclR HYPO CarboxypepD-regHYPO HYPO ATPaseHYPO Response-reg-HalXHYPO SRR6201721-C144

BPD-transp-1-BPD-transp-1ABC-tran-TOBE-2 PMT-2 Gluconate-2-dh3GMC-oxred-N-GMC-oxred-CResponse-reg-HisKA-HATPase-cResponse-regPAS-3-HisKA-HATPase-cHeliorhodopsinGlycos-transf-2 HTH-IclR CarboxypepD-regHYPO HYPO ATPaseHYPO Response-reg-HalXHYPOHYPO SRR6201724-C293

HYPOHYPOSHOCT Response-reg-PAS-4-PAS-4-GAF-2-4HBT DUF309 MF-00767-Succinylglutamate-desucci-nylaseCupin-2UPF0179HYPO HeliorhodopsinDUF433DUF5615 HpcH-HpaI COG1913 Putative-PNPOxHSP20HYPOHYPO Ion-trans-2 COG0613 HisKA-HATPase-c SRR6201724-C322

Response-reg-PAS-4-PAS-4-GAF-2-NifU-NAcetyltransf-10Methyltransf-31HYPO CDC48-N-CDC48-2-AAA-AAA-COG2108 HeliorhodopsinPGA-cap TrmB-Regulator-TrmBHYPOHYPOTRAMSigma70-r4-TFX-Cadh-short-C2 PKD-PKD-PKD-PKD HisKA-HATPase-c lid-3-AAA-AAA-lid-3 SRR6201724-C408 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Supplementary Figureperpetuity. 13. Histidine It is made kinases available underand arelatedCC-BY-NC-ND genes 4.0 (blue International labels) license in the. genomic neighborhood (within 10 kb) of MORN-Heliorhodopsins (red labels) originating from Firmicutes (phylum) MAGs (sediment and brine metagenomes).

PPDK-N ELFV-dehydrog-N-ELFV-dehydrogResponse-reg-Sigma54-activat-HTH-8HisKA-HATPase-c MORN-MORN-MORN-HeliorhodopsinFGase HYPOSpoVGAcetyltransf-10 AMARA-sed17-18411

GntR-FCDEamA-EamAHYPO HYPO HYPO Cys-Met-Meta-PP TIGR03852 PRK MORN-MORN-MORN-HeliorhodopsinDUF3159 Peptidase-U32-Peptidase-U32-CHYPO B1-Sed10-C1700

GDPD Hydrolase-6-Hydrolase-likeHYPOC-GCAxxG-C-CHYPO Peptidase-M42MF-00905 HYPO DUF3159HYPO MORN-MORN-MORN-HeliorhodopsinCHAP-Dockerin-1ELFV-dehydrog-N B1-Sed10-C3549

PC4 AFOR-N-AFOR-C HYPO Flavodoxin-5-Fer4 adh-short-C2HYPOAhpC-TSA ATC-hydrolaseAbhydrolase-6 MORN-MORN-MORN-HeliorhodopsinDctM CS-Sed10-C10030

PIN 2-Hacid-dh-2-Hacid-dh-CAldolase-II-Aldolase-IIAminotran-3 FGGY-N-FGGY-CCOG0701COG0701 MORN-MORN-MORN-HeliorhodopsinTspO-MBRPLDc-NABC-tran ABC2-membrane-3HYPO LGT Lactamase-BTetR-NHYPO NTP-transf-9 CS-Sed10-C4444

MORN-MORN-MORN-HeliorhodopsinDUF3159 Peptidase-U32-Peptidase-U32-CGlyco-hydro-130 PDZ-6-Peptidase-S41-PG-binding-1Toprim-Topoisom-bac Thiolase-N-Thiolase-C CS-Sed10-C7423

TIGR03852-sucrose-gtfA-sucrose-phosphorylasePRK CHAP-Dockerin-1DUF3159 MORN-MORN-MORN-HeliorhodopsinCys-Met-Meta-PP Peptidase-U32-Peptidase-U32-CIsochorismataseHYPO Glyco-hydro-130 SDF CSSed16-2cmA-14274

Alpha-amylase PRK Peptidase-M20-M20-dimerPeptidase-M20-M20-dimerDcuC cNMP-binding-Lyase-1-FumaraseC-CMORN-MORN-MORN-HeliorhodopsinCHAP-Dockerin-1DUF3159 Peptidase-U32-Peptidase-U32-CGlyco-hydro-130HYPO PDZ-6-Peptidase-S41-PG-binding-1HTH-30 CSSed16-2cmA-6208

SBP-bac-3HYPOBPD-transp-1ABC-tran MORN-MORN-MORN-HeliorhodopsinGYF-2 MFS-1 MF-00548-zupT-Zinc-transporter-ZupTCys-Met-Meta-PPDUF3159 Glyco-hydro-130Peptidase-U32 CSSed16-2cmB-19757

DUF772 CPBP DUF3267GerE MORN-MORN-MORN-Heliorhodopsin CSSed16-5cm-115175

TIGR01312COG0701COG0701 MORN-MORN-MORN-HeliorhodopsinTspO-MBRPLDc-NABC-tran ABC2-membrane-3HYPO LGT Lactamase-BTetR-NHYPO NTP-transf-9 CSSed16-5cm-5056

TetR-N ABC2-membrane-2MORN-MORN-MORN-ABC-tranECH-1HYPOHYPOHYPO adh-short-C2 HYPO MORN-MORN-MORN-HeliorhodopsinMF-01660SnoaL-2ATC-hydrolase COG2355 CSSed16-5cm-5924

MFS-1 GDPD Hydrolase-6-Hydrolase-likeC-GCAxxG-C-CHYPO Peptidase-M42COG2129 HYPO DUF3159HYPO MORN-MORN-MORN-Heliorhodopsin T1-Sed10-C3098

HYPOHYPO MORN-MORN-MORN-HeliorhodopsinHYPOAhpC-TSAHYPO T3-Sed10-C97774

HYPOHYPO EamA-EamA Na-H-antiporter Aminotran-3 PAS-Sigma54-activat MatE-MatE MORN-MORN-MORN-HeliorhodopsinHypD HupF-HypC Acylphosphatase-zf-HYPF-zf-HYPF-Sua5-yciO-yrdC-H-ypF-CcobW HypAHycI NiFeSe-HasesTIGR03295-frhA-coenzyme-F420-hydrogenase-subunit-alphaOxidored-q6-NiFe-hyd-SSU-CFer4-11 Tekir-sed17-143 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplementary Figure 14. Genomic neighborhood (within 10 kb) of Znf-Heliorhodopsins (red labels) origi- nating from archaeal contigs from the phylum Thermoplasmatales, class E2.

flux

UbiA Rhodanese TIGR03462-CarR-dom-SF-lycopene-cyclase-domainAmino-oxidase SQS-PSY DUF523-DUF1722HYPO UvdE Putative-PNPOxHYPO Znf-HeliorhodopsinCation-efHYPO Propeptide-C25-Peptidase-C25MTS MtrA MtrH Meth-synt-2DUF4346HYPO tRNA-anti-codon GCA-001595945-C1

Znf-HeliorhodopsinHYPODUF3335 Radical-SAM GCA-002496355-C126

Amino-oxidaseSQS-PSYPutative-PNPOxHYPO Znf-Heliorhodopsin GCA-002506745-C103

Heliorhodopsin SOUL HYPO Znf- NYN CSDAlbaHYPOHYPOeIF-1a GCA-002900535-C112

flux

UbiA Rhodanese TIGR03462-CarR-dom-SF-lycopene-cyclase-domainAmino-oxidase SQS-PSY DUF523-DUF1722UvdE Putative-PNPOxDNA-photolyaseHYPO Znf-HeliorhodopsinDUF3335 Radical-SAM Cation-ef PAS-PAS-9-HisKA-HATPase-cHYPO NosD HYPO Propeptide-C25-Peptidase-C25 SRR5506739-C331

Znf-Heliorhodopsin SRR6284161-C48644 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.01.429150; this version posted February 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

≥ 95 p. Actinobacteriota 1 o. Nanopelagicales (n = 110) 90-94 < 90 o. Microtrichales (n = 72) 2 p. Patescibacteria (n = 4)

3 o. Nanopelagicales f. S36-B12 (n = 12)

4 o. Actinomycetales (n = 62)

c. Coriobacteriia, Thermoleophilia (n = 12) 5 p. Chloroflexota c. Dehalococcoidia (n = 9)

6 c. Acidimicrobiia (n = 19)

7 o. Propionibacteriales, Microtrichales (n = 36)

p. Chloroflexota c. Anaerolineae (n = 11) 8 p. Firmicutes c. Bacilli (n = 32)

9 p. Firmicutes c. Dethiobacteria (n = 10)

p. Euryarchaeota c. Methanobacteria (n = 16) 10 p. Crenarchaeota c. Bathyarchaeia (n = 3) p. Asgardarchaeota c. Heimdallarchaeia (n = 2)

11 p. Actinobacteriota o. Actinomarinales (n = 95)

p. Halobacterota (n = 42) 12 p. Thermoplasmatota (n = 5) p. Thermotogota (n = 7)

13 p. Chloroflexota c. Anaerolineae (n = 43)

14 p. Thermoplasmatota o. Methanomassilliicoccales (n = 18)

15 p. Chloroflexota c. Anaerolineae (n = 8)

16 p. Chloroflexota c. Dehalococcoidia (n = 6)

p. Patescibacteria (n = 30) 17 p. Actinobacteriota c. Actinobacteria (n = 12)

18 p. Chloroflexota_A c. Ellin6529 (n = 12)

19 Eukaryotic ? (n = 8)

Supplementary Figure 15. Simplified representation (cladogram) of the HeR phylogenetic tree used for gene context analysis. Cluster numbers (defined in Supp. Fig. 4) are indicated on triangles at the tip of each branch. Actinobacteriota clusters are coloured yellow, Archaea - purple, Chloroflexota - green, Eukaryota - orange, Firmicutes - red, Patescibacteria - blue. Taxonomy and sequence counts are shown- only for representatives of each cluster. The blue rectangles highlight clusters where all or most mem- bers are anaerobic organisms. The outgroup (proteorhodopsins; n = 30) is depicted as a gray triangle at the bottom.