Bioinformatics, YYYY, 0–0 doi: 10.1093/bioinformatics/xxxxx Advance Access Publication Date: DD Month YYYY Discovery Note

Sequence analysis Diversity of rhodopsins in cultivated of the family Geodermatophilaceae associated with non-aquatic environments Sergey V. Tarlachkov1,2,*, Taras V. Shevchuk2, Maria del Carmen Montero- Calasanz3 and Irina P. Starodumova1 1G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, FRC Pushchino Scientific Center for Biological Research, Russian Academy of Sciences, Pushchino, 142290, Russia, 2Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, 142290, Russia and 3School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, Newcastle upon Tyne, NE1 7RU, UK

*To whom correspondence should be addressed.

Associate Editor: XXXXXXX Received on XXXXX; revised on XXXXX; accepted on XXXXX

Abstract Motivation: A small amount of research is focused on investigation of rhodopsins in cultivated bacteria isolated from non-aquatic environments. Furthermore, the abundance of these proteins in strains from hot and arid habitats was not reported previously. Since there is an insignificant amount of such isolates, the enigmatic role of the rhodopsins in dry ecological niches is still poorly understood. The members of the family Geodermatophilaceae could be used as interesting objects to search for new rhodopsin genes that will provide novel insights into versatility and importance of these proteins in non- aquatic conditions. Results: This is the first report of the abundance of different rhodopsins in cultivated bacteria isolated from hot and arid ecological niches. A total of 31 rhodopsin genes were identified in 51 analyzed genomes of strains belonging to the family Geodermatophilaceae. Overall, 88% of the strains harboring rhodopsins are isolated from non-aquatic environments. It was found that 82% of strains belonging to the genus Geodermatophilus have at least one gene as compared to 38% of strains of other genera which contain rhodopsins. Analysis of key amino acids revealed two types of the studied proteins: DTE type (putative proton pump) and NDQ type (putative sodium pump). Proton pumps were divided into two subtypes (DTEW and DTEF) according to phylogenetic analysis and the presence of highly conserved tryptophan or phenylalanine at position 182. Among all studied rhodopsins DTEF subtype is the most unique one, identified only in this family. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.

1 Introduction Govorunova et al., 2017; Sharma et al., 2006). The microbial rhodopsins are highly diverse retinal-binding membrane Microbial rhodopsins are broadly distributed in habitats where water is proteins which perform the function of light-gated ion channels or light abundant, in marine, sea ice, freshwater, hypersaline and brackish water sensors in photodetection processes as well as the function of ion pumps environments (Béjà et al., 2000; Finkel et al., 2013; Sharma et al., 2006). in light energy conversion processes. These proteins were first found in The presence of rhodopsins in non-aquatic environments has not been the archaea, and then in other microorganisms: the bacteria, the green widely reported. With the use of metagenomic approaches, microbial algae, the cryptomonads, the alveolates, and the fungi (Ernst et al., 2014; rhodopsins were found in phyllospheres of terrestrial plants (Atamna- Ismaeel et al., 2012; Finkel et al., 2013), in soil crust (Finkel et al., 2013), S.V.Tarlachkov et al. and in Antarctic Dry Valley edaphic systems (Guerrero et al., 2017). At the found rhodopsins were identified by BLASTp searches against the the same time, there are a scarce number of studied rhodopsins from NCBI non-redundant protein database (NCBI NR). Rare substitutions in strains isolated from non-aquatic ecological niches (Choi et al., 2014; rhodopsin sequences were detected based on the MicRhoDE database Petrovskaya et al., 2010). It is thereby of interest to further search for (Boeuf et al., 2015). Numbering of amino acid was in accordance with rhodopsins among cultivated bacteria associated with such habitats, and, bacteriorhodopsin sequence (1QM8). especially, in bacteria isolated from extremely hot and arid environments, for which the abundance of the rhodopsins was not previously shown. The family Geodermatophilaceae is Gram-positive, aerobic and 3 Results chemoheterotrophic which form rods or rudimentary 3.1 General characteristic of rhodopsins in the family hyphae (Normand, 2006). The family includes five genera, Geodermatophilaceae Geodermatophilus, Blastococcus, Modestobacter, Cumulibacter, and Of the 51 genomes studied, a total of 31 rhodopsin gene sequences were Klenkia (http://www.bacterio.net/). A majority of representatives of the identified in 29 genomes of strains belonging to the genera Blastococcus genera are adapted to extreme ecological niches and can grow as a pioneer (6 strains), Geodermatophilus (18 strains), Klenkia (4 strains) and on exiguous substrates. They are found in climates with extreme dry Modestobacter (1 strains) of the family Geodermatophilaceae conditions (Busarakam et al., 2016; Dobrovol’skaya et al., 1993; Eppard (Supplementary Tables S1 and S3, Fig. 1, Supplementary Fig. S1). All et al., 1996; Garrity et al., 1996; Mevs et al., 2000; Montero-Calasanz et these representatives contain 1 rhodopsin gene except strains G. al., 2012; Vasilenko et al., 2017). Some of them are recovered from desert dictyosporus DSM 43161T and G. pulveris DSM 46839T which harbor 2 soils (Dobrovol’skaya et al., 1993; Eppard et al., 1996; Garrity et al., genes each. Overall, 88% of the strains harboring rhodopsins are isolated 1996; Montero-Calasanz et al., 2013), other strains were isolated from from non-aquatic environments. At the same time, it was found that 56% various surfaces of rocks, stones, monuments, ancient walls, frescoes, of the strains from non-aquatic habitat have rhodopsin genes. These marble (Garcia-Vallés et al., 2000; Gurtner et al., 2000; Hezbri et al., proteins are much more abundant in Geodermatophilus than in other 2016; Mevs et al., 2000; Ortega-Morales et al., 2005; Urzì et al., 2001), genera: 82% of strains belonging to the genus Geodermatophilus have at which are characterized by water availability fluctuation and low nutrient least one gene as compared to 38% of strains of other genera which contain concentration but high solar irradiation. Only several strains of this family rhodopsin genes (Supplementary Table S1). were occasionally retrieved from deep-ocean sediments, plant leaves and rhizosphere soil (Bai et al., 2015; Montero-Calasanz et al., 2017; Xiao et al., 2011; Zhang et al., 2011). In view of wide ecological distribution of the Geodermatophilaceae members, they are chosen as interesting objects to search for new rhodopsins that will provide novel insights into versatility of these proteins and may enhance the next-generation optogenetic tools. In this study, we report the diversity of rhodopsins in Geodermatophilaceae family based on available genomic data for cultivated microorganisms.

2 Methods Complete and high-quality draft genome sequences of 51 bacteria of the family Geodermatophilaceae were downloaded from GenBank (Supplementary Table S1). Of these, sequence data for 34 strains were produced by the US Department of Energy Joint Genome Institute http://www.jgi.doe.gov/ in collaboration with the user community. Taxonomic identification of each genome was achieved by calculating 16S rRNA gene sequence similarity between the sequences retrieved from the genome assemblies and relevant sequences of type strains. Genome assemblies were annotated using Prokka 1.11 (Seemann, 2014) with the Barrnap 0.5 plug-in. Phylogenomic tree was inferred using JolyTree (Criscuolo, 2019). In order to search and identify rhodopsins present in genomes studied, the reference protein sequences (Supplementary Table S2) were used as query sequences in a BLASTp search against translated CDS which were extracted from each genome. A few best-hits with e-values lower than 0.01 were considered as potential rhodopsin proteins. Then, each found sequence was aligned using ClustalW (Thompson et al., 1994) against reference sequences to finally identify rhodopsins. Fig. 1. Maximum likelihood phylogenetic tree based on rhodopsin protein sequences Multiple sequence alignment of found rhodopsin proteins was found in bacteria of the family Geodermatophilaceae. Sequence accession numbers for each strain are given in the Supplementary Table S3. The tree is drawn to scale, with branch performed by ClustalW. Phylogenetic trees were constructed by lengths measured in the number of substitutions per site. Bootstrap values above 50% are maximum likelihood algorithm in MEGA7 (Kumar et al., 2016) using the indicated at the branch points. The sequence of bacteriorhodopsin (1QM8) served as an Jones-Taylor-Thornton model with 1000 bootstrap replications. The outgroup. pairwise identity between protein sequences was determined using TaxonDC 1.3 (Tarlachkov and Starodumova, 2017). Closest sequences to Rhodopsins in the family Geodermatophilaceae

Fig. 2. Protein alignment of key regions. Alignment includes one sequence from each rhodopsin groups found in the family Geodermatophilaceae and representatives of previously characterized microbial rhodopsins. BR, bacteriorhodopsin (1QM8); PR, proteorhodopsin (Q9F7P4.1); KR2, sodium pump (BAN14808.1); DTEW, DTEF and NDQ means rhodopsins from the corresponding groups found in strains G. dictyosporus DSM 43161T, G. obscurus DSM 43160T and M. caceresii KNN 45-2bT, respectively. Key amino acids identical for all aligned sequences are colored green. Key amino acids differing at least in one aligned sequence are colored yellow. Position 182, in which tryptophan is substituted by phenylalanine in rhodopsins of DTEF subtype, is blue colored. The identity of the amino acid sequences of rhodopsins varies in a wide 3.3 DTEF subtype of rhodopsins range from 24.0 to 97.9% (Supplementary Table S4). However, the A majority of DTEF subtype sequences are found in microorganisms presence of key conserved amino acids (Fig. 2, Supplementary Fig. S2) belonging to the genus Geodermatophilus (17 strains), however some of and a large number of genes suggest that all found rhodopsins are them are attributed to genera Blastococcus (6 strains), and Klenkia (3 potentially functional. An analysis of the amino acids in positions 85, 89 strains). and 96 (Fig. 2, Supplementary Fig. S2) has revealed that 30 sequences The DTEF rhodopsins are characterized by amino acid substitution of belong to the DTE type (putative proton pump) and 1 sequence is referred phenylalanine for tryptophan at position 182 in helix F. Tryptophan is to the NDQ type (putative sodium pump). Based on further analysis of highly conserved, and its replacement is quite rare. Among the 3,230 amino acids substitutions the DTE type has been divided into 2 subtypes: sequences from the MicRhoDE database which do not have a gap in the DTEW and DTEF which contain tryptophan and phenylalanine at position appropriate position, less than 0.8% of the sequences contain a substitution 182, respectively. All found rhodopsins bear leucine at position 93, and less than 0.1% have phenylalanine. This feature is typical of some suggesting their capacity to absorb green light according to Man et al. sequences of xenorhodopsins (Ugalde et al., 2011). However, the identity (2003) which is typical of representatives of the shallow marine of amino acid sequences between DTEF rhodopsins from environment. Geodermatophilaceae and xenorhodopsins is quite low and ranged from Phylogenetic analysis shows that the rhodopsin sequences form 3 15.4 to 25.5%, forming distinct clades on a tree (Supplementary Table S6, clades on the tree (DTEW and DTEF subtypes and NDQ type) with high Supplementary Fig. S4). In addition, as opposed to xenorhodopsins, there bootstrap values. However, close sequences not always occur in is no proline in position 212. Our findings suggest an independent origin phylogenetically related strains (Fig. 1, Supplementary Fig. S1). Despite of the substitution of tryptophan by phenylalanine in these groups. that DTEW and DTEF rhodopsins contain the same DTE-motif, they share The BLASTp search against the NCBI NR showed that DTEF only an average 28.2% of identity. For comparison, the identity of the rhodopsins are most closely related to protein WP_011981580.1 with amino acid sequences within the DTEW and DTEF clades varies in a average identity 50.7%. Interestingly, this protein is from Kineococcus range from 68.3 to 91.0% and 59.0 to 97.9%, respectively. Each of strains radiotolerans SRS30216 which is resistant to radiation (Phillips et al., T T G. dictyosporus DSM 43161 and G. pulveris DSM 46839 contains 2 2002). The identity for other found sequences is less than 48.1% rhodopsin proteins, belonging to DTEW and DTEF subtypes. (Supplementary Table S7). Moreover, closest sequences neither fall into clade of DTEF rhodopsins (Supplementary Fig. S4) nor contain a 3.2 DTEW subtype of rhodopsins substitution of Trp182. These facts demonstrate that this is a unique subtype of rhodopsins identified in the family Geodermatophilaceae. This subtype of rhodopsins are revealed in strains belonging to the genera Geodermatophilus (3 strains) and Klenkia (1 strain). These proteins are most closely related to xanthorhodopsins which have been divided into 3.4 NDQ type of rhodopsins two subgroups (designated as subgroup I and subgroup II) in the study by The NDQ rhodopsin was detected only in strain M. caceresii KNN 45- Vollmers et al. (2013). Phylogenetic analysis showed that rhodopsins of 2bT. Phylogenetic analysis showed that this protein falls into a cluster of the DTEW subtype are closer to the clade of actinorhodopsins of the known rhodopsins (Supplementary Fig. S5). The closest proteins, found subgroup I (Supplementary Fig. S3), specific for actinobacteria living in in the NCBI NR, are from the strains of the genus Micromonospora (order freshwater. ). The identity of the amino acid sequences of rhodopsins Closest proteins to the DTEW rhodopsins, found in the NCBI NR, are from Micromonospora and M. caceresii KNN 45-2bT varies from 66.3 to from the strains of the families Intrasporangiaceae (order 70.7% (Supplementary Table S8). Actinomycetales), and Kineosporiaceae (order Kineosporiales). The identity of the rhodopsins from these organisms and from Geodermatophilaceae family varies from 69.2 to 81.2%, and form a single 4 Discussion tight clade on a phylogenetic tree (Supplementary Table S5, In earlier studies, several rhodopsins were found using metagenomic Supplementary Fig. S3). This might indicate a possible horizontal gene methods in non-aquatic habitats (Atamna-Ismaeel et al., 2012; Finkel et transfer (HGT) of the DTEW rhodopsins between representatives of al., 2013; Guerrero et al., 2017). However, these studies have not always different orders. established exact relationships between the found sequences of rhodopsins and genomes, which harbor them. Only a limited number of rhodopsins were reported for a few strains belong to and isolated from non-aquatic environments (Choi et al., 2014; Petrovskaya et S.V.Tarlachkov et al. al., 2010). An insignificant amount of such cultivated microorganisms in a sodium-rich environment (Kwon et al., 2013). harboring rhodopsin genes restricts further genomic, physiological and On the other hand, studied rhodopsins may perform a sensory function biochemical studies. In our work we showed that different rhodopsins are by changes in membrane potential or via transducer protein. This function widely represented in cultivated bacteria of the family can be employed in the phototaxis of bacteria since some of them have Geodermatophilaceae isolated from moderate non-aquatic habitats (e.g. motile forms (Normand, 2006), as well as for activation of defense phyllosphere and rhizosphere of plants) and from harsh hot and arid mechanisms against excessive solar irradiation. However, the presence of ecological niches (e.g. arid soil, sand, limestone, marble). Moreover, more two rhodopsins of different subtypes in strains G. dictyosporus DSM than half of the studied strains contain at least one rhodopsin gene. It 43161T and G. pulveris DSM 46839T possibly indicate different functions makes microorganisms belonging to Geodermatophilaceae promising of DTEW and DTEF rhodopsins. objects for the study of the adaptive role of rhodopsins in non-aquatic This research is the first report of the wide presence of microbial habitats and may indicate the importance of rhodopsins for the survival of rhodopsins in cultivated bacteria isolated from harsh hot and arid habitats. microorganisms of this family in the mentioned ecological niches. Our analysis and conclusions open a prospect for further experimental As previously reported, rhodopsin genes are often subjected to HGT study of this group of proteins to expand the understanding of the role and between aquatic microorganisms (Frigaard et al., 2006). In addition, high significance of rhodopsins in adaptation to extreme living conditions. level of HGT was shown for members of the Geodermatophilaceae family (Sghaier et al., 2016). This suggests that the found rhodopsin genes are also highly likely to be the result of transfer between more distant Acknowledgements microorganisms. However, ages of these events could be different for all The authors thank Ye.V. Demina for her help in article preparation. types and subtypes of rhodopsins. Since DTEW subtype form a single tight clade with closest sequences represented in other orders, their genes Conflict of Interest: none declared. might have appeared in the Geodermatophilaceae after NDQ and DTEF rhodopsins. A similar picture is observed for the member of NDQ type: it falls into a cluster of well-known proteins, but a bit more divergent from References sequences found in Micromonospora, suggesting earlier gene transfer Atamna-Ismaeel,N. et al. 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