New Halonotius Species Provide Genomics-Based Insights Into Cobalamin Synthesis in Haloarchaea

Hypersaline aquatic and terrestrial ecosystems display a cosmopolitan distribution. These environments teem with microbes and harbor a plethora of prokaryotic lineages that evaded ecological characterization due to the prior inability to cultivate them or to access their genomic information. In order to close the current knowledge gap, we performed two sampling and isolation campaigns in the saline soils of the Odiel Saltmarshes and the salterns of Isla Cristina (Huelva, Spain). From the isolated haloarchaeal strains subjected to high-throughput phylogenetic screening, two were chosen (F15BT and F9-27T) for physiological and genomic characterization due of their relatedness to the genus Halonotius. Comparative genomic analyses were carried out between the isolated strains and the genomes of previously described species Halonotius pteroides CECT 7525T, Halonotius aquaticus F13-13T and environmentaly recovered metagenome-assembled representatives of the genus Halonotius. The topology of the phylogenomic tree showed agreement with the phylogenetic ones based on 16S rRNA and rpoB′ genes, and together with average amino acid and nucleotide identities suggested the two strains as novel species within the genus. We propose the names Halonotius terrestris sp. nov. (type strain F15BT = CECT 9688T = CCM 8954T) and Halonotius roseus sp. nov. (type strain F9-27T = CECT 9745T = CCM 8956T) for these strains. Comparative genomic analyses within the genus highlighted a typical salt-in signature, characterized by acidic proteomes with low isoelectric points, and indicated heterotrophic aerobic lifestyles. Genome-scale metabolic reconstructions revealed that the newly proposed species encode all the necessary enzymatic reactions involved in cobalamin (vitamin B12) biosynthesis. Based on the worldwide distribution of the genus and its abundance in hypersaline habitats we postulate that its members perform a critical function by being able to provide “expensive” commodities (i.e., vitamin B12) to the halophilic microbial communities at large.


INTRODUCTION
Haloarchaea are extremely halophilic archaea belonging to the phylum Euryarchaeota. They constitute a diverse group of archaea that are widely distributed in different extreme habitats with high salt concentrations, from aquatic systems (saline lakes, salterns) to saline soils, salt mines and salted foods (Ventosa, 2006). Haloarchaea are placed in the class Halobacteria, which comprises three orders: Halobacteriales, Haloferacales and Natrialbales, encompassing five families and more than 50 genera (Amoozegar et al., 2017). Despite their ecological role (in hypersaline habitats they constitute a dominant population) (Ghai et al., 2011;Fernández et al., 2014a,b;Ventosa et al., 2015;Vera-Gargallo et al., 2019) and their biotechnological potential (Amoozegar et al., 2017), little information is available on the genomic architecture and metabolic potential of cultured haloarchaea representatives (Bolhuis et al., 2006;Pfeiffer et al., 2008). The genus Halonotius belongs to the class Halobacteria, order Haloferacales, family Halorubraceae (Amoozegar et al., 2017) and currently it includes two species: the type species, Halonotius pteroides, isolated from a crystallizer of an Australian saltern, which represented up to 16% of the prokaryotic population of this habitat (Burns et al., 2004(Burns et al., , 2010, and Halonotius aquaticus, recently isolated from a marine saltern in Spain . Although the genus comprises only two described species, it has been previously reported in several other habitats, such as saline lakes in Australia and China (Podell et al., 2014;Han et al., 2017), solar salterns in Turkey and Spain (Çınar and Mutlu, 2016;Durán-Viseras et al., 2019) and food-grade salt samples (Henriet et al., 2014), indicative of a world-wide environmental distribution. Moreover, three metagenome-assembled genomes previously recovered from Lake Tyrell (Australia), appeared to be phylogenetically close to Halonotius (Podell et al., 2013).
In this work, we have carried out a comparative genomic analysis of the genus Halonotius, for which we used the genomes of the two species belonging to the genus (i.e., Halonotius pteroides and Halonotius aquaticus), the ones from the two new strains that we isolated, and two metagenomeassembled genomes mentioned above. Our data indicate that Halonotius spp., like many other haloarchaea [e.g., Haloquadratum (Bolhuis et al., 2006), Halobacterium (Pfeiffer et al., 2008)], are photoheterotrophs with a typical aerobic electron transport chain and harbor a rhodopsin-related gene pool, specifically haloarchaeal proton-pumps and sensory rhodopsins. Remarkably, all analyzed Halonotius genomes were found to encode a complete cobalamin biosynthesis pathway. Moreover, our results show that this genus is abundant and diverse in hypersaline environments and support the description of two new species, for which we propose the designations Halonotius roseus sp. nov., and Halonotius terrestris sp. nov., respectively. Additionally, the detailed study of the polar lipids profiles of these strains justify an emendation of the genus Halonotius.
Halonotius pteroides CECT 7525 T , type species of the genus Halonotius and Halonotius aquaticus F13-13 T were also used in this study. These strains were also grown in R2A 25% (w/v) medium as described above.
The 16S rRNA and the rpoB genes were PCR-amplified, using the primer pairs ArchF/ArchR (DeLong, 1992;Arahal et al., 1996) and rpobF/rpobR (Fullmer et al., 2014), respectively, and sequenced by Sanger technology at a commercial company (Stabvida, Oeiras, Portugal). The shotgun sequencing of strains F15B T and F9-27 T was also performed by the same commercial company (Stabvida, Oeiras, Portugal) on the Hiseq 4000 platform, using 150 bp paired-end sequencing. The genomes of the strains Halonotius aquaticus F13-13 T and Halonotius pteroides CECT 7525 T were recovered from a previous study .

Phylogenetic Analyses
The 16S rRNA gene-based phylogenetic analyses were performed using the maximum-likelihood and maximum-parsimony algorithms integrated in the ARB software package (Ludwig et al., 2004). The 16S rRNA gene sequences alignment was confirmed and checked against both the primary and secondary structures of the 16S rRNA sequence using the alignment tool of the ARB software package. In order to evaluate the robustness of the obtained phylogenetic tree, a bootstrap analysis (1000 replications) was performed.
The rpoB gene-based phylogenetic analyses were performed by MEGA 6.0 software (Tamura et al., 2013) using the maximum-likelihood. In order to evaluate the robustness of the obtained phylogenetic tree, a bootstrap analysis (1000 replications) was performed.
Additional 16S rRNA gene sequences, rpoB sequences and genomes used for phylogenetic analyses were obtained from the GenBank database, and their strain designations and accession numbers are shown in Figures 1, 2

Rhodopsins Tree
The genomes were scanned for the presence of rhodopsin sequences by using hmmsearch (Eddy, 2011) and a profile hidden Markov model (HMM) of the bacteriorhodopsinlike protein family (Pfam accession: PF01036). To align the highly similar identified sequences and a curated database composed of a collection of type-1 rhodopsins, MAFFT with the L-INS-i accuracy model was used (Katoh and Standley, 2013).
A maximum likelihood tree with 100 bootstrap replicates was constructed based on this protein alignment, by using FastTree2 (Price et al., 2010).

Abundance Estimation for Euryarchaeota and Halonotius
Preprocessed Illumina reads from public metagenomes were queried for putative RNA sequences by using UBLAST (Edgar, 2010) against the non-redundant SILVA (Pruesse et al., 2007) SSURef_NR99_132 database, that was clustered at 85% sequence identity by UCLUST (Edgar, 2010). Identified putative 16S rRNA sequences (e-value < 1e-5) were screened using SSU-ALIGN. Resulting bona fide 16S rRNA sequences were compared by blastn (Altschul et al., 1990) (e-value < 1e-5) against the curated SILVA (Pruesse et al., 2007) SSURef_NR99_132 database. Matches with identity ≥80% and alignment length ≥90 bp were considered for downstream analyses. Sequences assigned to the phylum Euryarchaeota and genus Halonotius were used to calculate the environmental abundances for these taxonomic categories.

Fragment Recruitment
The presence of haloarchaeal strains related (at species level) to the obtained Halonotius strains (F15B T , F9-27 T and Halonotius aquaticus F13-13 T ) was assessed by performing fragment recruitments with environmental metagenomic datasets ( Table 2). Briefly, in order to avoid analysis bias, we concatenated the contigs belonging to each genome and masked all the rRNA gene sequences present. Subsequently, blastn (with the cut-offs: alignment length ≥ 30 nt, identity > 95%, E value < = 1e-5) was used in order to align the metagenomic quality-filtered shotgun reads (

ANI, AAI, DDH and Protein Isoelectric Points
Average nucleotide identity (ANI) and average amino acid identity (AAI) were calculated as previously described by Konstantinidis and Tiedje (2005a,b), respectively. Digital DDH Sequence accession numbers are shown in parentheses. Red circles highlight Bootstrap/UFBootstrap values lower than 50, the yellow ones between 50 and 80 and green color is used to depict values higher than 80. The trees were rooted using the DPANN representatives Candidatus Nanosalinarum J07AB56 and Candidatus Nanosalina J07AB43. values were determined using the Genome-to-Genome Distance Calculator (GGDC) website using formula 2 as described by Auch et al. (2010) and Meier-Kolthoff et al. (2013). The isoelectric points of proteins were calculated using the iep program implemented in the EMBOSS package (Rice et al., 2000).

Chemotaxonomic Analysis
Cultures of the strains F15B T , F9-27 T , Halonotius aquaticus F13-13 T and Halonotius pteroides CECT 7525 T [obtained after 14 days of aerobic incubation in R2A with 25% (w/v) salts liquid medium under optimal conditions] were used for chemotaxonomic analyses. For lipid identification Halobacterium salinarum DSM 3754 T and Halorubrum saccharovorum DSM 1137 T were used as reference. The lipids of these two species have been previously experimentally identified (Kushwaha et al., 1982;Torreblanca et al., 1986). The strains used for comparisons were cultured according to the original descriptions for each species and standardized to the same incubation conditions. Lipid extraction was carried out following the method described by Corcelli and Lobasso (2006). Total lipid extracts were analyzed by highperformance TLC using HPTLC silica gel 60 plates crystal back (10 × 20 cm; Merck art. 5626); the plates were eluted in the solvent system chloroform/methanol/90% acetic acid (65: 4: 35, by vol.). For detection of all lipids, phospholipids and polar lipids, the plate was sprayed with sulfuric acid 5% in water, molybdenum blue spray reagent and phosphomolybdic acid, respectively and

Phylogenetic Analyses
After extensive haloarchaeal strains isolations from two hypersaline environments located in South west Spain (Odiel Saltmarshes and Isla Cristina), the strains F15B T and F9-27 T were selected for further analyses. Both strains proved to be closely related to members of the genus Halonotius, as shown by 16S rRNA gene sequence comparison with available sequences in the public databases and by phylogenetic reconstruction ( Figure 1A).  F15B T and F9-27 T grouped within the Halonotius cluster, but in independent branches from Halonotius pteroides CECT 7525 T and Halonotius aquaticus F13-13 T , respectively, suggesting that they could constitute different species within the genus Halonotius ( Figure 1A).
Limitations of the 16S rRNA gene sequence analysis to discriminate species of haloarchaea have been reported (Boucher et al., 2004;Sun et al., 2013), and thus a phylogenetic tree based on the comparison of the rpoB' gene sequences was reconstructed ( Figure 1B). In concordance to the 16S rRNA tree, both strains clustered with species of the genus Halonotius, but were located in different branches ( Figure 1B). Strain F15B T (1827 bp) showed a nucleotide similarity of 89.1% with Halonotius aquaticus F13-13 T and strain F9-27 T (1827 bp) shows a nucleotide similarity of 92.0% with Halonotius sp. J07HN4 and 91.8% with Halonotius aquaticus F13-13 T . The rpoB gene sequence similarity between strains F15B T and F9-27 T was 89.3%.

Genome Sequence Characteristics
The genomes of the isolated strains, F15B T and F9-27 T were successfully sequenced, and in conjunction with those of the two currently described species of the genus, Halonotius pteroides CECT 7525 T and Halonotius aquaticus F13-13 T , were assembled de novo. The characteristics of all these genomes are shown in Table 3. As most members of the class Halobacteria (phylum Euryarchaeota), all genomes had a high G + C content, between 59.5-62.7 mol%, with F9-27 T having the highest. All four Halonotius strains studied have similar genome size, ranging from 2.5-3.0 Mb, making them the smallest ones reported for members of the family Halorubraceae. Additional genomic features are shown in Supplementary Figure 1.

Phylogenomic Analysis
To confirm the phylogenomic relationships previously obtained by 16S rRNA and rpoB genes sequence comparison between Halonotius strains, Halonotius metagenome-assembled genomes (MAGs) and other related haloarchaeal species, a phylogenomic tree based on core orthologous genes was constructed. Candidatus Nanosalina J07AB43 (GCA_000220375) and Candidatus Nanosalinarum J07AB56 (GCA_000220355) MAGs were used as outgroups. A total of 257 core orthologous genes were shared between all genomes. The phylogenomic tree based on the concatenated alignment, showed that all Halonotius genomes clustered together in a single branch, well separated from other haloarchaeal groups and supported by high bootstrap values (Figure 2). Additionally, also supported by high bootstrap values, within the Halonotius cluster, all Halonotius strains appeared monophyletically separated, suggesting they represent different species within this genus. The results are in concordance with the initial phylogeny described by 16S rRNA and rpoB genes sequence comparison, which suggest that for this genus both genes could be used as reliable phylogenetical markers.

Ecological Distribution and Abundance of the Genus Halonotius
Metagenomic studies carried out in hypersaline environments have enabled the determination of the microbial diversity of these habitats, suggesting that a large proportion of haloarchaeal members, abundant in these systems, had not been isolated or characterized before (Ventosa et al., 2015). Based on 16S rRNA gene sequence reads abundances from different aquatic and terrestrial hypersaline systems with different salinities (see Materials and Methods), representatives of the genus Halonotius were found to be present in almost all metagenomes from habitats with intermediate to high salinity concentrations. The genus Halonotius comprised up to 9% of the prokaryotic community in aquatic saline environments ( Figure 3A) and up to 1.8% of the prokaryotic community in saline soils ( Figure 3B). The data confirm that members of the genus Halonotius are abundant microorganisms in hypersaline environments, especially at high salinities (from 19 to 37% total salts) but are completely absent in habitats without or with lower salt concentrations. In this sense, we can consider them as obligately extreme halophiles that prefer habitats with high salinities.
To assess the distribution of the cultured Halonotius strains, fragment recruitment was carried out against all those metagenomic datasets mentioned above. The metagenomes which provided significant recruitment values are shown in Figures 3C-H. All Halonotius strains used in this study showed high recruitment values in their environment of origin: the metagenomic dataset SMO2 reported from the Odiel Saltmarshes saline soil in Huelva, Spain for strain F15B T , and dataset IC21 obtained from Isla Cristina salterns in Huelva, Spain, for strains F13-13 T and F9-27 T , where strain F13-13 T seemed to be more abundant. Remarkably, highest recruitment values were obtained against the metagenomic dataset S7, which corresponds to a hypersaline meromictic pit lake located in Transylvanian Basin, Romania; suggesting a worldwide distribution for this genus. Even the highest abundance of 16S rRNA Halonotius reads was obtained in SS37 metagenome, none of the Halonotius isolates recruited significantly in that metagenome, only Halonotius pteroides CECT 7525 T seemed to be present in that metagenome dataset (Supplementary Figure 2). The abundance of reads above 95% similarity for Halonotius pteroides CECT 7525 T against metagenomic database SS37 and the above mentioned 16S rRNA reads abundance in this metagenome, indicates that there might be other Halonotius species present in significant amounts in this specific habitat.
Furthermore, to evaluate the presence of Halonotius worldwide, we have searched the SILVA database for 16S rRNA sequences belonging to this genus. Results indicate that members of the genus Halonotius are found in a variety of hypersaline environments from different countries, such as: Canada, United States, Bolivia, Tunisia, Spain, Romania, Turkey, Russia, China, and Australia (Supplementary Figure 3). All these data, in addition to the 16S rRNA reads abundance along the different metagenomes, support a worldwide distribution of this genus.

Metabolism
Halonotius strains were isolated on complex media for heterotrophic aerobic microbes (see Materials and Methods). Genome annotation of Halonotius members confirm their heterotrophic metabolism. No evidences for photosynthetic or chemolithotrophic capabilities were found in any of these genomes. In line with their heterotrophic capabilities, several ABC and other transporters for uptake of carbohydrates were identified, forming a large fraction of the genes in all Halonotius genomes (Figure 4).
Representative genes for central carbohydrate metabolism like the tricarboxylic acid cycle, pentose phosphate, gluconeogenesis and Entner-Doudoroff pathway were present (Figure 4). For the oxidation of pyruvate to acetyl CoA (Figure 4), only the anaerobic route via pyruvate ferredoxin oxidoreductase (porA and porB) seemed to be present, while the aerobic route via pyruvate dehydrogenase (PDH) was absent. Additionally, genes encoding an α-amylase (K21574), which can degrade complex carbohydrates to glucose were detected (Figure 4). Remarkably, in agreement with previous studies on haloarchaea Anderson et al., 2011), the standard Embden-Meyerhof pathway of glycolysis appears to be incomplete, with 6-phosphofructokinase (the key enzyme of the classical pathway), found absent in all studied genomes (Figure 4). This suggest that haloarchaea could utilize the Entner-Doudoroff pathway for glucose degradation or that alternative enzymes could be used for this step.
Regarding nitrogen metabolism, Halonotius genomes encode the essential genes involved in the assimilation of ammonia and amino acid metabolism, glutamine synthetase and glutamate synthase. Accordingly, all genomes also presented the gene encoding the high-affinity ammonium transporter Amt, indicating that nitrogen uptake occurs in its most reduced form, ammonia (Figure 4). Along similar lines, strains F15B T and F9-27 T showed a major facilitator superfamily (MFS) antiporter for nitrate/nitrite uptake (Figure 4). Genes encoding the enzymes for nitrate reduction assimilation to ammonia, nitrate reductase and nitrite reductase, were also present. Besides, several ABC transporters for nitrogen sources, such as branched-chain amino acids, urea, putrescine/spermidine and nitrate-nitrite/taurine were also found (Figure 4). In addition, Halonotius genomes showed other genes which could provide nitrogen rich compounds, by degradation of amino acids, such as L-glutamate, to ammonia generating NADPH (glutamate dehydrogenase) (Figure 4) or by urea degradation, which was only absent in strain F15B T . Most pathways for amino acid biosynthesis were also found in the studied genomes (Supplementary Table 1).
The presence of the high-affinity phosphate transport system (PstSCAB), suggests that this is the main route of uptake of inorganic phosphate (Figure 4). All studied genomes of Halonotius also contained the ABC transporter, PhnCDE, a high affinity uptake system for phosphonates (Figure 4), which are organophosphorous compounds with a carbonphosphorous bond that under phosphate starvation conditions could be used also as a nutritional source of phosphorous (Villarreal-Chiu et al., 2012).
In addition, several antimicrobial compounds and multidrugs transport systems were found and genes encoding archaella were also discovered in all Halonotius genomes.

Cobalamin (Vitamin B 12 ) Biosynthesis
Genome-scale metabolic reconstructions performed for the Halonotius members brought to light the complete cobalamin biosynthesis pathway (Figures 4, 5), suggesting the genetic capability of the strains of this genus for its de novo synthesis. Cobalamin (vitamin B 12 ) is a complex metabolite, produced only by a subset of bacteria and archaea, and essential cofactor required by many organisms from all the domains of life. Cobalamin can be synthesized by either an aerobic or anaerobic pathway from uroporphyrinogen III as precursor, involving 20 enzymatic steps. Several enzymes are homologous and shared by both pathways, while only few are specifically oxygen-requiring or oxygen-sensitive (Moore and Warren, 2012). The complex structure of cobalamin and the high metabolic toll of its biosynthesis pathway (Morris, 2012;Doxey et al., 2015) fueled a 'race' toward a genomic removal, thus designating the few taxa capable of its biosynthesis as 'losers' that perform a Black Queen function (Morris, 2015). This term alludes to the game of Hearts, where players try to avoid being stuck with the queen of spades, and has been used to describe costly genes or functions that members of the community benefit from losing. Thus, generating individual "beneficiaries" of reduced genomic content depending on luckless "helpers" that provide goods to the whole community . The role of representatives of the Archaea in cobalamin production has been poorly studied and limited to Thaumarchaeota, halophilic Crenarchaeaota and marine methanogenic Euryarchaeota (Rodionov et al., 2003;Doxey et al., 2015).
Halonotius genomes contained almost all genes involved in aerobic and anaerobic pathways, with only a few gaps (i.e., CobG and CobK were missing from the aerobic pathway; CbiD and CbiJ were missing from the anaerobic pathway) (Figure 4). Additionally, all genes encoding the conversion of cobyrinic acid a,c-diamide to vitamin B 12 were also found and are represented by a discontinuous line on the metabolic map (Figures 4, 5B). None of the absent genes were either detected in any known archaeal cobalamin producers and therefore should not be considered as essential genes for this pathway (Doxey et al., 2015). Furthermore, previous studies considered the presence of cbiA/cobB, cbiC/cobH FIGURE 4 | Metabolic reconstruction of the isolated strains Halonotius terrestris sp. nov. F15B T , Halonotius roseus sp. nov. F9-27 T and Halonotius aquaticus F13-13 T . The turquoise panel depicts the simplified metabolic pathway of vitamin B 12 biosynthesis; the gene names highlighted in boldface were present in the genomes, while the ones in gray are inferred to be absent in archaea. Horizontal arrows indicate homology between aerobic and anaerobic pathway enzymes. The discontinuous lines correspond to complete pathways not represented in the figure. The dark green box highlights glycolysis (Embden-Meyerhof-Parnas), while the light green one shows the Entner-Doudoroff pathway modified for haloarchaea. The orange depicts the tricarboxylic acid cycle (TCA); pink color corresponds to isoprenoid biosynthesis: C5 isoprenoid (dark) and C10-C20 isoprenoid (light). The light violet box corresponds to dTDP-L-rhamnose biosynthesis; the yellow color shows the assimilatory nitrate reduction pathway; dark purple is used for the L-glutamate degradation by glutamate dehydrogenase (GdhA). Alpha-amylase enzyme for glucose degradation is also represented by its KO identifier. Transparency was used to show the inferred components of the complex III of electron transport chain. Red boxes are used for genes not present in the studied genomes. BCAA, branched-chain amino acid; HMP, hydroxymethylpyrimidine; TCA, tricarboxylic acid cycle.
or cobT genes in a microorganism, as reliable indicators for the complete pathway (Bertrand et al., 2011). As far as the authors are aware, despite a previous report on Halobacterium salinarum NRC-1 cobamide production (Woodson et al., 2003), little is known about the cobalamin biosynthesis pathway in hypersaline environments, or halophilic archaea at large. Due to the detection of the cobalamin biosynthesis genes clusters in all the analyzed Halonotius genomes (Figure 5), and the widespread distribution and abundance (Figure 3 and Supplementary Figure 3) of this genus in hypersaline environments, we hypothesize that these organisms perform a critical role in the microbial community, where they contribute to the maintenance of the environmental cobalamin supply and modulate trophic interactions. Moreover, the detection of B 12 -dependent enzymes (e.g., methylmalonyl-CoA mutases; metH, MTR 5-methyltetrahydrofolate-homocysteine methyltransferases; nrdA/nrdE ribonucleoside-diphosphate reductases; pduC propanediol dehydratases) (Supplementary Table 2) in metagenomic datasets where Halonotius genomes were inferred to be present, pinpoints toward metabolic dependencies between the cobalamin suppliers and consumers.

Osmoregulation
The mechanisms used by microorganisms to deal with their cellular adjustment to high salinity environments are diverse and can be classified in salt-in and salt-out strategies (Galinski and Trüper, 1994;Wood et al., 2001), physiological osmostress adaptations which are not necessarily mutually exclusive (Deole et al., 2013;Oren, 2013;Youssef et al., 2014). The osmotic balance in microorganisms that use the salt-in strategy is achieved by accumulation of a large fraction of inorganic ions in the medium, as K + and Cl − via transport (Oren, 2011), while Na + ions are excluded as much as possible from cells ( Gunde-Cimerman et al., 2018). However, in order to keep the proteins solubility and to ensure the functionality of key cellular activities, the entire proteome of the microorganism required compensatory changes for the cytoplasm s adaptation to the high ionic strength (Oren, 2013;Saum et al., 2013;Talon et al., 2014;Warden et al., 2015). This has left an acidic signature on the proteome of those salt-in microorganisms, reducing its surface hydrophobicity and limiting these microorganisms to hypersaline habitats where the external salinity does not frequently fluctuate.
We have characterized the proteome of all available Halonotius genomes (Figure 6), and to analyze the differences in protein acidity we have compared them to proteomes from salt-in halophilic archaea [Haloquadratum walsbyi HBSQ00 (Bolhuis et al., 2006) and Halorubrum saccharovorum DSM 1137 T (Becker et al., 2014)], a salt-in halophilic bacterium [Salinibacter ruber DSM 13855 T (Mongodin et al., 2005)] and to that of a saltout bacterium [Spiribacter salinus M19-40 T (López-Pérez et al., 2013)] (Figure 6). The proteome pI plot showed a single peak around 4.0 for all haloarchaeal genomes and for Salinibacter ruber DSM 13855 T , which follow the trend of amino acid use in the hypersaline system (Fernández et al., 2014b) being acidic residues more frequently employed and thus suggesting a salt-in strategy for Halonotius strains. Only Spiribacter salinus M19-40 T , microorganism employing a salt-out strategy, resulted in a different single peak around 4.5.
Another method to determine the salt adaptation of a microorganism is the reliance on the Na + gradient to transport actively nutrients into the cell (Oren, 2011;Gunde-Cimerman et al., 2018). All Halonotius strains have several secondary transporters that catalyze the translocation of solutes across the cytoplasmic membrane using electrochemical ion gradients. Na + is extruded from the cells by Na + /H + and Na + /Ca 2+ antiporters systems while K + can enter the cells passively through K + channels in the membrane and by K + /H + symporters systems (Figure 4), consistent with salt-in adaptations to a hypersaline environment. The necessary energy is derived from the proton gradient over the membrane, generated by respiratory electron transport and from the light-dependent proton pumping rhodopsin (Figure 4).
In contrast, the salt-out strategy is based in keeping most of inorganic ions out and using organic compatible solutes to balance the high salinity of the external environment (Empadinhas and Da Costa, 2008;Gunde-Cimerman et al., 2018). In the case of Halonotius, ABC-type transport systems  or other transporters for compatible solutes uptaking were not observed in their genomes. In the same way, none of the genes encoding key enzymes for the synthesis of organic compatible solutes, such as glycine betaine, ectoine, hydroxyectoine, proline, biphosphoglycerate or trehalose were found in any of the studied Halonotius genomes. This suggests that members of the genus Halonotius do not use this mechanism to balance the high salinity of the environment.

Rhodopsin Analysis
Microbial rhodopsins are a family of photoactive retinylidene proteins widely distributed within the microbial world. They were first discovered in haloarchaea in the early's 1970 (Oesterhelt and Stoecknius, 1971;Oesterhelt and Stoeckenius, 1973), however, later genomic and metagenomic sequencing revealed homologs in many disparate eukaryotes and bacteria, and later in the marine group II euryarchaeota (Frigaard et al., 2006). These kind of proteins are characterized by their diversity of function, using variations of a shared seventransmembrane helix structure and similar photochemical reactions to carry out distinctly different light-driven energy and sensory traduction processes (Govorunova et al., 2017). Their biological functions fall into two categories: (a) photoenergy transducers that convert light into electrochemical potential to energize cells (light-driven ion pumps), which catalyze outward active transport of protons, and (b) photosensory receptors, that use light to gain information about the environment to regulate cell processes (Govorunova et al., 2017).
Rhodopsin-like sequences were found in all members of the genus Halonotius ( Figure 7B). Specifically, haloarchaeal proton-pumps were found in all sequenced Halonotius genomes, which indicates that ATP synthesis light-mediated is a typical feature of species of this genus. Additionally, strains F13-13 T and F15B T also encoded sensory rhodopsins, suggesting additional metabolic flexibility in illuminated conditions for these two strains.
According to major absorption light wavelength, rhodopsins could be classified as "green-absorbing" and "blue-absorbing." A single amino acid residue determines this preference. The leucine (L) and methionine (M) variants absorb maximally in the green spectrum while the glutamine (Q) variant absorbs maximally in the blue spectrum (Man et al., 2003). Alignment of rhodopsins sequences found in Halonotius genomes showed a leucine amino acid in this position, indicating that they show a green light absorption ( Figure 7A).
Amino Acid Identity (AAI), Average Nucleotide Identity (ANI) and Digital DNA-DNA Hybridization (DDH) The AAI, the ANI, and the digital DNA-DNA hybridization (DDH) parameters between strains F15B T , F9-27 T , Halonotius pteroides CECT 7525 T , Halonotius aquaticus F13-13 T and other environmental Halonotius MAGs, were calculated as described in the "Materials and Methods" and the results are shown in Figure 8 and in Table 4. A threshold AAI value of 65% has been established for genus delineation (Klappenbach et al., 2007;Konstantinidis et al., 2017). AAI values for all Halonotius genomes were in all cases higher than 75% (Figure 8), indicating that all of them belongs to the genus Halonotius.
For species delineation, a threshold ANI value of 95-96% ( Konstantinidis and Tiedje, 2004;Richter and Rosselló-Móra, 2009) and a DDH value of 70% (Stackebrandt and Goebel, 1994;Auch et al., 2010) has been established. ANI values for all four strains of Halonotius studied were in all cases lower than 88% (Figure 8 and Table 4) and DDH values lower than 35.7% ( Table 4), suggesting that they constitute four separate taxa at the species level. We can conclude that strains F9-27 T and F15B T represent two different species of the genus Halonotius, for which we propose the new designations Halonotius roseus sp. nov. and Halonotius terrestris sp. nov., respectively.

Phenotypic Characterization
The different phenotypic features between strains F9-27 T and F15B T and the other members of the genus Halonotius are shown in Table 5. More phenotypic features of strains F9-27 T and F15B T are detailed in their species descriptions given below.

Chemotaxonomic Characterization
An exhaustive comparative analysis of the total lipids, phospholipids and polar lipids, between strains F9-27 T and F15B T and the previously described Halonotius species, was carried out (Supplementary Figure 4). Results revealed that all of them possess phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS) and one glycolipid chromatographically identical to sulfated diglycosil diether (S-DGD-1) as major polar lipids. As the presence of PGS was not reported at the description of the genus Halonotius and Halonotius pteroides (Burns et al., 2010), here we also propose the emended description of this genus in order to include this and other features of species of the genus.

CONCLUSION
This study provides a complete comparative genomic analysis of the genus Halonotius, showing evidence of the ubiquity and abundance of species of this genus in different hypersaline environments and significantly expanding our knowledge of this almost unknown haloarchaeal genus. Results from Halonotius 16S rRNA read abundances from different aquatic and terrestrial hypersaline systems and Halonotius genomes recruitments against these systems, confirm Halonotius abundance in these environments. Remarkably, results from the complete metabolic analysis of this genus brought to light that Halonotius members present the complete cobalamin biosynthesis cluster, suggesting their capability to produce cobalamin. Thus, we suggest that species of this genus could play a relevant environmental role for the community in de novo cobalamin synthesis on these saline environments, and thus, the members of Halonotius could perform a Black Queen function among members of the haloarchaea in hypersaline habitats Mas et al., 2016).
Additionally, this study indicates that members of the genus Halonotius might have a osmoregulatory salt-in strategy, based on their acidic proteomes, the presence of typically salt-in transporters and by the absence of compatible solutes transporters or pathways for their biosynthesis which could suggest a salt-out strategy. We could find proton-pumping rhodopsins in all Halonotius genomes and sensory rhodopsins in some, all absorbing in the green spectrum.
Finally, this study has also permitted the emended description of the genus Halonotius and the identification of the isolates as two novel species of this genus, for which we propose the new names Halonotius terrestris sp. nov. and Halonotius roseus sp. nov., whose descriptions are given below.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA and rpoB gene sequences of Halonotius terrestris F15B T are MH465135 and MH454087, respectively, and that of the complete genome is RKLU00000000.
Description of Halonotius roseus sp. nov.
The type strain is F9-27 T (= CECT 9745 T = CCM 8956 T ), isolated from the water of a pond from Isla Cristina saltern (Huelva, Spain).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA and rpoB' gene sequences of Halonotius roseus F9-27 T are MK478811 and MK491894, respectively, and that of the complete genome is SESI00000000.

Emended Description of the Genus
Halonotius Burns et al. (2010) Characteristics are as given in the description of the genus by Burns et al. (2010). The genus belongs phylogenetically to the family Halorubraceae, within the order Haloferacales, class Halobacteria. The major polar lipids are PG, PGP-Me, PGS and one glycolipid chromatographically identical to sulfated diglycosil diether (S-DGD-1). The DNA G + C content ranges from 59.5 to 62.7 mol% (genome).

DATA AVAILABILITY
The datasets generated for this study can be found in GenBank, RKLU00000000 and SESI00000000.

AUTHOR CONTRIBUTIONS
AV, CS-P, and RG conceived and designed the study. AD-V, CS-P, and AV designed and performed the acquisition of environmental isolates. AD-V performed the microbial experiments and obtained the genomes. AD-V performed the bioinformatic analyses under close guidance of A-SA and RG. AD-V, A-SA, RG, CS-P, and AV analyzed and interpreted the data. AD-V, CS-P, and AV drafted the manuscript. AD-V, A-SA, RG, CS-P, and AV critically revised the manuscript. All authors read and approved the final manuscript.

FUNDING
This study was supported by projects from the Spanish Ministry of Economy and Competitiveness (MINECO) (Project CGL2017-83385-P) and from the Junta de Andalucía (BIO-213), both with FEDER funds. AD-V was recipient of a predoctoral fellowship (FPU14/05128) from the Spanish Ministry of Education, Culture and Sports and of a shortstay FEMS research and training grant (FEMS-GO-2018-139). RG was supported by the research grant 17-04828S (Grant Agency of the Czech Republic). A-SA was supported by the research grants 17-04828 S (Grant Agency of the Czech Republic) and MSM200961801 (Academy of Sciences of the Czech Republic).