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The genus Natronomonas is classified on the family Haloarculaceae, within the class Halobacteria and currently includes six species isolated from salterns, saline or soda lakes, and salt mines. All are extremely halophilic (optimal growth at 20–25% [w/v] NaCl) and neutrophilic, except Natronomonas pharaonis, the type species of the genus, that is haloalkaliphilic (showing optimal growth at pH 9.0) and possesses distinct phenotypic features, such as a different polar lipid profile than the rest of species of the genus. We have carried out a genome-based study in order to determine the phylogenetic structure of the genus Natronomonas and elucidate its current taxonomic status. Overall genomic relatedness indexes, i.e., OrthoANI (Average Nucleotide Identity), dDDH (digital DNA–DNA hybridization), and AAI (Average Amino acid Identity), were determined with respect to the species of Natronomonas and other representative taxa of the class Halobacteria. Our data show that the six species of Natronomonas constitute a coherent cluster at the genus level. Besides, we have characterized a new haloarchaeon, strain F2-12T, isolated from the brine of a pond of a saltern in Isla Cristina, Huelva, Spain, and we determined that it constitutes a new species of Natronomonas, for which we propose the name Natronomonas aquatica sp. nov. Besides, the metabolic analysis revealed a heterotrophic lifestyle and a versatile nitrogen metabolism for members of this genus. Finally, metagenomic fragment recruitments from a subset of hypersaline habitats, indicated that the species of Natronomonas are widely distributed in saline lakes and salterns as well as on saline soils. Species of this haloarchaeal genus can be considered as ubiquitous in intermediate to high salinity habitats.

Metagenomic studies on prokaryotic diversity of hypersaline soils from the Odiel saltmarshes, South-west Spain, revealed a high proportion of genomic sequences not related to previously cultivated taxa, that might be related to haloarchaea with a high environmental and nutritional flexibility. In this study, we used a culturomics approach in order to isolate new haloarchaeal microorganisms from these hypersaline soils. Four haloarchaeal strains, designated strains F24AT, F28, F27T, and F13T, phylogenetically related to the genus Halomicroarcula, were isolated and characterized in detail. The phylogenomic tree based on the 100 orthologous single-copy genes present in the genomes of these four strains as well as those of the type strains of the species Halomicroarcula pellucida CECT 7537T, Halomicroarcula salina JCM 18369T and Halomicroarcula limicola JCM 18640T, that were determined in this study, revealed that these four new isolates clustered on three groups, with strains F24AT and F28 within a single cluster, and altogether with the species of Halomicroarcula. Additionally, Orthologous Average Nucleotide Identity (OrthoANI), digital DNA-DNA hybridization (dDDH) and Average Amino-acid Identity (AAI) values, likewise phenotypic characteristics, including their polar lipids profiles, permitted to determine that they represent three new species, for which we propose the names Halomicroarcula rubra sp. nov. (type strain F13T), Halomicroarcula nitratireducens sp. nov. (type strain F27T) and Halomicroarcula salinisoli sp. nov. (type strain F24AT). An in deep comparative genomic analysis of species of the genus Halomicroarcula, including their metabolism, their capability to biosynthesize secondary metabolites and their osmoregulatory adaptation mechanisms was carried out. Although they use a salt-in strategy, the identification of the complete pathways for the biosynthesis of the compatible solutes trehalose and glycine betaine, not identified before in any other haloarchaea, might suggest alternative osmoadaptation strategies for this group. This alternative osmoregulatory mechanism would allow this group of haloarchaea to be versatile and eco-physiologically successful in hypersaline environments and would justify the capability of the species of this genus to grow not only on environments with high salt concentrations [up to 30% (w/v) salts], but also under intermediate to low salinities.

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.

A halophilic archaeon, strain F20-122T, was isolated from a marine saltern of Isla Bacuta (Huelva, Spain). Cells were Gram-stain-negative, aerobic, and coccoid in morphology. It grew at 25–50 °C (optimum 37 °C), pH 6.5–9.0 (optimum pH 8.0), and 10–30% (w/v) total salts (optimum 25% salts). The phylogenetic analyses based on the 16S rRNA and rpoB’ genes showed its affiliation with the genus Natronomonas and suggested its placement as a new species within this genus. The in silico DNA–DNA hybridization (DDH) and average nucleotide identity (ANI) analyses of this strain against closely related species supported its placement in a new taxon. The DNA G + C content of this isolate was 63.0 mol%. The polar lipids of strain F20-122T were phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol (PG), and phosphatidylglycerol sulfate (PGS). Traces of biphosphatidylglycerol (BPG) and other minor phospholipids and unidentified glycolipids were also present. Based on the phylogenetic, genomic, phenotypic, and chemotaxonomic characterization, we propose strain F20-122T (= CCM 8891T = CECT 9564T = JCM 33320T) as the type strain of a new species within the genus Natronomonas, with the name Natronomonas salsuginis sp. nov. Rhodopsin-like sequence analysis of strain F20-122T revealed the presence of haloarchaeal proton pumps, suggesting a light-mediated ATP synthesis for this strain and a maximum wavelength absorption in the green spectrum.

A halophilic archaeal strain, designated F16-60T, was isolated from Isla Cristina marine saltern in Huelva, Spain. Cells were pleomorphic, irregular, non-motile, and Gram-stain-negative. It produced red-pigmented colonies on agar plates. Strain F16-60T was extremely halophilic (optimum at 30% (w/v) NaCl) and neutrophilic (optimum pH 7.5). Phylogenetic tree reconstructions based on 16S rRNA and rpoB´ gene sequences revealed that strain F16-60T was distinct from species of the related genera Natronomonas, Halomarina, and Halomicrobium, of the order Halobacteriales. The polar lipids are phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS), and one glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1). The DNA G+C content is 68.0 mol%. The taxonomic study, based on a combination of phylogenetic, genomic, chemotaxonomic, and phenotypic analyses, suggest that strain F16-60T (= CECT 9635T = JCM 33318T), represents a novel species of a new genus within the family Haloarculaceae and the order Halobacteriales, for which the name Haloglomus irregulare gen. nov., sp. nov. is proposed. Metagenomic fragment recruitment analysis revealed the worldwide distribution of members of this genus and suggested the existence of other closely related species to be isolated.