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 (F15B T and F9-27 T ) 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 7525 T , Halonotius aquaticus F13-13 T 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 F15B T = CECT 9688 T = CCM 8954 T ) and Halonotius roseus sp. nov. (type strain F9-27 T = CECT 9745 T = CCM 8956 T ) 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 B 12 ) 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 B 12 ) to the halophilic microbial communities at large.

Increasing salinization in wetland systems is a major threat to ecosystem services carried out by microbial communities. Thus, it is paramount to understand how salinity drives both microbial community structures and their diversity. Here we evaluated the structure and diversity of the prokaryotic communities from a range of highly saline soils (EC1:5 from 5.96 to 61.02 dS/m) from the Odiel Saltmarshes and determined their association with salinity and other soil physicochemical features by analyzing 16S rRNA gene amplicon data through minimum entropy decomposition (MED). We found that these soils harbored unique communities mainly composed of halophilic and halotolerant taxa from the phyla Euryarchaeota, Proteobacteria, Balneolaeota, Bacteroidetes and Rhodothermaeota. In the studied soils, several site-specific properties were correlated with community structure and individual abundances of particular sequence variants. Salinity had a secondary role in shaping prokaryotic communities in these highly saline samples since the dominant organisms residing in them were already well-adapted to a wide range of salinities. We also compared ESV-based results with OTU-clustering derived ones, showing that, in this dataset, no major differences in ecological outcomes were obtained by the employment of one or the other method.

The development of culture-independent techniques has revolutionized our understanding of microbial ecology, especially through the illustration of the vast gap between the environmentally abundant microbial diversity and that accessible through cultivation. However, culture-based approaches are not only crucial for understanding the evolutionary, metabolic and ecological milieu of microbial diversity but also for the development of novel biotechnological applications. In this study, we used a culturomics-based approach in order to isolate novel microbial taxa from hypersaline environments (i.e. Isla Cristina and Isla Bacuta salterns in Huelva, Spain). We managed to obtain axenic cultures of four haloarchaeal strains that belong to a new haloarchaeal genus and to obtain their genomic sequences. The phylogenomic and phylogenetic analyses (together with AAI, ANI and digital DDH indices) showed that the isolates constitute two new species, for which we propose the names Halosegnis longus sp. nov. and Halosegnis rubeus sp. nov. The genomic-based metabolic reconstructions indicated that members of this new haloarchaeal genus have photoheterotrophic aerobic lifestyle with a typical salt-in signature. 16S rRNA gene sequence reads abundance profiles and genomic recruitment analyses revealed that the Halosegnis genus has a worldwide geographical distribution, reaching high abundance (up to 8%) in habitats with intermediate salinities.

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.

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.