Search

Abstract The genus Spiribacter encompasses halophilic bacteria widely distributed in hypersaline environments worldwide. Despite their ecological significance, initially isolating Spiribacter species under laboratory settings was challenging due to the lack of knowledge of their growth and cultivation requirements. However, with improved understanding of their ecological niche and metabolic pathways, additional species of Spiribacter have been successfully isolated and identified from diverse locations around the globe. Enriched media with sodium pyruvate as carbon source facilitated the isolation of twelve new strains closely related to the genus Spiribacter from hypersaline environments in Spain. Genome sequencing and analysis of these new strains and previously described Spiribacter species provided insights into their genomic features and phylogenomic relationships, supporting the delineation of three distinct new species within this genus, designated as Spiribacter insolitus sp. nov., Spiribacter onubensis sp. nov., and Spiribacter pallidus sp. nov. In Spiribacter species, streamlined genomes enhance survival in hypersaline environments by reducing non-essential genes and optimizing resource utilization. Key genes involved in osmoprotectant mechanisms, including those for the metabolism of myo-inositol, hydroxyproline, and L-proline, were identified and numerous transporters were noted, ensuring efficient nutrient acquisition and osmotic balance. Notably, these new species, along with other Spiribacter strains, exhibit metabolic diversity in utilizing inorganic sulfur compounds, including thiosulfate and tetrathionate, for energy production and adaptation to hypersaline environments. The presence of thiosulfate dehydrogenase (TsdA) genes suggests their capability to oxidize thiosulfate to tetrathionate, potentially influencing both aerobic and anaerobic respiration. Furthermore, the prevalence of the sqr gene indicates a role for sulfide oxidation in Spiribacter metabolism, underlining their metabolic versatility in saline habitats. These adaptations allow Spiribacter to thrive in nutrient-limited, high-salinity habitats. Moreover, genome mining analysis and physiological disparities observed in the already described species Spiribacter halobius raise significant challenges to its classification within the genus Spiribacter.

Recent advances in molecular and metagenomic analyses have enhanced the ability to precisely determine the microbiota of hypersaline environments of marine origin, such as solar salterns, saline lakes, and hypersaline soils, uncovering numerous yet-to-be-isolated prokaryotic groups. Our research focused on the hypersaline ecosystems within the Odiel Saltmarshes, a natural tidal wetland situated at the confluence of the Odiel and Tinto rivers in Huelva province, Southwestern Spain. Employing culture-dependent techniques, we aimed to isolate and characterize novel halophilic prokaryotes from this area. Two haloarchaeal strains, designated S1BR25-6T and S3BR25-2T were classified within the genus Halogeometricum based on Overall Genome Related Indexes (OGRIs) such as Orthologous Average Nucleotide Identity, digital DNA-DNA hybridization, and Average Amino Acid Identity as standard criteria for species delineation. Moreover, this study embarks on an exhaustive genome-based comparative analysis of the haloarchaeal genus Halogeometricum, delineating the metabolic capacities, osmoregulatory adaptations, and resistance to certain heavy metals of its species. The dual osmoregulatory mechanism observed by in-silico analysis of the Halogeometricum species combines “salt-in” and “salt-out” strategies which highlights the adaptive flexibility of these haloarchaea. In addition, capability for de novo thiamine biosynthesis of strain S1BR25-6T along with other Halogeometricum species underscores their metabolic complexity and resilience, offering insights into their role in ecosystem dynamics and potential biotechnological applications. Wet lab experimental analysis of strains S1BR25-6T and S3BR25-2T confirmed their resistance to heavy metals, particularly to arsenic, zinc, and cadmium, emphasizing their potential for bioremediation applications. Furthermore, conducting fragment recruitment analysis across different metagenomic datasets revealed a predominant recruitment of species from the genus Halogeometricum in hypersaline soils of Odiel Saltmarshes (especially the two novel strains), and in the brines of marine saltern ponds with high salt concentrations. These results contribute to a reinforced understanding of the extremely halophilic characteristics inherent to the genus Halogeometricum. Finally, taxogenomic analysis has substantiated that strains S1BR25-6T (= CCM 9250T = CECT 30624T), and S3BR25-2T (= CCM 9253T = CECT 30622T) denote two previously unidentified species within the genus Halogeometricum, for which we propose the names Halogeometricum salsisoli sp. nov., and Halogeometricum luteum sp. nov., respectively.

The family Halomonadaceae is the largest family composed of halophilic bacteria, with more than 160 species with validly published names as of July 2023. Several classifications to circumscribe this family are available in major resources, such as those provided by the List of Prokaryotic names with Standing in Nomenclature (LPSN), NCBI Taxonomy, Genome Taxonomy Database (GTDB), and Bergey’s Manual of Systematics of Archaea and Bacteria (BMSAB), with some degree of disagreement between them. Moreover, regardless of the classification adopted, the genus Halomonas is not phylogenetically consistent, likely because it has been used as a catch-all for newly described species within the family Halomonadaceae that could not be clearly accommodated in other Halomonadaceae genera. In the past decade, some taxonomic rearrangements have been conducted on the Halomonadaceae based on ribosomal and alternative single-copy housekeeping gene sequence analysis. High-throughput technologies have enabled access to the genome sequences of many type strains belonging to the family Halomonadaceae; however, genome-based studies specifically addressing its taxonomic status have not been performed to date. In this study, we accomplished the genome sequencing of 17 missing type strains of Halomonadaceae species that, together with other publicly available genome sequences, allowed us to re-evaluate the genetic relationship, phylogeny, and taxonomy of the species and genera within this family. The approach followed included the estimate of the Overall Genome Relatedness Indexes (OGRIs) such as the average amino acid identity (AAI), phylogenomic reconstructions using amino acid substitution matrices customized for the family Halomonadaceae, and the analysis of clade-specific signature genes. Based on our results, we conclude that the genus Halovibrio is obviously out of place within the family Halomonadaceae, and, on the other hand, we propose a division of the genus Halomonas into seven separate genera and the transfer of seven species from Halomonas to the genus Modicisalibacter, together with the emendation of the latter. Additionally, data from this study demonstrate the existence of various synonym species names in this family.

The genus Halomicroarcula, classified within the family Haloarculaceae, presently comprises eight haloarchaeal species isolated from diverse saline habitats, such as solar salterns, hypersaline soils, marine salt, and marine algae. Here, a detailed taxogenomic study and comparative genomic analysis of the genus Halomicroarcula was carried out. In addition, two strains, designated S1CR25-12T and S3CR25-11T, that were isolated from hypersaline soils located in the Odiel Saltmarshes in Huelva (Spain) were included in this study. The 16S rRNA and rpoB’ gene sequence analyses affiliated the two strains to the genus Halomicroarcula. Typically, the species of the genus Halomicroarcula possess multiple heterogeneous copies of the 16S rRNA gene, which can lead to misclassification of the taxa and overestimation of the prokaryotic diversity. In contrast, the application of overall genome relatedness indexes (OGRIs) augments the capacity for the precise taxonomic classification and categorization of prokaryotic organisms. The relatedness indexes of the two new isolates, particularly digital DNA–DNA hybridization (dDDH), orthologous average nucleotide identity (OrthoANI), and average amino acid identity (AAI), confirmed that strains S1CR25-12T (= CECT 30620T = CCM 9252T) and S3CR25-11T (= CECT 30621T = CCM 9254T) constitute two novel species of the genus Halomicroarcula. The names Halomicroarcula saliterrae sp. nov. and Halomicroarcula onubensis sp. nov. are proposed for S1CR25-12T and S3CR25-11T, respectively. Metagenomic fragment recruitment analysis, conducted using seven shotgun metagenomic datasets, revealed that the species belonging to the genus Halomicroarcula were predominantly recruited from hypersaline soils found in the Odiel Saltmarshes and the ponds of salterns with high salt concentrations. This reinforces the understanding of the extreme halophilic characteristics associated with the genus Halomicroarcula. Finally, comparing pan-genomes across the twenty Halomicroarcula and Haloarcula species allowed for the identification of commonalities and differences between the species of these two related genera.

The hypersaline soils of the Odiel Saltmarshes Natural Area are an extreme environment with high levels of some heavy metals; however, it is a relevant source of prokaryotic diversity that we aim to explore. In this study, six strains related to the halophilic genus Pseudidiomarina were isolated from this habitat. The phylogenetic study based on the 16S rRNA gene sequence and the fingerprinting analysis suggested that they constituted a single new species within the genus Pseudidiomarina. Comparative genomic analysis based on the OGRIs indices and the phylogeny inferred from the core genome were performed considering all the members of the family Idiomarinaceae. Additionally, a completed phenotypic characterization, as well as the fatty acid profile, were also carried out. Due to the characteristics of the habitat, genomic functions related to salinity and high heavy metal concentrations were studied, along with the global metabolism of the six isolates. Last, the ecological distribution of the isolates was studied in different hypersaline environments by genome recruitment. To sum up, the six strains constitute a new species within the genus Pseudidiomarina, for which the name Pseudidiomarina terrestris sp. nov. is proposed. The low abundance in all the studied hypersaline habitats indicates that it belongs to the rare biosphere in these habitats. In silico genome functional analysis suggests the presence of heavy metal transporters and pathways for nitrate reduction and nitrogen assimilation in low availability, among other metabolic traits.

Hypersaline soils are a source of prokaryotic diversity that has been overlooked until very recently. The phylum Bacillota, which includes the genus Aquibacillus, is one of the 26 phyla that inhabit the heavy metal contaminated soils of the Odiel Saltmarshers Natural Area (Southwest Spain), according to previous research. In this study, we isolated a total of 32 strains closely related to the genus Aquibacillus by the traditional dilution-plating technique. Phylogenetic studies clustered them into two groups, and comparative genomic analyses revealed that one of them represents a new species within the genus Aquibacillus, whereas the other cluster constitutes a novel genus of the family Bacillaceae. We propose the designations Aquibacillus salsiterrae sp. nov. and Terrihalobacillus insolitus gen. nov., sp. nov., respectively, for these two new taxa. Genome mining analysis revealed dissimilitude in the metabolic traits of the isolates and their closest related genera, remarkably the distinctive presence of the well-conserved pathway for the biosynthesis of molybdenum cofactor in the species of the genera Aquibacillus and Terrihalobacillus, along with genes that encode molybdoenzymes and molybdate transporters, scarcely found in metagenomic dataset from this area. In-silico studies of the osmoregulatory strategy revealed a salt-out mechanism in the new species, which harbor the genes for biosynthesis and transport of the compatible solutes ectoine and glycine betaine. Comparative genomics showed genes related to heavy metal resistance, which seem required due to the contamination in the sampling area. The low values in the genome recruitment analysis indicate that the new species of the two genera, Terrihalobacillus and Aquibacillus, belong to the rare biosphere of representative hypersaline environments.

ABSTRACT The class Halobacteria is one of the most diverse groups within the Euryarchaeota phylum, whose members are ubiquitously distributed in hypersaline environments, where they often constitute the major population. Here, we report the discovery and isolation of a new halophilic archaeon, strain F3-133T exhibiting ≤86.3% 16S rRNA gene identity to any previously cultivated archaeon, and, thus, representing a new order. Analysis of available 16S rRNA gene amplicon and metagenomic data sets showed that the new isolate represents an abundant group in intermediate-to-high salinity ecosystems and is widely distributed across the world. The isolate presents a streamlined genome, which probably accounts for its ecological success in nature and its fastidious growth in culture. The predominant osmoprotection mechanism appears to be the typical salt-in strategy used by other haloarchaea. Furthermore, the genome contains the complete gene set for nucleotide monophosphate degradation pathway through archaeal RuBisCO, being within the first halophilic archaea representatives reported to code this enzyme. Genomic comparisons with previously described representatives of the phylum Euryarchaeota were consistent with the 16S rRNA gene data in supporting that our isolate represents a novel order within the class Halobacteria for which we propose the names Halorutilales ord. nov., Halorutilaceae fam. nov., Halorutilus gen. nov. and Halorutilus salinus sp. nov. IMPORTANCE The discovery of the new halophilic archaeon, Halorutilus salinus, representing a novel order, family, genus, and species within the class Halobacteria and phylum Euryarchaeota clearly enables insights into the microbial dark matter, expanding the current taxonomical knowledge of this group of archaea. The in-depth comparative genomic analysis performed on this new taxon revealed one of the first known examples of an Halobacteria representative coding the archaeal RuBisCO gene and with a streamlined genome, being ecologically successful in nature and explaining its previous non-isolation. Altogether, this research brings light into the understanding of the physiology of the Halobacteria class members, their ecological distribution, and capacity to thrive in hypersaline environments.

The fraction of low-abundance microbiota in the marine environment is a promising target for discovering new bioactive molecules with pharmaceutical applications. Phenomena in the ocean such as diel vertical migration (DVM) and seasonal dynamic events influence the pattern of diversity of marine bacteria, conditioning the probability of isolation of uncultured bacteria. In this study, we report a new marine bacterium belonging to the rare biosphere, Leeuwenhoekiella parthenopeia sp. nov. Mr9T, which was isolated employing seasonal and diel sampling approaches. Its complete characterization, ecology, biosynthetic gene profiling of the whole genus Leeuwenhoekiella, and bioactivity of its extract on human cells are reported. The phylogenomic and microbial diversity studies demonstrated that this bacterium is a new and rare species, barely representing 0.0029% of the bacterial community in Mediterranean Sea metagenomes. The biosynthetic profiling of species of the genus Leeuwenhoekiella showed nine functionally related gene cluster families (GCF), none were associated with pathways responsible to produce known compounds or registered patents, therefore revealing its potential to synthesize novel bioactive compounds. In vitro screenings of L. parthenopeia Mr9T showed that the total lipid content (lipidome) of the cell membrane reduces the prostatic and brain tumor cell viability with a lower effect on normal cells. The lipidome consisted of sulfobacin A, WB 3559A, WB 3559B, docosenamide, topostin B-567, and unknown compounds. Therefore, the bioactivity could be attributed to any of these individual compounds or due to their synergistic effect. Beyond the rarity and biosynthetic potential of this bacterium, the importance and novelty of this study is the employment of sampling strategies based on ecological factors to reach the hidden microbiota, as well as the use of bacterial membrane constituents as potential novel therapeutics. Our findings open new perspectives on cultivation and the relationship between bacterial biological membrane components and their bioactivity in eukaryotic cells, encouraging similar studies in other members of the rare biosphere.

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.

Hypersaline soils are extreme environments that have received little attention until the last few years. Their halophilic prokaryotic population seems to be more diverse than those of well-known aquatic systems. Among those inhabitants, representatives of the family Balneolaceae (phylum Balneolota) have been described to be abundant, but very few members have been isolated and characterized to date. This family comprises the genera Aliifodinibius and Fodinibius along with four others. A novel strain, designated 1BSP15-2V2T, has been isolated from hypersaline soils located in the Odiel Saltmarshes Natural Area (Southwest Spain), which appears to represent a new species related to the genus Aliifodinibius. However, comparative genomic analyses of members of the family Balneolaceae have revealed that the genera Aliifodinibius and Fodinibius belong to a single genus, hence we propose the reclassification of the species of the genus Aliifodinibius into the genus Fodinibius, which was first described. The novel strain is thus described as Fodinibius salsisoli sp. nov., with 1BSP15-2V2T (=CCM 9117T = CECT 30246T) as the designated type strain. This species and other closely related ones show abundant genomic recruitment within 80–90% identity range when searched against several hypersaline soil metagenomic databases investigated. This might suggest that there are still uncultured, yet abundant closely related representatives to this family present in these environments. In-depth in-silico analysis of the metabolism of Fodinibius showed that the biotin biosynthesis pathway was present in the genomes of strain 1BSP15-2V2T and other species of the family Balneolaceae, which could entail major implications in their community role providing this vitamin to other organisms that depend on an exogenous source of this nutrient.

Hypersaline environments are mainly represented by aquatic systems, such as solar salt ponds or natural salt lakes, as well as by the sediments of these hypersaline aquatic ecosystems and soils with high salt content [...]

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.

Actinobacteria are prokaryotes with a large biotechnological interest due to their ability to produce secondary metabolites, produced by two main biosynthetic gene clusters (BGCs): polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS). Most studies on bioactive products have been carried out on actinobacteria isolated from soil, freshwater or marine habitats, while very few have been focused on halophilic actinobacteria isolated from extreme environments. In this study we have carried out a comparative genomic analysis of the actinobacterial genus Saccharomonospora, which includes species isolated from soils, lake sediments, marine or hypersaline habitats. A total of 19 genome sequences of members of Saccharomonospora were retrieved and analyzed. We compared the 16S rRNA gene-based phylogeny of this genus with evolutionary relationships inferred using a phylogenomic approach obtaining almost identical topologies between both strategies. This method allowed us to unequivocally assign strains into species and to identify some taxonomic relationships that need to be revised. Our study supports a recent speciation event occurring between Saccharomonospora halophila and Saccharomonospora iraqiensis. Concerning the identification of BGCs, a total of 18 different types of BGCs were detected in the analyzed genomes of Saccharomonospora, including PKS, NRPS and hybrid clusters which might be able to synthetize 40 different putative products. In comparison to other genera of the Actinobacteria, members of the genus Saccharomonospora showed a high degree of novelty and diversity of BGCs.

In the genomic era phylogenetic relationship among prokaryotes can be inferred from the core orthologous genes (OGs) or proteins in order to elucidate their evolutionary history and current taxonomy should benefits of that. The genus Salinivibrio belongs to the family Vibrionaceae and currently includes only five halophilic species, in spite the fact that new strains are very frequently isolated from hypersaline environments. Species belonging to this genus have undergone several reclassifications and, moreover, there are many strains of Salinivibrio with available genomes which have not been affiliated to the existing species or have been wrongly designated. Therefore, a phylogenetic study using the available genomic information is necessary to clarify the relationships of existing strains within this genus and to review their taxonomic affiliation. For that purpose, we have also sequenced the first complete genome of a Salinivibrio species, Salinivibrio kushneri AL184T, which was employed as a reference to order the contigs of the draft genomes of the type strains of the current species of this genus, as well as to perform a comparative analysis with all the other available Salinivibrio sp. genomes. The genome of S. kushneri AL184T was assembled in two circular chromosomes (with sizes of 2.84 Mb and 0.60 Mb, respectively), as typically occurs in members of the family Vibrionaceae, with nine complete ribosomal operons, which might explain the fast growing rate of salinivibrios cultured under laboratory conditions. Synteny analysis among the type strains of the genus revealed a high level of genomic conservation in both chromosomes, which allow us to hypothesize a slow speciation process or homogenization events taking place in this group of microorganisms to be tested experimentally in the future. Phylogenomic and orthologous average nucleotide identity (OrthoANI)/average amino acid identity (AAI) analyses also evidenced the elevated level of genetic relatedness within members of this genus and allowed to group all the Salinivibrio strains with available genomes in seven separated species. Genome-scale attribute study of the salinivibrios identified traits related to polar flagellum, facultatively anaerobic growth and osmotic response, in accordance to the phenotypic features described for species of this genus.

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.

The genus Cyclobacterium belongs to the phylum Bacteroidetes and includes eight species. Our study, based on the genomic parameters in silico DNA–DNA hybridization (GGDC), average nucleotide identity (OrthoANI), and average amino acid identity (AAI), confirmed that all current species of Cyclobacterium belong to this genus and constitute a coherent phylogenomic group, but with species forming two separate branches. In addition, the genome-based analyses revealed that Cyclobacterium xiamenense and Cyclobacterium halophilum are members of the same species. Besides, we carried out a taxonomic characterization of the new strain GBPx2T, isolated from the halophytic plant Salicornia sp. Analysis of its 16S rRNA gene sequence showed the highest sequence similarity (97.5%) to Cyclobacterium lianum HY9T. Percentages of GGDC and OrthoANI between strain GBPx2T and species of the genus Cyclobacterium were lower than the threshold value for species delineation. The DNA G+C content was 43.0 mol%. The polar lipids included phosphatidylethanolamine as well as one unidentified phospholipid and four unidentified lipids, and its major cellular fatty acids were iso-C15:0 and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). The only quinone present was menaquinone 7. Based on a combination of phenotypic, chemotaxonomic, and phylogenomic features, the GBPx2T strain represents a novel species of the genus Cyclobacterium, for which the name Cyclobacterium plantarum sp. nov. is proposed. The type strain of Cyclobacterium plantarum is GBPx2T (= IBRC-M 10634T = LMG 28551T).

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.

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related Halorubrum strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge.

Members of the genus Spiribacter are found worldwide and are abundant in ecosystems possessing intermediate salinities between seawater and saturated salt concentrations. Spiribacter salinus M19-40 is the type species of this genus and its first cultivated representative. In the habitats of S. salinus M19-40, high salinity is a key determinant for growth and we therefore focused on the cellular adjustment strategy to this persistent environmental challenge. We coupled these experimental studies to the in silico mining of the genome sequence of this moderate halophile with respect to systems allowing this bacterium to control its potassium and sodium pools, and its ability to import and synthesize compatible solutes. S. salinus M19-40 produces enhanced levels of the compatible solute ectoine, both under optimal and growth-challenging salt concentrations, but the genes encoding the corresponding biosynthetic enzymes are not organized in a canonical ectABC operon. Instead, they are scrambled (ectAC; ectB) and are physically separated from each other on the S. salinus M19-40 genome. Genomes of many phylogenetically related bacteria also exhibit a non-canonical organization of the ect genes. S. salinus M19-40 also synthesizes trehalose, but this compatible solute seems to make only a minor contribution to the cytoplasmic solute pool under osmotic stress conditions. However, its cellular levels increase substantially in stationary phase cells grown under optimal salt concentrations. In silico genome mining revealed that S. salinus M19-40 possesses different types of uptake systems for compatible solutes. Among the set of compatible solutes tested in an osmostress protection growth assay, glycine betaine and arsenobetaine were the most effective. Transport studies with radiolabeled glycine betaine showed that S. salinus M19-40 increases the pool size of this osmolyte in a fashion that is sensitively tied to the prevalent salinity of the growth medium. It was amassed in salt-stressed cells in unmodified form and suppressed the synthesis of ectoine. In conclusion, the data presented here allow us to derive a genome-scale picture of the cellular adjustment strategy of a species that represents an environmentally abundant group of ecophysiologically important halophilic microorganisms.

The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria.

The genus Salinivibrio includes obligatory halophilic bacteria and is commonly isolated from hypersaline habitats and salted food products. They grow optimally between 7.5 and 10% salts and are facultative anaerobes. Currently, this genus comprises four species, one of them, S. costicola, with three subspecies. In this study we isolated and characterized an additional 70 strains from solar salterns located in different locations. Comparative 16S rRNA gene sequence analysis identified these strains as belonging to the genus Salinivibrio but could not differentiate strains into species-like groups. To achieve finer phylogenetic resolution, we carried out a MultiLocus Sequence Analysis (MLSA) of the new isolates and the type strains of the species of Salinivibrio based on the individual as well as concatenated sequences of four housekeeping genes: gyrB, recA, rpoA, and rpoD. The strains formed four clearly differentiated species-like clusters called phylogroups. All of the known type and subspecies strains were associated with one of these clusters except S. sharmensis. One phylogroup had no previously described species coupled to it. Further DNA–DNA hybridization (DDH) experiments with selected representative strains from these phylogroups permitted us to validate the MLSA study, correlating the species level defined by the DDH (70%) with a 97% cut-off for the concatenated MLSA gene sequences. Based on these criteria, the novel strains forming phylogroup 1 could constitute a new species while strains constructing the other three phylogroups are members of previously recognized Salinivibrio species. S. costicola subsp. vallismortis co-occurs with S. proteolyticus in phylogroup 4, and separately from other S. costicola strains, indicating its need for reclassification. On the other hand, genome fingerprinting analysis showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. In future studies regarding the classification and identification of new Salinivibrio strains we recommend the following strategy: (i) initial partial sequencing of the 16S rRNA gene for genus-level identification; (ii) sequencing and concatenation of the four before mentioned housekeeping genes for species-level discrimination; (iii) DDH experiments, only required when the concatenated MLSA similarity values among a new isolate and other Salinivibrio strains are above the 97% cut-off.

The genus Salinivibrio belongs to the family Vibrionaceae and includes Gram-stain-negative, motile by a polar flagellum, and facultatively anaerobic curved rods. They are halophilic bacteria commonly found in hypersaline aquatic habitats and salted foods. This genus includes five species and two subspecies. A presumed novel species, strain S35T, was previously isolated from the high-altitude volcanic, alkaline, and saline lake Socompa (Argentinean Andes). In this study we carried out a complete taxonomic characterization of strain S35T, including the 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANIb, ANIm, and orthoANI), and in silico DNA−DNA hybridization (GGDC), as well as the phenotypic and chemotaxonomic characterization. It grew at 3%−20% (w/v) NaCl, pH 6−10, and 10−42 °C, with optimum growth at 7.0%−7.5% (w/v) NaCl, pH 8.0, and 37 °C, respectively. Strain S35T was oxidase- and catalase-positive, able to produce acid from D-glucose and other carbohydrates. Hydrolysis of DNA, methyl red test, and nitrate and nitrite reduction were positive. Its main fatty acids were C16:0, C16:1 ω7c and C16:1 ω6c, and C18:1 ω7c and/or C18:1 ω6c. ANI, GGDC, and core-genome analysis determined that strain S35T constitutes a novel species of the genus Salinivibrio, for which the name Salinivibrio socompensis sp. nov. is proposed. The type strain is S35T (= CECT 9634T = BNM 0535T).

Five strains (1126-1H-08(T), 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a temperature range between 15°C to 30°C, but they did not grow at 37°Cor 42°C. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08(T) exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678(T) and Flavobacterium pectinovorum DSM 6368(T) (98.5% and 97.9% sequence similarity, respectively). DNA-DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H-08(T), selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678(T) and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08(T) was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C15∶0 and C15∶0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126-1H-08(T) ( = CECT 7844(T) = CCUG 60112(T)).

BackgroundExtensive use of phenolic compounds in industry has resulted in the generation of saline wastewaters that produce significant environmental contamination; however, little information is available on the degradation of phenolic compounds in saline conditions. Halomonas organivorans G-16.1 (CECT 5995(T)) is a moderately halophilic bacterium that we isolated in a previous work from saline environments of South Spain by enrichment for growth in different pollutants, including phenolic compounds. PCR amplification with degenerate primers revealed the presence of genes encoding ring-cleaving enzymes of the β-ketoadipate pathway for aromatic catabolism in H. organivorans.FindingsThe gene cluster catRBCA, involved in catechol degradation, was isolated from H. organivorans. The genes catA, catB, catC and the divergently transcribed catR code for catechol 1,2-dioxygenase (1,2-CTD), cis,cis-muconate cycloisomerase, muconolactone delta-isomerase and a LysR-type transcriptional regulator, respectively. The benzoate catabolic genes (benA and benB) are located flanking the cat genes. The expression of cat and ben genes by phenol and benzoic acid was shown by RT-PCR analysis. The induction of catA gene by phenol and benzoic acid was also probed by the measurement of 1,2-CTD activity in H. organivorans growth in presence of these inducers. 16S rRNA and catA gene-based phylogenies were established among different degrading bacteria showing no phylogenetic correlation between both genes.Conclusions/significanceIn this work, we isolated and determined the sequence of a gene cluster from a moderately halophilic bacterium encoding ortho-pathway genes involved in the catabolic metabolism of phenol and analyzed the gene organization, constituting the first report characterizing catabolic genes involved in the degradation of phenol in moderate halophiles, providing an ideal model system to investigate the potential use of this group of extremophiles in the decontamination of saline environments.