Reclassification of Halomicroarcula saliterrae Straková et al. 2024 and Halomicroarcula onubensis Straková et al. 2024 into the genus Haloarcula, as Haloarcula saliterrae comb. nov. and Haloarcula onubensis comb. nov., respectively.

The haloarchaeal genera Halomicroarcula and Haloarcula, belonging to the family Haloarculaceae, order Halobacteriales, class Halobacteria, within the phylum Methanobacteriota, have previously exhibited significant phylogenetic and taxonomic overlaps. This issue was recently resolved by merging the two genera into a single genus, Haloarcula. However, Halomicroarcula saliterrae and Halomicroarcula onubensis were described almost simultaneously with the proposal to unify the genera Haloarcula and Halomicroarcula. Their names were validly published under the International Code of Nomenclature of Prokaryotes (ICNP) according to Validation List no. 217, alongside six Haloarcula species and the transfer of the existing Halomicroarcula species into the genus Haloarcula. Therefore a phylogenetic, phylogenomic, and comparative genomic analysis was carried out to clarify the taxonomic status of these two haloarchaeal species, Halomicroarcula saliterrae and Halomicroarcula onubensis, with lower priority than the six new species of the genus Haloarcula. Phylogenetic studies of 16S rRNA and rpoB' gene sequences, along with phylogenomic reconstructions using single-copy core-orthologous proteins, indicated that the two species clustered with the members of the genus Haloarcula. The overall genome relatedness indexes (OGRIs), comparative analyses of phenotypic features, and polar lipid profiles further supported their taxonomic reassignment as two separate species within the genus Haloarcula. Consequently, we propose the reclassification of Halomicroarcula saliterrae Straková et al. 2024 and Halomicroarcula onubensis Straková et al. 2024 into the genus Haloarcula, as Haloarcula saliterrae comb. nov. and Haloarcula onubensis comb. nov., respectively, in accordance with the ICNP.

Halobacterium hubeiense sp. nov., a haloarchaeal species isolated from a bore core drilled in Hubei Province, PR China.

Eight colonies of live microbes were isolated from an extensively surface-sterilized halite sample which had been retrieved from a depth of 2000 m from a salt mine in the Qianjiang Depression, Hubei Province, PR China. The eight colonies, obtained after 4 weeks of incubation, were named JI20-1T-JI20-8 and JI20-1T was selected as the type strain. The strains have been previously described, including a genomic analysis based on the complete genome for strain JI20-1T and draft genomes for the other strains. In that study, the name Halobacterium hubeiense was suggested, based on the location of the drilling site. Previous phylogenomic analysis showed that strain JI20-1T is most closely related to the Permian isolate Halobacterium noricense from Alpine rock salt. The orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridization (dDDH) percentages between the eight strains are 100-99.6 % and 99.8-96.4 %, respectively. The orthoANI and dDDH values of these strains with respect to the type strains of species of the genus Halobacterium are 89.9-78.2 % and 37.3-21.6 %, respectively, supporting their placement in a novel extremely halophilic archaeal species. The phylogenomic tree based on the comparison of sequences of 632 core-orthologous proteins confirmed the novel species status for these haloarchaea. The polar lipid profile includes phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and sulfated galactosyl mannosyl galactosyl glucosyl diether, a profile compatible with that of Halobacterium noricense. Based on genomic, phenotypic, and chemotaxonomic characterization, we propose strain JI20-1T (=DSM 114402T = HAMBI 3616T) as the type strain of a novel species in the genus Halobacterium, with the name Halobacterium hubeiense sp. nov.

Proposed minimal standards for description of new taxa of the class Halobacteria.

Halophilic archaea of the class Halobacteria are the most salt-requiring prokaryotes within the domain Archaea. In 1997, minimal standards for the description of new taxa in the order Halobacteriales were proposed. From then on, the taxonomy of the class Halobacteria provides an excellent example of how changing concepts on prokaryote taxonomy and the development of new methods were implemented. The last decades have witnessed a rapid expansion of the number of described taxa within the class Halobacteria coinciding with the era of genome sequencing development. The current members of the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteria propose these revisions to the recommended minimal standards and encourage the use of advanced technologies in the taxonomic description of members of the Halobacteria. Most previously required and some recommended minimal standards for the description of new taxa in the class Halobacteria were retained in the present revision, but changes have been proposed in line with the new methodologies. In addition to the 16S rRNA gene, the rpoB' gene is an important molecular marker for the identification of members of the Halobacteria. Phylogenomic analysis based on concatenated conserved, single-copy marker genes is required to infer the taxonomic status of new taxa. The overall genome relatedness indexes have proven to be determinative in the classification of the taxa within the class Halobacteria. Average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values should be calculated for rigorous comparison among close relatives.

Natrinema salsiterrestre sp. nov., an extremely halophilic archaeon isolated from a hypersaline soil.

An extremely halophilic archaeal strain, designated S1CR25-10T, was isolated from hypersaline soil sampled in the Odiel Saltmarshes Natural Area in Southwestern Spain (Huelva) and subjected to a polyphasic taxonomic characterization. The cells were Gram-stain-negative, motile and their colonies were pink-pigmented. It was a strictly aerobic haloarchaeon that could grow at 25-55 °C (optimum, 37 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 12-30 % (w/v) total salts (optimum, 20-25 %, w/v). The phylogenetic analysis based on the comparison of the 16S rRNA gene sequences revealed that strain S1CR25-10T belongs to the genus Natrinema, with 98.9 % similarity to Natrinema salinisoli SLN56T. In addition, the values of orthologous average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity were below the threshold limits accepted for prokaryotic species delineation, with N. salinisoli SLN56T showing the highest relatedness values (92.6 % and 48.4 %, respectively). The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a glycolipid chromatographically identical to sulfated diglycosyl diether. The DNA G+C content of the isolate was 63.8 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic characterization and the whole genome results, strain S1CR25-10T represents a new species within the genus Natrinema, for which the name Natrinema salsiterrestre sp. nov., with type strain S1CR25-10T (=CECT 30623T=CCM 9251T), is proposed.

Characterization of Haloarcula terrestris sp. nov. and reclassification of a Haloarcula species based on a taxogenomic approach.

An extremely halophilic archaeon, strain S1AR25-5AT, was isolated from a hypersaline soil sampled in Odiel Saltmarshes Natural Area (Huelva, Spain). The cells were Gram-stain-negative, motile, pleomorphic rods. Cell growth was observed in the presence of 15-30 % (w/v) NaCl [optimum, 25 % (w/v) NaCl], at pH 6.0-9.0 (optimum, pH 6.5-7.5) and at 25-50 °C (optimum, 37 °C). Based on the 16S rRNA and rpoB' gene sequence comparisons, strain S1AR25-5AT was affiliated to the genus Haloarcula. Taxogenomic analysis, including comparison of the genomes and the phylogenomic tree based on the core-orthologous proteins, together with the genomic indices, i.e., orthologous average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, confirmed that strain S1AR25-5AT (=CCM 9249T=CECT 30619T) represents a new species of the genus Haloarcula, for which we propose the name Haloarcula terrestris sp. nov. The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate and an unidentified glycolipid, which correlated with the lipid profile of species of the genus Haloarcula. In addition, based on the modern approach in description of species in taxonomy of prokaryotes, the above mentioned genomic indexes indicated that the species Haloarcula tradensis should be considered as a heterotypic synonym of Haloarcula argentinensis.

Helicobacter ibis sp. nov., isolated from faecal droppings of black-faced ibis (Theristicus melanopis).

As part of a larger study on Epsilonproteobacteria carried by wild birds in the city of Valdivia (southern Chile), two curved rod-shaped Gram-stain-negative strains (A82T and WB-40) were recovered from faecal samples and subjected to a taxonomic study. Results of a genus-specific PCR showed that these isolates belonged to the genus Helicobacter. Further identification by 16S rRNA and hsp60 (60 kDa heat-shock protein) gene sequence analysis revealed that they formed a separate phylogenetic clade, different from other known Helicobacter species with 'Helicobacter burdigaliensis' CNRCH 2005/566HT and Helicobacter valdiviensis WBE14T being the most closely related species. This was confirmed by core-genome phylogeny as well as digital DNA-DNA hybridization and average nucleotide identity analyses between the genomes of strains A82T and WB-40 and all other Helicobacter species. The draft genome sequences of A82T and WB-40, obtained by Illumina NextSeq 2000 sequencing, consisted of 1.6 Mb with a G+C content of 31.9-32.0 mol%. The results obtained from the phylogenetic and genomic characterization, together with their different morphological and biochemical features, revealed that these two strains represent a novel species, for which we propose the name Helicobacter ibis sp. nov. with A82T (=LMG 32718T=CCCT 22.04T) as the type strain.

Genome-based analyses reveal a synonymy among Halorubrum distributum Zvyagintseva and Tarasov 1989; Oren and Ventosa 1996, Halorubrum terrestre Ventosa et al. 2004, Halorubrum arcis Xu et al. 2007 and Halorubrum litoreum Cui et al. 2007. Emended description of Halorubrum distributum Zvyagintseva and Tarasov 1989; Oren and Ventosa 1996.

A comparative taxonomic study of Halorubrum distributum, Halorubrum terrestre, Halorubrum arcis and Halorubrum litoreum was carried out using different approaches, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), phylogenomic analysis based on the comparison of the core genome, orthologous average nucleotide identity (OrthoANI), Genome-to-Genome Distance Calculator (GGDC), synteny plots and polar lipid profile (PLP). The MLSA study, using the five concatenated housekeeping genes atpB, EF-2, glnA, ppsA and rpoB', and the phylogenomic analysis based on 1347 core translated gene sequences obtained from their genomes showed that Halorubrum distributum JCM 9100T, Halorubrum terrestre JCM 10247T, Halorubrum arcis JCM 13916T and Halorubrum litoreum JCM 13561T formed a robust cluster, clearly separated from the rest of species of the genus Halorubrum. The OrthoANI and digital DDH values, calculated by the GGDC, showed percentages among Hrr. distributum JCM 9100T, Hrr. terrestre JCM 10247T, Hrr. arcis JCM 13916T and Hrr. litoreum JCM 13561T that ranged from 98.1 to 97.5 %, and 84.0 to 78.0 %, respectively, while these values among those strains and the type strains of their most related species of Halorubrum were equal or lower than 90.8 and 41.2 %, respectively. Moreover, degree of synteny across the four genomes was very high, especially between the genomes of Halorubrum litoreum JCM 13561T and Halorubrum arcis JCM 13916T. In addition, the PLP is quite similar among the four strains studied, showing a common pattern typical of the neutrophilic species of the genus Halorubrum. Overall, these data show that Hrr. distributum, Hrr. terrestre, Hrr. arcis and Hrr. litoreum constitute a single species. Thus, the latter three should be considered as later, heterotypic synonyms of Hrr. distributum based on the rules for priority of names. We propose an emended description of Hrr. distributum, including the features of Hrr. terrestre, Hrr. arcis and Hrr. litoreum.

Emended description of Salinivibrio proteolyticus, including Salinivibrio costicola subsp. vallismortis and five new isolates.

We carried out a comparative taxonomic study of Salinivibrio proteolyticus and Salinivibrio costicola subsp. vallismortis, as well as of five halophilic strains (IB574, IB872, PR5, PR919 and PR932), isolated from salterns in Spain and Puerto Rico that were closely related to these bacteria. Multilocus sequence analysis of concatenated gyrB, recA, rpoA and rpoD housekeeping genes showed that they constituted a single cluster separate from the other species and subspecies of Salinivibrio. Experimental and in silico DNA-DNA hybridization studies indicated that they are members of the same species, with relatedness of 100-74 % and 97.8-70.0 %, respectively. The average nucleotide identity (ANI) determined for these strains was 99.7-95.6 % for ANIb and 99.7-95.7 % for OrthoANI. However, the ANI values for S. costicolasubsp.vallismortis DSM 8285T with respect to S. costicolasubsp.costicola DSM 11403T and S. costicolasubsp.alcaliphilus DSM 16359T were 78.7 and 78.9 % (ANIb) and 79.4 and 79.4 % (OrthoANI), respectively. The phylogenomic tree based on 1072 concatenated orthologous single-copy core genes confirmed that S. proteolyticus, S. costicolasubsp.vallismortis and the five new isolates constitute a coherent single phylogroup, separated from the other species and subspecies of Salinivibrio. All these data indicate that S. costicolasubsp.vallismortis is a heterotypic synonym of S. proteolyticus and we propose an emended description of this species.

Halorubrum chaoviator Mancinelli et al. 2009 is a later, heterotypic synonym of Halorubrum ezzemoulense Kharroub et al. 2006. Emended description of Halorubrum ezzemoulense Kharroub et al. 2006.

A polyphasic comparative taxonomic study of Halorubrum ezzemoulense Kharroub et al. 2006, Halorubrum chaoviator Mancinelli et al. 2009 and eight new Halorubrum strains related to these haloarchaeal species was carried out. Multilocus sequence analysis using the five concatenated housekeeping genes atpB, EF-2, glnA, ppsA and rpoB', and phylogenetic analysis based on the 757 core protein sequences obtained from their genomes showed that Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator Halo-G*T (=DSM 19316T) and the eight Halorubrum strains formed a robust cluster, clearly separated from the remaining species of the genus Halorubrum. The orthoANI value and digital DNA-DNA hybridization value, calculated by the Genome-to-Genome Distance Calculator (GGDC), showed percentages among Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator DSM 19316T and the eight Halorubrum strains ranging from 99.4 to 97.9 %, and from 95.0 to 74.2 %, respectively, while these values for those strains and the type strains of the most closely related species of Halorubrum were 88.7-77.4 % and 36.1-22.3 %, respectively. Although some differences were observed, the phenotypic and polar lipid profiles were quite similar for all the strains studied. Overall, these data show that Hrr. ezzemoulense, Hrr. chaoviator and the eight new Halorubrum isolates constitute a single species. Thus, Hrr. chaoviator should be considered as a later, heterotypic synonym of Hrr. ezzemoulense. We propose an emended description of Hrr. ezzemoulense, including the features of Hrr. chaoviator and those of the eight new isolates.

Soortia roseihalophila gen. nov., sp. nov., a new taxon in the order Balneolales isolated from a travertine spring, and description of Soortiaceae fam. nov.

A novel Gram-stain-negative, slightly halophilic, motile, curved rod with a horseshoe shape, designated strain Bsw-2bT, was isolated from Badab-Soort travertine spring in Iran. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Bsw-2bT belongs to the order Balneolales, showing 84.6 % sequence similarity to Gracilimonastropica DSM 19535T and 84.4 % and 83.9 % sequence similarity to Gracilimonas rosea CL-KR2T and Balneola vulgaris DSM 17893T, respectively. In addition, phenotypic and physiological features could clearly differentiate strain Bsw-2bT from species of the most closely related genera, Gracilimonas, Balneola, Aliifodinibius and Fodinibius. The strain was able to grow with 1-3 % (w/v) (optimum at 2 %) NaCl, at temperatures of 28-34 °C (optimum at 30 °C) and between pH 6.0 and 8.0 (optimum at pH 7.0). The major cellular fatty acids of strain Bsw-2bT were iso-C15 : 0, iso-C13 : 0 and iso-C14 : 0. The polar lipid profile of strain Bsw-2bT was composed predominantly of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, an unknown glycolipid and four unknown phospholipids. The DNA G+C content was 40.5 mol%. Based on the evidence from the polyphasic study, strain Bsw-2bT represents a novel species in a novel genus within a new family, for which the name Soortia roseihalophila gen. nov., sp. nov. is proposed, within the new family Soortiaceae fam. nov. The type strain is strain Bsw-2bT (=IBRC-M 10915T=LMG 28547T).

Halorubrum halodurans sp. nov., an extremely halophilic archaeon isolated from a hypersaline lake.

Two extremely halophilic archaea, strains Cb34T and C170, belonging to the genus Halorubrum, were isolated from the brine of the hypersaline lake Aran-Bidgol in Iran. Cells of the two strains were motile, pleomorphic rods, stained Gram-variable and produced red-pigmented colonies. Strains Cb34T and C170 required 25 % (w/v) salts, pH 7.0 and 37 °C for optimal growth under aerobic conditions; 0.3 M Mg2+ was required. Cells of both isolates were lysed in distilled water and hypotonic treatment with < 10 % NaCl provoked cell lysis. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that these two strains were closely related to Halorubrum cibi B31T (98.8 %) and other members of the genus Halorubrum. In addition, studies based on the rpoB' gene revealed that strains Cb34T and C170 are placed among the species of Halorubrum and are closely related to Halorubrum cibi B31T, with rpoB' gene sequence similarity less than or equal to 95.7 %. The polar lipid patterns of both strains consisted of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The DNA G+C content was 62.1-62.4 mol%. DNA-DNA hybridization studies confirmed that strains Cb34T and C170 constitute a distinct species. Data obtained in this study show that the two strains represent a novel species, for which the name Halorubrum halodurans sp. nov. is proposed. The type strain is Cb34T ( = CECT 8745T = IBRC-M 10233T).

Pseudorhodoplanes sinuspersici gen. nov., sp. nov., isolated from oil-contaminated soil.

Strain RIPI 110T was isolated from a soil sample collected from an oil-contaminated site on Siri Island, Persian Gulf, Iran. Cells of the novel isolate were Gram-stain-negative, facultatively anaerobic, non-motile and rod-shaped. Cells divided asymmetrically by budding and formed rosette-like clusters. The optimum pH and temperature for growth were pH 7 and 30 °C, while the strain was able to grow at pH 5.5-8 and 15-35 °C. Strain RIPI 110T utilized only complex carbon sources and pyruvate as the sole carbon source and could not grow under photoautotrophic conditions. The highest 16S rRNA gene sequence similarities, 93.9, 93.9 and 93.5 %, were obtained with Variibacter gotjawalensis GJW-30T, Rhodoplanes roseus 941T and Rhodoplanes elegans AS130T, respectively. The major cellular fatty acids were summed feature 8 (C18 : 1ω7c/ω6c), C16 : 0 and C19 : 0 cyclo ω8c. Polar lipid analyses revealed that strain RIPI 110T contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, an unknown aminophospholipid and four unknown phospholipids. Ubiquinone-10 was the predominant quinone component. The DNA G+C content was 59.4 mol%. On the basis of the 16S rRNA gene sequence analysis, in combination with chemotaxonomic and physiological data, the novel isolate could not be classified in any recognized genera. Strain RIPI 110T is thus considered to represent a novel species of a new genus within the order Rhizobiales, for which the name Pseudorhodoplanes sinuspersici gen. nov., sp. nov. is proposed. The type strain of the type species is RIPI 110T ( = IBRC-M 10770T = CECT 8374T).

Halorubrum persicum sp. nov., an extremely halophilic archaeon isolated from sediment of a hypersaline lake.

An extremely halophilic archaeon belonging to the genus Halorubrum, strain C49T, was isolated from sediment of the hypersaline lake Aran-Bidgol in Iran. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain C49T was closely related to Halorubrum saccharovorum JCM 8865T (99.5 %) and other species of the genus Halorubrum. Studies based on multilocus sequence analysis revealed that strain C49T is placed among the species of Halorubrum; the strain constituted a defined branch in comparison with the type strains of species of Halorubrum, while the 16S rRNA gene sequence divergence could not define the status of the newly isolated strain. For optimum growth, strain C49T required 20 % (w/v) salts at pH 7.0 and 37 °C under aerobic conditions. Mg2+ was not required. The cells were pleomorphic rods, motile and stained Gram-variable. Colonies of the strain were pink. Hypotonic treatment with <12 % NaCl provoked cell lysis. The polar lipid pattern of strain C49T consisted of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester derived from both C20C20 and C20C25 archaeol, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The DNA G+C content was 64.2 mol%. DNA-DNA hybridization studies and average nucleotide identity confirmed that strain C49T constitutes a distinct genospecies. Data obtained in this study show that strain C49T represents a novel species, for which the name Halorubrum persicum sp. nov. is proposed. The type strain is C49T ( = IBRC-M 10232T = JCM 30541T).

Salininema proteolyticum gen. nov., sp. nov., a halophilic rare actinomycete isolated from wetland soil, and emended description of the family Glycomycetaceae.

A Gram-stain-positive actinobacterial strain, Miq-4T, was isolated from soil around Meighan wetland in the centre of Iran. Strain Miq-4T was strictly aerobic, catalase- and oxidase-positive. The isolate grew in the presence of 3­15 % (w/v) NaCl, at 20­40 °C and pH 6.0­11.0. The optimum NaCl, temperature and pH for growth were 7.0 %, 30 °C and 7.0­8.5, respectively. The cell wall of strain Miq-4T contained meso-diaminopimelic acid as the diamino acid and glucose and ribose as the whole-cell sugars. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Strain Miq-4T synthesized cellular fatty acids of anteiso- and iso-branched types, including anteiso-C17 : 0, anteiso- C15 : 0 and iso-C16 : 0, and the major respiratory quinone was MK-9(H4). The G+C content of the genomic DNA was 68.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences and characteristic patterns of 16S rRNA gene signature nucleotides revealed that strain Miq-4T belongs to the family Glycomycetaceae and showed the closest phylogenetic similarity with Haloglycomyces albus YIM 92370T (94.1 % 16S rRNA gene sequence similarity). On the basis of phylogenetic analysis and phenotypic and chemotaxonomic characteristics, strain Miq-4T represents a novel species of a new genus in the family Glycomycetaceae, for which the name Salininema proteoliyticum gen. nov., sp. nov. is proposed. The type strain of the type species is Miq-4T ( = IBRC-M 10908T = LMG 28391T). An emended description of the family Glycomycetaceae is also proposed in order to include features of the new genus.

Decoding the Genomic Profile of the Halomicroarcula Genus: Comparative Analysis and Characterization of Two Novel Species.

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 as a Source for Uncovering a New Taxon: Pseudidiomarina terrestris sp. nov.

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.

Metagenomic insights into the prokaryotic communities of heavy metal-contaminated hypersaline soils.

Saline soils and their microbial communities have recently been studied in response to ongoing desertification of agricultural soils caused by anthropogenic impacts and climate change. Here we describe the prokaryotic microbiota of hypersaline soils in the Odiel Saltmarshes Natural Area of Southwest Spain. This region has been strongly affected by mining and industrial activity and feature high levels of certain heavy metals. We sequenced 18 shotgun metagenomes through Illumina NovaSeq from samples obtained from three different areas in 2020 and 2021. Taxogenomic analyses demonstrate that these soils harbored equal proportions of archaea and bacteria, with Methanobacteriota, Pseudomonadota, Bacteroidota, Gemmatimonadota, and Balneolota as most abundant phyla. Functions related to the transport of heavy metal outside the cytoplasm are among the most relevant features of the community (i.e., ZntA and CopA enzymes). They seem to be indispensable to avoid the increase of zinc and copper concentration inside the cell. Besides, the archaeal phylum Methanobacteriota is the main arsenic detoxifier within the microbiota although arsenic related genes are widely distributed in the community. Regarding the osmoregulation strategies, "salt-out" mechanism was identified in part of the bacterial population, whereas "salt-in" mechanism was present in both domains, Bacteria and Archaea. De novo biosynthesis of two of the most universal compatible solutes was detected, with predominance of glycine betaine biosynthesis (betAB genes) over ectoine (ectABC genes). Furthermore, doeABCD gene cluster related to the use of ectoine as carbon and energy source was solely identified in Pseudomonadota and Methanobacteriota.

'Altruistic' cooperation among the prokaryotic community of Atlantic salterns assessed by metagenomics.

Hypersaline environments are extreme habitats with a limited prokaryotic diversity, mainly restricted to halophilic or halotolerant archaeal and bacterial taxa adapted to highly saline conditions. This study attempts to analyze the taxonomic and functional diversity of the prokaryotes that inhabit a solar saltern located at the Atlantic Coast, in Isla Cristina (Huelva, Southwest Spain), and the influence of salinity on the diversity and metabolic potential of these prokaryotic communities, as well as the interactions and cooperation among the individuals within that community. Brine samples were obtained from different saltern ponds, with a salinity range between 19.5 % and 39 % (w/v). Total prokaryotic DNA was sequenced using the Illumina shotgun metagenomic strategy and the raw sequence data were analyzed using supercomputing services following the MetaWRAP and SqueezeMeta protocols. The most abundant phyla at moderate salinities (19.5-22 % [w/v]) were Methanobacteriota (formerly "Euryarchaeota"), Pseudomonadota and Bacteroidota, followed by Balneolota and Actinomycetota and Uroviricota in smaller proportions, while at high salinities (36-39 % [w/v]) the most abundant phylum was Methanobacteriota, followed by Bacteroidota. The most abundant genera at intermediate salinities were Halorubrum and the bacterial genus Spiribacter, while the haloarchaeal genera Halorubrum, Halonotius, and Haloquadratum were the main representatives at high salinities. A total of 65 MAGs were reconstructed from the metagenomic datasets and different functions and pathways were identified in them, allowing to find key taxa in the prokaryotic community able to synthesize and supply essential compounds, such as biotin, and precursors of other bioactive molecules, like ß-carotene, and bacterioruberin, to other dwellers in this habitat, lacking the required enzymatic machinery to produce them. This work shed light on the ecology of aquatic hypersaline environments, such as the Atlantic Coast salterns, and on the dynamics and factors affecting the microbial populations under such extreme conditions.

Synergistic antifungal activity against Candida albicans between voriconazole and cyclosporine a loaded in polymeric nanoparticles.

The goal of this work is to investigate if the synergistic antifungal activity between cyclosporine A, CsA, and voriconazole, VRZ, increases when both drugs are encapsulated in a nanocarrier as compared when they are free. The preparation and characterization of blank and VRZ and CsA loaded polymeric based PLGA nanoparticles (PLGA, PLGA-PEG, and PLGA+PEG) was a necessary previous step. Using the more suitable NPs, those of PLGA, the antifungal susceptibility tests performed with VRZ-loaded PLGA NPs, show no significant increase of the antifungal activity in comparison to that of free VRZ. However, the synergistic behavior found for the (VRZ+CsA)-loaded PLGA NPs was fourfold stronger than that observed for the two free drugs together. On the other hand, the investigation into the suppression of C. albicans biofilm formation showed that blank PLGA NPs inhibit the biofilm formation at high NPs concentrations. However, a minor effect or even a slight biofilm increase formation was observed at low and moderate NPs concentrations. Therefore, the enhancement of the biofilm inhibition found for the three tested treatments (CsA alone, VRZ alone, and VRZ+CsA) when comparing free and encapsulated drugs, within the therapeutic window, can be attributed to the drug encapsulation approach. Indeed, polymeric PLGA NPs loaded with CsA, VRZ, or VRZ+CsA are more effective at inhibiting the C. albicans biofilm growth than their free counterparts.