Halomicrococcus gelatinilyticus sp. nov. and Halosimplex aquaticum sp. nov., halophilic archaea isolated from saline soil and an inland solar saltern.

Two extremely halophilic archaeal strains, GSLN9T and XZYJT29T, were isolated from the saline soil in different regions of western China. Both strains GSLN9T and XZYJT29T have two 16S rRNA genes with similarities of 95.1 and 94.8 %, respectively. Strain GSLN9T was mostly related to the genus Halomicrococcus based on 16S rRNA (showing 91.0-96.0 % identities) and rpoB' genes (showing 92.0 % identity). Strain XZYJT29T showed 92.1-97.6 % (16S rRNA gene) and 91.4-93.1 % (rpoB' gene) sequence similarities to its relatives in the genus Halosimplex, respectively. The polar lipid profile of strain GSLN9T included phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulphate (PGS), sulphated mannosyl glucosyl diether (S-DGD-1) and sulphated galactosyl mannosyl glucosyl diether (S-TGD-1), mostly similar to that of Halomicrococcus hydrotolerans H22T. PA, PG, PGP-Me, S-DGD-1 (S-DGD-PA), S2-DGD, S-TGD-1 and an unidentified glycolipid were detected in strain XZYJT29T; this polar lipid composition is similar to those of members of the genus Halosimplex. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between these two strains and their relatives of the genera Halomicrococcus and Halosimplex were no more than 82, 27 and 80 %, respectively, much lower than the thresholds for species demarcation. Other phenotypic characterization results indicated that strains GSLN9T and XZYJT29T can be differentiated from the current species of the genera Halomicrococcus and Halosimplex, respectively. These results revealed that strains GSLN9T (=CGMCC 1.15215T=JCM 30842T) and XZYJT29T (=CGMCC 1.15828T=JCM 31853T) represent novel species of Halomicrococcus and Halosimplex, for which the names Halomicrococcus gelatinilyticus sp. nov. and Halosimplex aquaticum sp. nov. are proposed.

Halospeciosus flavus gen. nov., sp. nov. and Haladaptatus caseinilyticus sp. nov., halophilic archaea isolated from saline soil of an inland solar saltern and offshore sediment.

Two novel halophilic archaeal strains (XZGYJ-43T and ZJ1T) were isolated from Mangkang ancient solar saltern (Tibet, PR China) and Zhujiang river inlet (Guangdong, PR China), respectively. The comparison of the 16S rRNA gene sequences revealed that strain XZGYJ-43T is related to the current species of the family Halobacteriaceae (89.2-91.7% similarity) and strain ZJ1T showed 94.7-98.3% similarity to the current species of the genus Haladaptatus. Phylogenetic analyses based on 16S rRNA genes, rpoB' genes and genomes indicated that strain XZGYJ-43T is separate from the related genera, Halocalculus, Salarchaeum and Halarchaeum of the family Halobacteriaceae, and strain ZJ1T tightly clusters with the current species of the genus Haladaptatus. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between strain XZGYJ-43T and the current species of the family Halobacteriaceae were 71-75, 20-25 and 59-68 %, and these values between strain ZJ1T and the current species of the genus Haladaptatus were 77-81, 27-32 and 76-82 %, respectively, clearly below the thresholds for prokaryotic species demarcation. These two strains could be distinguished from their relatives according to differential phenotypic characteristics. The major polar lipids of strain XZGYJ-43T were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), mannosyl glucosyl diether (DGD-1; DGD-PA) and sulphated mannosyl glucosyl diether (S-DGD-1; S-DGD-PA), and those of strain ZJ1T were PA, PG, PGP-Me, DGD-PA, S-DGD-1 (S-DGD-PA) and sulphated galactosyl mannosyl glucosyl diether. Based on phenotypic, phylogenetic and genomic data, strain XZGYJ-43T (=CGMCC 1.13890T=JCM 33735T) represents a novel species of a new genus within the family Halobacteriaceae, and strain ZJ1T (=CGMCC 1.18785T=JCM 34917T) represents a novel species of the genus Haladaptatus, for which the names Halospeciosus flavus gen. nov., sp. nov. and Haladaptatus caseinilyticus sp. nov. are proposed, respectively.

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.

Halobacterium yunchengense sp. nov., Natronomonas amylolytica sp. nov., Halorientalis halophila sp. nov., Halobellus salinisoli sp. nov., halophilic archaea isolated from a saline lake and inland saline soil.

Four halophilic archaeal strains YCN1T, YCN58T, LT38T, and LT62T were isolated from Yuncheng Salt Lake (Shanxi, China) and Tarim Basin (Xinjiang, China), respectively. Phylogenetic and phylogenomic analyses showed that these four strains tightly cluster with related species of Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively. The AAI, ANI, and dDDH values between these four strains and their related species of respective genera were lower than the proposed threshold values for species delineation. Strains YCN1T, YCN58T, LT38T, and LT62T could be differentiated from the current species of Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively, based on the comparison of diverse phenotypic characteristics. The polar lipid profiles of these four strains were closely similar to those of respective relatives within the genera Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively. The phenotypic, phylogenetic, and genome-based analyses indicated that strains YCN1T, YCN58T, LT38T, and LT62T represent respective novel species within the genera Halobacterium, Natronomonas, Halorentalis, and Halobellus, for which the names Halobacterium yunchengense sp. nov., Natronomonas amylolytica sp. nov., Halorientalis halophila sp. nov., and Halobellus salinisoli sp. nov. are proposed, respectively.

Genome-based classification of the family Haloferacaceae and description of five novel species of Halobaculum.

The taxonomic status of some species of Halobellus, Haloferax, Halogranum, and Haloplanus within the family Haloferacaceae was elucidated by phylogenetic, phylogenomic, and comparative genomic analyses. The relative species of each genus should constitute a single species based on the overall genome-related indexes proposed for species demarcation. The cutoff values of AAI (72.1%), ANI (82.2%), and rpoB' gene similarity (90.7%) were proposed to differentiate genera within the family Haloferacaceae. According to these standards, a novel genus related to the genus Halobaculum was proposed to accommodate Halobaculum halophilum Gai3-2 T and Halobaculum salinum NJ-3-1 T. Five halophilic archaeal strains, DT31T, DT55T, DT92T, SYNS20T, and YSMS11T, isolated from a tidal flat and a marine solar saltern in China, were subjected to polyphasic classification. The phenotypic, phylogenetic, phylogenomic, and comparative genomic analyses revealed that strains DT31T (= CGMCC 1.18923 T = JCM 35417 T), DT55T (= CGMCC 1.19048 T = JCM 36147 T), DT92T (= CGMCC 1.19057 T = JCM 36148 T), SYNS20T (= CGMCC 1.62628 T = JCM 36154 T), and YSMS11T (= CGMCC 1.18927 T = JCM 34912 T) represent five novel species of the genus Halobaculum, for which the names, Halobaculum lipolyticum sp. nov., Halobaculum marinum sp. nov., Halobaculum litoreum sp. nov., Halobaculum halobium sp. nov., and Halobaculum limi sp. nov., are proposed.

Genome-based taxonomy of genera Haloarcula and Halomicroarcula, and description of six novel species of Haloarcula.

The genera Haloarcula and Halomicroarcula are the most closely related genera within the family Haloarculaceae (class Halobacteria). The respective 16S rRNA genes of type strains from the genus Haloarcula showed 94.7-96.5% similarities to their homologous genes of type strains from the genus Halomicroarcula. The Haloarcula species showed 89.3-92.8% rpoB' gene similarities to Halomicroarcula species. These similarities were higher than the proposed genus boundary. Phylogenomic analysis revealed that these two genera formed a tight cluster separated from Halomicrobium with high bootstrap confidence. The average amino acid identity (AAI) values among Haloarcula and Halomicroarcula were 70.1-74.5%, higher than the cutoff value (67.0%) to differentiate the genera Haloarcula and Halomicroarcula from Halomicrobium. These results indicated that the genus Halomicroarcula should be merged with Haloarcula. Then, six novel species are described based on strains DFY41T, GDY20T, SHR3T, XH51T, YJ-61-ST, and ZS-22-S1T isolated from coarse sea salt, marine solar saltern, and salt lake (China). These six strains formed separate clades (90.1-99.3% 16S rRNA and 89.0-94.9% rpoB' gene similarities) and then clustered with current Haloarcula and Halomicroarcula species (89.4-99.1% 16S rRNA and 87.6-95.0% rpoB' gene similarities), as revealed by phylogenetic analyses. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and AAI values among these six strains and current Haloarcula and Halomicroarcula species were 76.2-89.8%, 25.3-46.0%, and 70.3-89.7%, respectively, clearly below the species demarcation threshold. These six strains were distinguished from current Haloarcula and Halomicroarcula species according to differential phenotypic characteristics. Six novel species, Haloarcula halophila sp. nov., Haloarcula litorea sp. nov., Haloarcula rara sp. nov., Haloarcula halobia sp. nov., Haloarcula pelagica sp. nov., and Haloarcula ordinaria sp. nov., are proposed to accommodate strains DFY41T, GDY20T, SHR3T, XH51T, YJ-61-ST, and ZS-22-S1T, respectively.

Prognostic value of left ventricular trabeculae fractal analysis in patients with dilated cardiomyopathy.

BACKGROUND: The prognostic value of left ventricular (LV) myocardial trabecular complexity on cardiovascular magnetic resonance (CMR) in dilated cardiomyopathy (DCM) remains unknown. This study aimed to evaluate the prognostic value of LV myocardial trabecular complexity using fractal analysis in patients with DCM. METHODS: Consecutive patients with DCM who underwent CMR between March 2017 and November 2021 at two hospitals were prospectively enrolled. The primary endpoints were defined as the combination of all-cause death and heart failure hospitalization. The events of cardiac death alone were defined as the secondary endpoints.LV trabeculae complexity was quantified by measuring the fractal dimension (FD) of the endocardial border based on fractal geometry on CMR. Cox proportional hazards regression and Kaplan-Meier survival analysis were used to examine the association between variables and outcomes. The incremental prognostic value of FD was assessed in nested models. RESULTS: A total of 403 patients with DCM (49.31 ± 14.68 years, 69% male) were recruited. After a median follow-up of 43 months (interquartile range, 28-55 months), 87 and 24 patients reached the primary and secondary endpoints, respectively. Age, heart rate, New York Heart Association functional class >II, N-terminal pro-B-type natriuretic peptide, LV ejection fraction, LV end-diastolic volume index, LV end-systolic volume index, LV mass index, presence of late gadolinium enhancement, global FD, LV mean apical FD, and LV maximal apical FD were univariably associated with the outcomes (all P < 0.05). After multivariate adjustment, LV maximal apical FD remained a significant independent predictor of outcome [hazard ratio = 1.179 (1.116, 1.246), P < 0.001]. The addition of LV maximal apical FD in the nested models added incremental prognostic value to other common clinical and imaging risk factors (all <0.001; C-statistic: 0.84-0.88, P < 0.001). CONCLUSION: LV maximal apical FD was an independent predictor of the adverse clinical outcomes in patients with DCM and provided incremental prognostic value over conventional clinical and imaging risk factors.

Genome-based classification of genera Halosegnis and Salella, and description of four novel halophilic archaea isolated from a tidal flat.

The current species of Halosegnis and Salella within the class Halobacteria are closely related based on phylogenetic, phylogenomic, and comparative genomic analyses. The Halosegnis species showed 99.8-100.0% 16S rRNA and 96.6-99.6% rpoB' gene similarities to the Salella species, respectively. Phylogenetic and phylogenomic analyses showed that Salella cibi CBA1133T, the sole species of Salella, formed a single tight cluster with Halosegnis longus F12-1T, then with Halosegnis rubeus F17-44T. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values between Salella cibi CBA1133T and Halosegnis longus F12-1T were 99.2, 94.2, and 98.6%, respectively, much higher than the thresholds for species demarcation. This genome-based classification revealed that the genus Salella should be merged with Halosegnis, and Salella cibi should be a later heterotypic synonym of Halosegnis longus. Halophilic archaeal strains DT72T, DT80T, DT85T, and DT116T, isolated from the saline soil of a tidal flat in China, were subjected to polyphasic taxonomic characterization. The phenotypic, chemotaxonomic, phylogenetic, and phylogenomic features indicated that strains DT72T (= CGMCC 1.18925T = JCM 35418T), DT80T (= CGMCC 1.18926T = JCM 35419T), DT85T (= CGMCC 1.19049T = JCM 35605T), and DT116T (= CGMCC 1.19045T = JCM 35606T) represent four novel species of the genera Halorussus, Halosegnis and Haloglomus, respectively, for which the names, Halorussus caseinilyticus sp. nov., Halorussus lipolyticus sp. nov., Halosegnis marinus sp. nov., and Haloglomus litoreum sp. nov., are proposed.

Halomarina litorea sp. nov., Halomarina pelagica sp. nov., Halomarina halobia sp. nov., and Halomarina ordinaria sp. nov., Halophilic Archaea Isolated from Coastal and Inland Saline Soil.

Four halophilic archaeal strains, BCD28T, BND7T, PSR21T, and PSRA2T, were isolated from coastal and inland saline soil, respectively. The 16S rRNA and rpoB' gene sequence similarities among these four strains and current species of Halomarina were 95.9-96.6% and 86.9-90.3%, respectively. Phylogenetic and phylogenomic analyses revealed that these four strains tightly cluster with the current species of the genus Halomarina. The AAI, ANI, and dDDH values among these four strains and current species of Halomarina were 65.3-68.4%, 75.8-77.7%, and 20.3-22.0%, respectively, clearly below the threshold values for species demarcation. Strains BCD28T, BND7T, PSR21T, and PSRA2T could be differentiated from the current species of Halomarina based on the comparison of diverse phenotypic characteristics. The major polar lipids of these four strains were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), and four to five glycolipids. Phosphatidylglycerol sulfate (PGS) was only detected in strain BND7T. The phenotypic, phylogenetic, and genome-based analyses suggested that strains BCD28T (= CGMCC 1.18776T = JCM 34908T), BND7T (= CGMCC 1.18778T = JCM 34910T), PSR21T (= CGMCC 1.17027T = JCM 34147T), and PSRA2T (= CGMCC 1.17214T = JCM 34148T) represent four novel species of the genus Halomarina, for which the names Halomarina litorea sp. nov., Halomarina pelagica sp. nov., Halomarina halobia sp. nov., and Halomarina ordinaria sp. nov. are proposed.

Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS.

BACKGROUND: Spermatozoa have the task of delivering an intact paternal genome to the oocyte and supporting successful embryo development. The detection of sperm DNA fragmentation (SDF) has been emerging as a complementary test to conventional semen analysis for male infertility evaluation, but the mechanism leading to SDF and its impact on assisted reproduction remain unclear. Therefore, the study identified and analyzed the differentially expressed proteins of sperm with high and low SDF. METHODS: Semen samples from men attended the infertility clinic during June 2020 and August 2020 were analyzed, and sperm DNA fragmentation index (DFI) was detected by the sperm chromatin structure assay. Semen samples with low DFI (< 30%, control group) and high DFI (≥ 30%, experimental group) were optimized by density gradient centrifugation (DGC), and the differentially expressed proteins of obtained sperm were identified by the Sequential Window Acquisition of All Theoretical Mass Spectra Mass Spectrometry (SWATH-MS) and performed GO and KEGG analysis. RESULTS: A total of 2186 proteins were identified and 1591 proteins were quantified, of which 252 proteins were identified as differentially expressed proteins, including 124 upregulated and 128 downregulated. These differentially expressed proteins were involved in metabolic pathways, replication/recombination/repair, acrosomal vesicles, kinase regulators, fertilization, tyrosine metabolism, etc. Western blotting results showed that the expression levels of RAD23B and DFFA proteins and the levels of posttranslational ubiquitination and acetylation modifications in the experimental group were significantly higher than those in the control group, which was consistent with the results of proteomics analysis. CONCLUSIONS: Proteomic markers of sperm with high DNA fragmentation can be identified by the SWATH-MS and bioinformatic analysis, and new protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. Our findings may improve our understanding of the basic molecular mechanism of sperm DNA damage.

Investigation of the mechanisms leading to human sperm DNA damage based on transcriptome analysis by RNA-seq techniques.

RESEARCH QUESTION: What are the molecular mechanisms leading to human sperm DNA damage? DESIGN: Semen samples were collected and the sperm DNA fragmentation index (DFI) was assessed. Differentially expressed RNA in spermatozoa with a high (DFI ≥30%, experimental group) or normal (DFI <30%, control group) DFI were identified by RNA-sequencing (RNA-seq) technology, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. Three differentially expressed RNA related to sperm DNA damage and repair, namely PMS1, TP53BP1 and TLK2, were validated using real-time quantitative (RT-qPCR). RESULTS: A total of 19,970 expressed RNA were detected in the two groups. Compared with the control group, the expression levels of 189 RNA in the experimental group were significantly increased and those of 163 genes decreased. Gene Ontology enrichment analysis showed that these RNA were mainly concentrated in the ATPase-dependent transmembrane transport complex, extracellular exosome, somatic cell DNA recombination, protein binding, cytoplasm and regulation of localization. KEGG pathway analysis showed that these RNA were mainly related to the PI3K-Akt signalling pathway, endocytosis, p53 signalling pathway and cGMP-PKG signalling pathway. The RT-qPCR results showed that the expression levels of PMS1, TP53BP1 and TLK2 in the experimental group were significantly lower than in the control group (P = 0.01, 0.015 and 0.004, respectively), which was identical to the results of RNA sequencing. CONCLUSIONS: Differentially expressed RNA related to sperm DNA damage and repair may be identified by RNA-seq technology, which provides new insights into the understanding of sperm DNA damage and repair, and will help to discover new biomarkers related to sperm DNA damage.

Genome-based classification of the class Halobacteria and description of Haladaptataceae fam. nov. and Halorubellaceae fam. nov.

Currently, there are four mainstream taxonomic opinions on the classification of the class Halobacteria at the family and order levels. The International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteria (ICSP), List of Prokaryotic names with Standing in Nomenclature (LPSN) and National Centre for Biotechnology Information (NCBI) adopted taxonomies have three to four orders and up to eight families, while the Genome Taxonomy Database (GTDB) taxonomy proposes only one order with nine families. To resolve the taxonomic inconsistency, phylogenomic analyses based on concatenated single-copy orthologous proteins and 122 concatenated conserved single-copy marker proteins were conducted to infer the taxonomic status of the current representatives of the class Halobacteria at the family and order levels. The current 76 genera with validly published names of the class Halobacteria were able to be assigned into eight families in one order. On the basis of these results, it is proposed that the current species with validly published names of the class Halobacteria should be remerged into the order Halobacteriales, then assigned to eight families, Haladaptataceae, Haloarculaceae, Halobacteriaceae, Halococcaceae, Haloferacaceae, Natronoarchaeaceae, Natrialbaceae and Halorubellaceae. Thus, Haladaptataceae fam. nov. is described based on Haladaptatus, Halomicrococcus and Halorussus and Halorubellaceae fam. nov. is proposed incorporating Haloarchaeobius and Halorubellus, respectively.

Halomicroarcula laminariae sp. nov. and Halomicroarcula marina sp. nov., extremely halophilic archaea isolated from salted brown alga Laminaria and coastal saline-alkali lands.

Four extremely halophilic archaeal strains, LYG-108T, LYG-24, DT1T and YSSS71, were isolated from salted Laminaria produced in Lianyungang and saline soil from the coastal beach at Jiangsu, PR China. The four strains were found to be related to the current species of Halomicroarcula (showing 88.1-98.5% and 89.3-93.6% similarities, respectively) as revealed by phylogenetic analysis based on 16S rRNA and rpoB' genes. These phylogenies were fully supported by the phylogenomic analysis, and the overall genome-related indexes (average nucleotide identity, DNA-DNA hybridization and average amino acid identity) among these four strains and the Halomicroarcula species were 77-84 %, 23-30 % and 71-83 %, respectively, clearly below the threshold values for species demarcation. Additionally, the phylogenomic and comparative genomic analyses revealed that Halomicroarcula salina YGH18T is related to the current species of Haloarcula rather than those of Halomicroarcula, Haloarcula salaria Namwong et al. 2011 is a later heterotypic synonym of Haloarcula argentinensis Ihara et al. 1997, and Haloarcula quadrata Oren et al. 1999 is a later heterotypic synonym of Haloarcula marismortui Oren et al. 1990. The major polar lipids of strains LYG-108T, LYG-24, DT1T and YSSS71 were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate, sulphated mannosyl glucosyl diether and additional glycosyl-cardiolipins. All these results showed that strains LYG-108T (=CGMCC 1.13607T=JCM 32950T) and LYG-24 (=CGMCC 1.13605=JCM 32949) represent a new species of the genus Halomicroarcula, for which the name Halomicroarcula laminariae sp. nov. is proposed; strains DT1T (=CGMCC 1.18928T=JCM 35414T) and YSSS71 (=CGMCC 1.18783=JCM 34915) also represent a new species of the genus Halomicroarcula, for which the name Halomicroarcula marina sp. nov. is proposed.

Halobacterium wangiae sp. nov. and Halobacterium zhouii sp. nov., two extremely halophilic archaea isolated from sediment of a salt lake and saline soil of an inland saltern.

Two novel halophilic archaeal strains, Gai3-17T and XZYJT26T, were isolated from the sediment of Gaize salt lake and the saline soil of Mangkang ancient solar saltern in Tibet, PR China, respectively. Strains Gai3-17T and XZYJT26T were related to each other (96.5 and 89.7% similarity, respectively) and showed 97.5-95.4 and 91.5-87.7% similarities to the current members of Halobacterium based on 16S rRNA and rpoB' genes. The phylogenomic analysis indicated that strains Gai3-17T and XZYJT26T formed two distinct clades and clustered with the Halobacterium species. The two strains can be differentiated from the type strains of the six species with validly published names based on several phenotypic characteristics. The phospholipids of the two strains were phosphatidic acid, phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. One major glycolipid, sulphated galactosyl mannosyl glucosyl diether, was detected in strain Gai3-17T, while four glycolipids, mannosyl glucosyl diether, sulphated mannosyl glucosyl diether, disulphated mannosyl glucosyl diether and sulphated galactosyl mannosyl glucosyl diether were observed in strain XZYJT26T. The average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values among the two strains and the members of Halobacterium were no more than 81, 25 and 77 %, respectively. These overall genome-related indices were below the threshold values for species boundary, indicating that strains Gai3-17T and XZYJT26T represent two novel species of Halobacterium. Thus, two novel species, Halobacterium wangiae sp. nov. and Halobacterium zhouii sp. nov., are proposed to accommodate strains Gai3-17T (=CGMCC 1.16101T=JCM 33551T) and XZYJT26T (=CGMCC 1.16682T=JCM 33556T), respectively.

Salinigranum marinum sp. nov. and Halohasta salina sp. nov., halophilic archaea isolated from sediment of a marine saltern and inland saline soil.

Two halophilic archaeal strains, ZS-10T and GSL13T, were isolated from the Zhoushan marine saltern in Zhejiang, and an inland saline soil from the Tarim Basin, Xinjiang, PR China, respectively. The cells of strain ZS-10T were pleomorphic while those of strain GSL13T were rod-shaped. Both of them stained Gram-negative and formed red-pigmented colonies on agar plates and their cells lysed in distilled water. The optimum growth of strain ZS-10T was observed at 40 °C, 3.4 M NaCl, 0.03 M MgCl2 and pH 7.5, while that of strain GSL13T was at 37 °C, 3.1 M NaCl, 0.5 M MgCl2 and pH 7.5. Phylogenetic and phylogenomic analyses indicated that these two strains were related to Salinigranum and Halohasta, respectively. Strains ZS-10T and GSL13T could be differentiated from the current members of Salinigranum and Halohasta based on the comparison of diverse phenotypic characteristics. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values among strain ZS-10T and current species of Salinigranum were 75.8-78.6 %, 80.6-81.9 % and 24.3-26.1 %, respectively. These values between strain GSL13T and current species of Halohasta were 78.4-80.8 %, 79.8-82.8% and 22.7-25.7 %, respectively, clearly below the threshold values for species demarcation. The polar lipids of strain ZS-10T were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulphated mannosyl glucosyl diether (S-DGD-1), while those of strain GSL13T were phosphatidic acid, PG, PGP-Me, phosphatidylglycerol sulphate and S-DGD-1. The polar lipid profile of strain GSL13T was identical to those of Halohasta, whereas strain ZS-10T did not contain the minor glycolipids detected in the current Salinigranum species. The phenotypic, phylogenetic and genome-based results suggested that strains ZS-10T (=CGMCC 1.12868T=JCM 30241T) and GSL13T (=CGMCC 1.15214T=JCM 30841T) represent two novel species, for which the names Salinigranum marinum sp. nov. and Halohasta salina sp. nov. are proposed.

Natronosalvus halobius gen. nov., sp. nov., Natronosalvus caseinilyticus sp. nov., Natronosalvus vescus sp. nov., Natronosalvus rutilus sp. nov. and Natronosalvus amylolyticus sp. nov., halophilic archaea isolated from salt lakes and soda lakes.

Five halophilic archaeal strains (AGai3-5T, KZCA101T, CGA3T, WLHS1T and WLHSJ1T) were isolated from salt lakes and soda lakes in PR China. These strains had low 16S rRNA gene similarities (91.3-96.0 %) to closely related species of the family Natrialbaceae and may represent a new genus of the family. Phylogenetic and phylogenomic analyses revealed that these strains formed a distinct clade, separate from the nearby genera Natronobiforma and Saliphagus. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity (AAI) values among these five strains and the current members of the family Natrialbaceae were 72-90, 20-42 and 62-91 %, respectively, clearly below the threshold values for species demarcation. According to the critical value of AAI (≤76 %) proposed to differentiate genera within the family Natrialbaceae, it was further indicated that these strains represented a novel genus within the family. These strains could be distinguished from the related genera according to differential phenotypic characteristics. The major lipids of these strains were phosphatidic acid (PA), phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, mannosyl glucosyl diether (DGD-PA), sulphated DGD-1 (S-DGD-PA) and sulphated galactosyl mannosyl glucosyl diether. The phenotypic, chemotaxonomic, phylogenetic and phylogenomic features indicated that strains AGai3-5T (=CGMCC 1.16078T=JCM 33549T), KZCA101T (=CGMCC 1.17431T=JCM 35074T), CGA3T (=CGMCC 1.17463T=JCM 34318T), WLHS1T (=CGMCC 1.13780T=JCM 33562T) and WLHSJ1T (=CGMCC 1.13784T=JCM 33563T) represent five novel species of a new genus within the family Natrialbaceae, named Natronosalvus halobius gen. nov., sp. nov., Natronosalvus caseinilyticus sp. nov., Natronosalvus vescus sp. nov., Natronosalvus rutilus sp. nov. and Natronosalvus amylolyticus sp. nov., respectively.

Salinilacihabitans rarus gen. nov., sp. nov., Natrononativus amylolyticus gen. nov., sp. nov., Natronobeatus ordinarius gen. nov., sp. nov., and Halovivax gelatinilyticus sp. nov., halophilic archaea, isolated from a salt lake and soda lakes.

Four halophilic archaea strains, AD-4T, CGA30T, CGA73T, and WLHSJ27T, were isolated from a salt lake and two soda lakes located in different regions of China. The 16S rRNA and rpoB' gene sequence similarities among strains AD-4T, CGA30T, CGA73T, WLHSJ27T, and the current species of the family Natrialbaceae were 90.9-97.5% and 83.1-91.8%, respectively. The phylogenetic and phylogenomic analyses revealed that these four strains separated from existing genera in the family Natrialbaceae and formed distant branches. The ANI, isDDH, and AAI values among these four strains and the current members of the family Natrialbaceae were 72-79%, 20-25%, and 63-73%, respectively, much lower than the threshold values for species demarcation. Strains AD-4T, CGA73T, and WLHSJ27T may represent three novel genera of the family Natrialbaceae according to the cutoff value of AAI (≤ 76%) proposed to differentiate genera within the family Natrialbaceae. These four strains could be distinguished from the related genera according to differential phenotypic characteristics. The major phospholipids of these four strains were identical while their glycolipid profiles were diverse. DGD-1 is a major glycolipid found in strain AD-4T, trace glycolipids, DGD-1, and S-DGD-1, and (or) S-TGD-1 was found in the other three strains. The major respiratory quinones detected in the four strains were menaquinone MK-8 and MK-8(H2). This polyphasic classification indicated that strains AD-4T, CGA73T, and WLHSJ27T represent three novel species of three new genera with the family Natrialbaceae, and strain CGA30T represents a novel species of Halovivax.

Halocatena marina sp. nov., a novel filamentous halophilic archaeon isolated from marine tidal flat and emended description of the genus Halocatena.

Three novel filamentous halophilic archaea, strains DFN5T, RDMS1, and QDMS1, were isolated from the coastal saline soil samples of the intertidal zones located in different regions of Jiangsu Province, China. The colonies of these strains were pinkish-white due to the presence of white spores. These three strains are extremely halophilic and grew optimally at 35-37 °C and pH 7.0-7.5. Based on 16S rRNA and rpoB' gene analysis, strains DFN5T, RDMS1, and QDMS1 gathered together in phylogenetic trees and then clustered with the current species of the genus Halocatena showing 96.9-97.4% and 82.2-82.5% similarities, respectively. Both the 16S rRNA gene-based and rpoB' gene-based phylogenies were fully supported by the phylogenomic analysis, and the overall genome-related indexes indicated that strains DFN5T, RDMS1, and QDMS1 should be a novel species of the genus Halocatena. Genome mining revealed that there are considerable differences in the genes related to ß-carotene synthesis among these three strains and the current species of Halocatena. The major polar lipids of strains DFN5T, RDMS1, and QDMS1 are PA, PG, PGP-Me, S-TGD-1, TGD-1, and TGD-2. The minor polar lipids, S-DGD-1, DGD-1, S2-DGD, and S-TeGD may be detected. According to the phenotypic characteristics, phylogenetic analysis, genomic and chemotaxonomic features, strains DFN5T (= CGMCC 1.19401 T = JCM 35422 T), RDMS1 (= CGMCC 1.19411) and QDMS1 (= CGMCC 1.19410) were classified as a novel species of the genus Halocatena with the proposed name, Halocatena marina sp. nov. This is the first report of the description of a novel filamentous haloarchaeon isolated from marine intertidal zones.

Natrinema caseinilyticum sp. nov., Natrinema gelatinilyticum sp. nov., Natrinema marinum sp. nov., Natrinema zhouii sp. nov., extremely halophilic archaea isolated from marine environments and a salt mine.

Four extremely halophilic archaeal strains (ZJ2T, BND6T, DT87T, and YPL30T) were isolated from marine environments and a salt mine in China. The 16S rRNA and rpoB' gene sequence similarities among strains ZJ2T, BND6T, DT87T, YPL30T and the current species of Natrinema were 93.2-99.3% and 89.2-95.8%, respectively. Both phylogenetic and phylogenomic analyses revealed that strains ZJ2T, BND6T, DT87T, and YPL30T cluster with the Natrinema members. The overall genome-related indexes (ANI, isDDH, and AAI) among these four strains and the current species of genus Natrinema were 70-88%, 22-43% and 75-89%, respectively, clearly below the threshold values for species boundary. Strains ZJ2T, BND6T, DT87T, and YPL30T could be distinguished from the related species according to differential phenotypic characteristics. The major polar lipids of the four strains were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), sulfated mannosyl glucosyl diether (S-DGD-1), and disulfated mannosyl glucosyl diether (S2-DGD). The phenotypic, chemotaxonomic, phylogenetic and phylogenomic features indicated that strains ZJ2T (= CGMCC 1.18786 T = JCM 34918 T), BND6T (= CGMCC 1.18777 T = JCM 34909 T), DT87T (= CGMCC 1.18921 T = JCM 35420 T), and YPL30T (= CGMCC 1.15337 T = JCM 31113 T) represent four novel species of the genus Natrinema, for which the names, Natrinema caseinilyticum sp. nov., Natrinema gelatinilyticum sp. nov., Natrinema marinum sp. nov., and Natrinema zhouii sp. nov., are proposed.

Halorubrum hochsteinianum sp. nov., an ancient haloarchaeon from a natural experiment.

A single extremely halophilic strain was isolated from salt brine produced when a fresh water lake flooded a large salt mine located beneath the lake. The water that entered this mine contained less than 0.34 M NaCl, but over time, this sealed brine became saturated by Cenozoic age salt (121-125 million-year BCE). The isolated strain requires at least 1.7 M NaCl for survival and grows optimally in 3.1 M NaCl. Therefore, it could not have survived or been present in the waters that flooded this salt mine. The strain grows at a pH range from 6.5 to 9.0 and has a wide tolerance to temperatures from 25 ℃ to at least 60 ℃. The comparison of 16S rRNA and rpoB' genes revealed that strain 1-13-28T is related to Halorubrum tebenquichense DSM 14210T showing 98.6% and 98.1% similarities, respectively. Phylogenetic analyses based on 16S rRNA, rpoB' genes and 122 concatenated archaeal genes show that the strain 1-13-28T consistently forms a cluster with Halorubrum tebenquichense of the genus Halorubrum. Strain 1-13-28T contained sulfated mannosyl glucosyl diether, and the polar lipid profile was identical to those of most Halorubrum species. Based on the overall combination of physiological, phylogenetic, polar lipids and phylogenomic characteristics, strain 1-13-28T (= ATCC 700083T = CGMCC 1.62627T) represents a newly identified species within the genus Halorubrum for which the name Halorubrum hochsteinianum is proposed.