Phosphatidylglycerol Phosphate Methyl Ester Research Articles

Genome-based classification of Halobellus rubicundus sp. nov., a novel extremely halophilic archaeon isolated from a Korean solar saltern.

A novel extremely halophilic archaeon designated, MBLA0158T, was isolated from a solar saltern in Sorae, Republic of Korea. The colonies are red-pigmented, Gram-stain-negative, pleomorphic, non-motile, and lysed in distilled water. The strain grows at 25-45°C (optimum, 37°C), in 15-30% (w/v) NaCl (optimum, 20%) and 0.1-1.0M Mg2+ (optimum, 0.2-0.3M) at pH 6.0-10.0 (optimum, 7.0-8.0). Comparative analysis based on the 16S rRNA gene sequence revealed that this strain is most closely related to the Halobellus inordinatus YC20T with a sequence identity of 96.0%. Strain MBLA0158T contained phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester as major polar lipids. The genome size is 3.29Mb and the DNA G + C content is 66.9mol%. Phylogenomic analysis confirmed that strain MBLA0158T is distinct from previously reported type strains of the genus Halobellus. Pan-genome analysis showed that strain MBLA0158T contains 419 genes that are not present in other type strains of the genus Halobellus. Based on overall analyses, strain MBLA0158T is considered to represent a new species of the genus Halobellus, for which the name Halobellus rubicundus sp. nov. is proposed. The type strain is MBLA0158T (= KCTC 4318T = JCM 36642T).

Genome-Based Classification of 'Haloarcula aidinensis' and Description of Three Novel Halophilic Archaea Isolated from an Inland Saline Lake, Coastal Saline Soil, and a Marine Solar Saltern.

'Haloarcula aidinensis' was described by a pioneer Chinese scientist focused on halophilic archaea thirty years ago, and the type strain of 'Haloarcula aidinensis' was recently classified based on phylogenetic, phylogenomic, and comparative genomic analyses. Other three novel halophilic archaeal strains, CK38T, DT43T, and SYNS111T, isolated from diverse saline environments in China, were simultaneously subjected to polyphasic classification. 'Haloarcula aidinensis' A5 was found to be related to Haloarcula amylolytica, while strains CK38T, DT43T, and SYNS111T represented three novel species of Haloarcula based on phylogenetic, phylogenomic, and comparative genomic analyses. These strains can be distinguished from other species within the genus Haloarcula based on multiple phenotypic characteristics. The major phospholipids, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate can be detected in these strains, while the glycolipid profiles of these strains are diverse. Strains A5 and DT43T contained glucosyl mannosyl glucosyl diether and a diglycosyl diether, while other strains CK38T and SYNS111T had sulfated mannosyl glucosyl diether and mannosyl glucosyl diether. Thus, strain A5 should be a reference strain of Har. amylolytica and three novel species of Haloarcula, Haloarcula sediminis sp. nov., Haloarcula brevis sp. nov., and Haloarcula regularis sp. nov. are proposed to accommodate strains CK38T (= CGMCC 1.62732T = JCM 36675T), DT43T (= CGMCC 1.18924T = JCM 36146T), and SYNS111T (= CGMCC 1.62601T = JCM 36149T).

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.

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.

Halomontanus rarus gen. nov., sp. nov., a novel halophilic archaeon of the family Natrialbaceae from salt lakes on the Qinghai-Xizang Plateau

Two halophilic archaeal strains TS33T and KZCA124 were isolated from two distant salt lakes on the Qinghai-Xizang Plateau, respectively. Culture-independent analysis indicated that these two strains were original inhabitants but low abundant taxa in respective salt lakes. Strains TS33T and KZCA124 were able to grow at 20–60 °C (optimum were 42 and 35 °C, respectively), with 0.9–4.8 M NaCl (optimum were 3.0 and 2.6 M, respectively), with 0–0.7 M MgCl2 (optimum, 0.3 M) and at pH 5.0–9.5 (optimum were pH 7.5 and pH 7, respectively). The 16S rRNA and rpoB' gene similarities between these two strains were 99.7% and 99.4%, and these two similarities among strains TS33T, KZCA124, and existing species of the family Natrialbaceae were 90.6–95.5% and 84.4–89.3%, respectively. Phylogenetic and phylogenomic analyses indicated that strains TS33T and KZCA124 formed an independent branch separated from neighboring genera, Saliphagus, Natronosalvus, and Natronobiforma. The averagenucleotideidentity (ANI), digital DNA-DNAhybridization (dDDH), and average amino acid identity (AAI) values between strains TS33T and KZCA124 were 96.4%, 73.1%, and 96.7%, respectively, higher than the thresholds for species demarcation. The overall genome-related indexes between these two strains and existing species of family Natrialbaceae were 73–77%, 21–25%, and 63–70%, respectively, significantly lower than the species boundary thresholds. Strains TS33T and KZCA124 may represent a novel species of a new genus within the family Natrialbaceae judged by the cutoff value of AAI (≤76%) proposed to differentiate genera within the family Natrialbaceae. The major polar lipids of strains TS33T and KZCA124 were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated mannosyl glucosyl diether, and sulfated galactosyl mannosyl glucosyl diether. These two strains could be distinguished from the related genera according to differential phenotypic characteristics. These phenotypic, phylogenetic, and genomic analyses revealed that strains TS33T (=KCTC 4310T = MCCC 4K00132T) and KZCA124 (=CGMCC 1.17432 = JCM 34316) represent a novel species of a new genus of the family Natrialbaceae and were named Halomontanus rarus gen. nov., sp. nov.

Genomic, Physiological, Biochemical, and Phenotypic Evidences Reveal a New Species, Halomicroarcula salaria sp. nov.

An extremely halophilic archaeon strain named FL173T was isolated from a salt mine (Anhui Province, China). Colonies on agar plate are orange-red, moist, and opaque. Cells are motile, Gram-stain-negative, polymorphic, and lyse in distilled water. Cells are able to grow at temperatures, NaCl concentrations, and pH ranging from 20 to 50°C (optimum 42°C), 2.6 to 5.1M NaCl concentration (optimum 3.4M), and 5.5 to 9.5 pH (optimum 7.0), respectively. Mg2+ is not necessary for growth. The major polar lipids of strain FL173T were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfonate (PGS), sulfonated mannosyl glycolipid (S-DGD-1). It has two copies of the 16S rRNA gene, which share the highest sequence similarity (93.04-99.02% sequence similarity) to the 16S rRNA genes of Halomicroarcula salinisoli F24AT, respectively. The rpoB' gene of strain FL173T showed the highest sequence similarity (93.76%) to that of H. salinisoli F24AT. The genome-based analysis showed that the average amino-acid identity (AAI), orthologous average nucleotide identity (ANI) and in silico DNA-DNA hybridization values between strains FL173T and H. salinisoli F24AT were 84.80%, 85.29%, and 29.70%, respectively, which are far below the threshold for the delineation of a prokaryotic new species. The DNA G+C content of strain FL173T is 64.9%. Genomic, physiological, biochemical, and phenotypic evidences showed that strain FL173T (CGMCC 1.18851=NBRC 114260) represents a new species of the genus Halomicroarcula, for which the name Halomicroarcula salaria sp. nov. is proposed.

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.

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.

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.

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.

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.

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.

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 1.18786T = JCM 34918T), BND6T (= CGMCC 1.18777T = JCM 34909T), DT87T (= CGMCC 1.18921T = JCM 35420T), and YPL30T (= CGMCC 1.15337T = JCM 31113T) 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.

Combined effect of the head groups and alkyl chains of archaea lipids when interacting with bacteriorhodopsin

Bacteriorhodopsin (bR), a transmembrane protein with seven α-helices, is highly expressed in the purple membrane (PM) of archaea such as Halobacterium salinarum. It is well known that bR forms two-dimensional crystals with acidic lipids such as phosphatidylglycerol phosphate methyl ester (PGP-Me)—a major component of PM lipids bearing unique chemical structures—methyl-branched alkyl chains, ether linkages, and divalent anionic head groups with two phosphodiester groups. Therefore, we aimed to determine which functional groups of PGP-Me are essential for the boundary lipids of bR and how these functionalities interact with bR. To this end, we compared various well-known phospholipids (PLs) that carry one of the structural features of PGP-Me, and evaluated the affinity of PLs to bR using the centerband-only analysis of rotor-unsynchronized spin echo (COARSE) method in solid-state NMR measurements and thermal shift assays. The results clearly showed that the branched methyl groups of alkyl chains and double negative charges in the head groups are important for PL interactions with bR. We then examined the effect of phospholipids on the monomer–trimer exchange of bR using circular dichroism (CD) spectra. The results indicated that the divalent negative charge in a head group stabilizes the trimer structure, while the branched methyl chains significantly enhance the PLs' affinity for bR, thus dispersing bR trimers in the PM even at high concentrations. Finally, we investigated the effects of PL on the proton-pumping activity of bR based on the decay rate constant of the M intermediate of a bR photocycle. The findings showed that bR activities decreased to 20% in 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), and in 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers as compared to that in PM. Meanwhile, 1,2-Diphytanoyl-sn-glycero-3-phosphate (DPhPA) bilayers bearing both negative charges and branched methyl groups preserved over 80% of the activity. These results strongly suggest that the head groups and alkyl chains of phospholipids are essential for boundary lipids and greatly influence the biological function of bR.

Halocatena salina sp. nov., a filamentous halophilic archaeon isolated from Aiding Salt Lake.

A filamentous cell-shaped halophilic archaeon (strain AD-1T) was isolated from Aiding Salt Lake, PR China. Its colonies on HCM7 agar plates were pinkish white, 1-4 mm (diameter), elevated and round. The optimum conditions for growth were observed at 42 °C, 4.3 M NaCl, 0.01 M MgCl2 and pH 7. Strain AD-1T could hydrolyse Tween 60, Tween 80, starch and gelatin. Phylogenetic analysis based on 16S rRNA gene, rpoB' and the concatenated 484 single-copy orthologous proteins revealed that strain AD-1T formed a clade with Halocatena pleomorpha SPP-AMP-1T. The average nucleotide identity and in silico DNA-DNA hybridization values between strain AD-1T and Halocatena pleomorpha SPP-AMP-1T were both below the species delineation thresholds (95~96 and 70 %, respectively). The major phospholipids of strain AD-1T were phosphatidic acid, phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, while the major glycolipids were sulphated galactosyl mannosyl glucosyl diether, galactosyl mannosyl glucosyl diether and glucosyl mannosyl glucosyl diether. The phenotypic, phylogenetic and genome-based analyses suggested that strain AD-1T (=CGMCC 1.13724T=JCM 32960T) represents a novel species, for which the name Halocatena salina sp. nov. is proposed.

Description of Halosolutus amylolyticus gen. nov., sp. nov., Halosolutus halophilus sp. nov. and Halosolutus gelatinilyticus sp. nov., and genome-based taxonomy of genera Natribaculum and Halovarius.

Three extremely halophilic archaeal strains (LT55T, SQT-29-1T and WLHS5T) were isolated from Gobi saline soil and a salt lake, China. These strains were most related to the genera Natribaculum and Halovarius (92.6-95.1 % similarities), and showed low similarities with other genera within the family Natrialbaceae based on 16S rRNA genes. Phylogenomic analysis confirmed that the three strains formed a distinct clade separated from the related genera Halostagnicola and Natronococcus, which indicated that they may represent a novel genus of the family Natrialbaceae. The average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH) and average amino acid identity (AAI) values among the three strains were no more than 87, 34 and 85 %, respectively, much lower than the threshold values for species demarcation. The major phospholipids of the three strains were phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylglycerol phosphate methyl ester (PGP-Me). The glycolipid profiles of the three strains were diverse; sulfated mannosyl glucosyl diether (S-DGD-1) and disulfated mannosyl glucosyl diether (S2-DGD) were found in strains LT55T and WLHS5T, while mannosyl glucosyl diether (DGD-1) and S-DGD-1 in strain SQT-29-1T. The combination of phenotypic, chemotaxonomic, phylogenetic and genomic analyses suggested that strains WLHS5T (=CGMCC 1.13781T = JCM 33558T), SQT-29-1T (=CGMCC 1.16065T = JCM 33554T) and LT55T (=CGMCC 1.15188T = JCM 30838T) represent three novel species of a new genus within the family Natrialbaceae, for which the names, Halosolutus amylolyticus gen. nov., sp. nov., Halosolutus gelatinilyticus sp. nov. and Halosolutus halophilus sp. nov., are proposed. Genome-based classification of genera Natribaculum and Halovarius revealed that Halovarius luteus should be transferred to the genus Natribaculum as Natribaculum luteum comb. nov. and Natribaculum longum as a heterotypic synonym of Natribaculum breve Liu et al. 2015.

Haladaptatus halobius sp. nov. and Haladaptatus salinisoli sp. nov., two extremely halophilic archaea isolated from Gobi saline soil.

Two extremely halophilic archaeal strains, PSR5T and PSR8T, were isolated from a saline soil sample collected from the Tarim Basin, Xinjiang, PR China. Both strains had two copies of the 16S rRNA genes rrn1 and rrn2, showing 2.6 and 3.9% divergence, respectively. The rrn1 gene of PSR5T showed 98.4 and 95.3% similarity to the rrn1 and rrn2 genes of strain PSR8T; the rrn2 gene of PSR5T displayed 97.4 and 96.7% similarity to those of strain PSR8T, respectively. Phylogenetic analyses based on the 16S rRNA and rpoB' genes revealed that strains PSR5T and PSR8T formed a single cluster, and then tightly clustered with the current four Haladaptatus species (93.5-97.1% similarities for the 16S rRNA gene and 89.3-90.9% similarities for the rpoB' gene, respectively). Several phenotypic characteristics differentiate strains PSR5T and PSR8T from current Haladaptatus members. The polar lipids of the two strains are phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester phosphatidylglycerol sulphate and three glycolipids. One of the glycolipids is sulphated mannosyl glucosyl diether, and the remaining two glycolipids are unidentified. The average nucleotide identity, in silico DNA-DNA hybridization, amino acid identity and percentage of conserved proteins values between the two strains were 88.5, 39.1, 89.3 and 72.8 %, respectively, much lower than the threshold values proposed as a species boundary. These values among the two strains and Haladaptatus members were 77.9-79.2, 22.0-23.5, 75.1-78.2 and 56.8-69.9 %, respectively, much lower than the recommended threshold values for species delimitation. These results suggested that strains PSR5T and PSR8T represent two novel species of Haladaptatus. Based on phenotypic, chemotaxonomic, genomic and phylogenetic properties, strains PSR5T (=CGMCC 1.16851T=JCM 34141T) and PSR8T (=CGMCC 1.17025T=JCM 34142T) represent two novel species of the genus Haladaptatus, for which the names Haladaptatus halobius sp. nov. and Haladaptatus salinisoli sp. nov. are proposed.

Halorussus vallis sp. nov., Halorussus aquaticus sp. nov., Halorussus gelatinilyticus sp. nov., Halorussus limi sp. nov., Halorussus salilacus sp. nov., Halorussus salinisoli sp. nov.: six extremely halophilic archaea isolated from solar saltern, salt lake and saline soil.

Six novel halophilic archaeal strains of XZYJT10T, XZYJ18T, XZYJT40T, XZYJT49T, YCN54T and LT46T were isolated from a solar saltern in Tibet, a salt lake in Shanxi, and a saline soil in Xinjiang, China. Sequence similarities of 16S rRNA and rpoB' genes among strains XZYJT10T, XZYJ18T, XZYJT40T, XZYJT49T, YCN54T, LT46T and current members of Halorussus were 90.6-97.8% and 87.8-96.4%, respectively. The average nucleotide identity and in silico DNA-DNA hybridization values among these six strains and current Halorussus members were in the range of 76.5-87.5% and 21.0-33.8%, respectively. These values were all below the species boundary threshold values. The phylogenomic tree based on 122 conserved archaeal protein marker genes revealed that the six novel strains formed individual distinct branches and clustered tightly with Halorussus members. Several phenotypic characteristics distinguished the six strains from current Halorussus members. Polar lipid analysis showed that the six novel strains contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and two to three glycolipids. Phenotypic, chemotaxonomic and phylogenetic properties showed that the six strains represented six novel species within the genus Halorussus, for which the names Halorussus vallis sp. nov., Halorussus aquaticus sp. nov., Halorussus gelatinilyticus sp. nov., Halorussus limi sp. nov., Halorussus salilacus sp. nov., and Halorussus salinisoli sp. nov. are proposed.

Halorientalis salina sp. nov., Halorientalis marina sp. nov., Halorientalis litorea sp. nov.: three extremely halophilic archaea isolated from a salt lake and coarse sea salt.

Three halophilic archaeal strains, NEN8T, GDY88T and ZY14T, were isolated from a salt lake in Tibet and coarse sea salt samples from Guangdong and Hebei, China, respectively. These strains formed three separate clades (showing 94.4-95.8% and 87.1-89.4% similarities, respectively) and then clustered with the current Halorientalis members (showing 90.7-97.6% and 87.0-91.2% similarities, respectively), as revealed by phylogenetic analyses based on 16S rRNA and rpoB' genes. The overall genome-related index, average nucleotide identity (ANI), in silico DNA-DNA hybridization (DDH), average amino acid identity (AAI) and the percentage of conserved proteins (POCP) values, among the three strains and members of the genus Halorientalis were 76.0-88.0%, 21.3-37.2%, 69.0-88.3% and 57.7-78.1%, clearly below the threshold values for species demarcation. Strains NEN8T, GDY88T and ZY14T could be distinguished from current Halorientalis species according to differential phenotypic characteristics. The major polar lipids of the three strains were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), sulfated mannosyl glucosyl diether (S-DGD-1) and disulfated mannosyl glucosyl diether (S2-DGD). In addition, mannosyl glucosyl diether (DGD-1) was detected in strain NEN8T and phosphatidic acid (PA), posssulfated galactosyl mannosyl glucosyl diether (S-TGD-1) and sulfated mannosyl glucosyl diether-phosphatidic acid (S-DGD-PA) were observed in strain ZY14T. These results revealed that strains NEN8T (= CGMCC 1.17213T = JCM 34155T), GDY88T (= CGMCC 1.18548T = JCM 34481T) and ZY14T (= CGMCC 1.17178T = JCM 34154T) represent three novel species of the genus Halorientalis, for which the names Halorientalis salina sp. nov., Halorientalis marina sp. nov. and Halorientalis litorea sp. nov. are proposed.