Halorubellus salinus gen. nov., sp. nov. and Halorubellus litoreus sp. nov., novel halophilic archaea isolated from a marine solar saltern

Two extremely halophilic archaeal strains GX3(T) and GX26(T) were isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells from the two strains were pleomorphic and stained Gram-negative, colonies were red-pigmented. Strains GX3(T) and GX26(T) were able to grow at 25-50 °C (optimum 37 °C), at 1.4-5.1M NaCl (optimum 3.1M), at pH 5.5-9.5 (optimum pH 7.0) and neither strain required Mg(2+) for growth. Cells lyse in distilled water and the minimal NaCl concentration to prevent cell-lysis was 8% (w/v). The major polar lipids of the two strains were PA (phosphatidic acid), PG (phosphatidylglycerol), PGP-Me (phosphatidylglycerol phosphate methyl ester) and three major glycolipids (GL1, GL2 & GL3) chromatographically identical to S-TGD-1 (sulfated galactosyl mannosy glucosyl diether), S-DGD-1 (sulfated mannosyl glucosyl diether), and DGD-1 (mannosyl glucosyl diether) respectively, an unidentified lipid (GL4) was also detected in strain GX26(T). Phylogenetic analysis based on 16S rRNA gene revealed that strain GX3(T) and strain GX26(T) formed a distinct clade with the closest relative, Haladaptatus paucihalophilus (89.9-92.4% and 90.4-92.7, respectively). The rpoB' gene similarities between strains GX3(T) and GX26(T), and between the two strains and the closest relative, Halorussus rarus TBN4(T) are 96.5%, 84.3% and 83.9%, respectively. The DNA G+C contents of strain GX3(T) and strain GX26(T) are 67.3 mol% and 67.2 mol%, respectively. The DNA-DNA hybridization value between strain GX3(T) and strain GX26(T) was 44%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX3(T) and strain GX26(T) represent two novel species in a new genus within the family Halobacteriaceae, Halorubellus salinus gen. nov., sp. nov. (type strain GX3(T)=CGMCC 1.10384(T)=JCM 17115(T)) and Halorubellus litoreus sp. nov. (type strain GX26(T)=CGMCC 1.10386(T)=JCM 17117(T)).     

Ultrastructure and phylogenetic placement within Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate Pleurostomum flabellatum (Ruinen 1938)

Although Pleurostomum was described almost a century ago, flagellates assigned to this taxon have been recorded only in very occasional faunistic studies of highly saline habitats, and their phylogenetic position has remained uncertain. We report the cultivation, ultrastructure, and phylogenetic relationships of Pleurostomum flabellatum isolated from a Korean saltern pond of 313 per thousand salinity. This isolate is biflagellated with a cytostomal groove, and is not distinguishable from previous accounts of P. flabellatum from saturated brines in India and Australia. Pleurostomum flabellatum shows ultrastructural features characteristic of many Heterolobosea: (1) a striated rhizoplast, (2) an absence of stacked Golgi bodies, (3) parallel basal bodies and flagella, and (4) a large number of peripheral microtubules supporting a rostrum. 18S rRNA gene phylogenies strongly confirm the affinities of P. flabellatum within Heterolobosea. Furthermore, the 18S rRNA gene of P. flabellatum has the heterolobosean-specific helix 17_1, and a group I intron in the same position as in Acrasis rosea. Within Heterolobosea, the 'amoeboflagellate' genera Naegleria and Willaertia were its closest relatives with high bootstrap support and posterior probability. P. flabellatum was observed only as a flagellate, and never as an amoeba. Since light microscopy and electron microscopy observations indicate that P. flabellatum flagellates are capable both of feeding and division, there might be no amoeba stage. Being morphologically distinct from its closest relatives and phylogenetically distant from other flagellate-only Heterolobosea, P. flabellatum cannot be moved into any previously described heterolobosean genus. Instead, we move Pleurostomum into Heterolobosea, and assign as the type species Pleurostomum salinum Namyslowski 1913, a species that closely resembles P. flabellatum. The optimal temperature for growth of P. flabellatum is 40 degrees C. Interestingly, P. flabellatum grows optimally at 300 per thousand salinity and fails to grow below 200 per thousand salinity, indicating that it is an 'extreme halophile'. The optimal salinity for growth is the highest for any eukaryote examined to date.     

A new class of the stramenopiles,Placididea Classis nova: description of Placidia cafeteriopsis gen. et sp. nov

A marine flagellate resembling Cafeteria roenbergensis (bicosoecids, stramenopiles) in cell shape and behavior of the cell while attached to substratum was collected from the coast of Japan. The flagellate was examined by light and electron microscopy, and the 18S rDNA was sequenced to elucidate its taxonomic and phylogenetic position. Ultrastructural features suggested that the flagellate is not a bicosoecid, but a relative of the recently described stramenopile, Wobblia lunata. 18S rDNA phylogenetic trees also revealed that the flagellate forms a monophyletic clade with W. lunata and that it is distantly related to Cafeteria and other bicosoecids. The flagellate differs from W. lunata due to its lack of wobbling motion as well as intracellular features such as the number of mitochondria, flagellar apparatus architecture, the presence of a paranuclear body and cytoplasmic microtubules. The similarity of 18S rDNA sequences was 81% between the flagellate and W. lunata. This new flagellate was described as Placidia cafeteriopsis gen. et sp. nov. Because the phylogenetic lineage comprised of W. lunata and P. cafeteriopsis was one of the major, deep-branching clades of the stramenopiles, the class Placididea (= Placidiophyceae) classis nova was proposed.     

海滨盐薄片菌——分离自海洋盐田的一个嗜盐古菌新种

【目的】研究分离自我国华南地区阳江盐田的一株嗜盐古菌菌株YJ-41^T,探究其分类学地位。【方法】运用多相分类学方法即通过表型和遗传型特征鉴定,研究菌株YJ-41^T 的分类学地位。【结果】菌株YJ-41^T 的细胞为杆状、革兰氏染色阴性、菌落呈红色。菌株YJ-41^T 的生长温度范围20–50 ℃ (最适为37 ℃)、NaCl 浓度范围2.1–4.8 mol/L (最适为3.1 mol/L)、MgCl₂浓度范围0–1.0 mol/L (最适为0.05 mol/L)、pH 范围5.0–9.0 (最适为pH 7.0)。细胞在蒸馏水中会裂解,维持细胞形态的最低NaCl 浓度为10% (质量体积比)。菌株YJ-41^T 的极性脂为磷脂酸(Phosphatidic acid,PA)、磷脂酰甘油(Phosphatidyl glycerol,PG)、磷脂酰甘油磷酸甲基酯(Phosphatidyl glycerol phosphate methyl ester,PGP-Me)、磷脂酰甘油硫酸酯(Phosphatidylglycerol sulphate,PGS)和8 种糖脂;其中3 种糖脂为硫酸甘露糖苷葡萄糖二醚(Sulfated mannosylglucosyl diether,S-DGD-1)、半乳糖苷甘露糖苷葡萄糖二醚(Galactosyl mannosy glucosyl diether,TGD-1)和甘露糖苷葡萄糖二醚(Mannosyl glucosyl diether,DGD-1),其余为未知糖脂。菌株YJ-41^T 的16S rRNA 基因和rpoB''基因与盐薄片菌属(Halolamina)的成员相关基因相似性分别为97.5%–98.4%和93.1%–94.4%。菌株YJ-41^T 的G+C mol%为61.4 mol%。【结论】表型、化学分类和系统发育的特性表明,菌株YJ-41^T (=CGMCC 1.12859^T=JCM 30237^T)代表Halolamina 属的一个新种,建议命名为海滨盐薄片菌(Halolamina litorea)。     

The Extremophile Endolithella mcmurdoensis gen. et sp. nov. (Trebouxiophyceae,Chlorellaceae), A New Chlorella-like Endolithic Alga From Antarctica

The McMurdo Dry Valleys constitute the largest ice-free region of Antarctica and one of the most extreme deserts on Earth. Despite the low temperatures, dry and poor soils and katabatic winds, some microbes are able to take advantage of endolithic microenvironments, inhabiting the pore spaces of soil and constituting photosynthesis-based communities. We isolated a green microalga, Endolithella mcmurdoensis gen. et sp. nov, from an endolithic sandstone sample collected in the McMurdo Dry Valleys (Victoria Land, East Antarctica) during the K020 expedition, in January 2013. The single non-axenic isolate (E. mcmurdoensis LEGE Z-009) exhibits cup-shaped chloroplasts, electron-dense bodies, and polyphosphate granules but our analysis did not reveal any diagnostic morphological characters. On the basis of phylogenetic analysis of the 18S rRNA (SSU) gene, the isolate was found to represent a new genus within the family Chlorellaceae.     

Natronomonas gomsonensis sp. nov., isolated from a solar saltern

A halophilic archaeal strain, SA3^T, was isolated from sediment of a solar saltern in Gomso Bay, Republic of Korea. Cells of strain SA3^T were observed to be coccoid-shaped, to lyse in distilled water, Gram stain-negative and to form red-pigmented colonies. Strain SA3^T was found to require at least 18 % (w/v) NaCl for growth. Optimal growth was observed at 24 % (w/v) NaCl and 6 % (w/v) MgCl₂. The optimum pH and temperature for growth were determined to be pH 7.0 and 40 °C, respectively, while the strain was found to grow within pH and temperature ranges of 5.5–8.0 and 20–45 °C, respectively. The polar lipids were determined to consist of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, unidentified phosphoglycolipids and unidentified phospholipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SA3^T was most closely related to the members of the genus Natronomonas, Natronomonas moolapensis JCM 14361^T (95.2 %) and Natronomonas pharaonis JCM 8858^T (95.1 %). The genomic DNA G+C content (61.8 mol%) determined for strain SA3^T was slightly lower than those of N. moolapensis JCM 14361^T (63.4 mol%) and N. pharaonis JCM 8858^T (64.3 mol%). DNA–DNA hybridization values between N. moolapensis JCM 14361^T and N. pharaonis JCM 8858^T and strain SA3^T were <20 %. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe a new species of the genus Natronomonas, represented by strain SA3^T (=JCM 17867^T = KCTC 4088^T), for which we propose the name Natronomonas gomsonensis sp. nov.     

Haloplanus rubicundus sp. nov., an extremely halophilic archaeon isolated from solar salt

Two extremely halophilic archaea strains, CBA1112^T and CBA1113, were isolated from solar salt in Korea. The genome sizes and G + C content of CBA1112^T and CBA1113 were 3.77 and 3.53 Mb, and 66.0 and 66.5 mol%, respectively. Phylogenetic analysis based on closely related taxa and environmental Haloplanus sequences indicated that both CBA1112^T and CBA1113 strains are grouped within the genus Haloplanus. OrthoANI and in silico DNA–DNA hybridization values were below the species delineation threshold. Pan-genomic analysis showed that the two novel strains and four reference strains had 6203 pan-orthologous groups in total. Six Haloplanus strains shared 1728 core pan-genome orthologous groups, which were mainly associated with amino acid transport and metabolism and translation, ribosomal structure and biogenesis categories, and amino acid metabolism and carbohydrate metabolism related categories. The novel strain-specific pan-genome orthologous groups were mainly involved with replication, recombination and repair category and replication and repair pathway or amino acid metabolism pathway. Cells of both strains were Gram-negative and pleomorphic, and colonies were red-pigmented. The major polar lipids of both strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and one glycolipid, sulfated mannosyl glucosyl diether. Based on genomic, phylogenetic, phenotypic, and chemotaxonomic features, strains CBA1112^T and CBA1113 are described as novel species of the genus Haloplanus. Thus, we propose the name Haloplanus rubicundus sp. nov. The type strain is CBA1112^T (=KCCM 43224^T = JCM 30475^T).     

Halobellus ruber sp. nov., a deep red-pigmented extremely halophilic archaeon isolated from a Korean solar saltern

A novel halophilic archaeon, strain MBLA0160^T, was isolated from a solar saltern in Sorae, Republic of Korea. The cells are deep-red pigmented, Gram-negative, rod shaped, motile, and lysed in distilled water. The strain MBLA0160^T grew at 25–45 °C (optimum 37 °C), in 15–30% (w/v) NaCl (optimum 20%) and 0.1–1.0 M MgCl₂ (optimum 0.3–0.5 M) at pH 5.0–9.0 (optimum 7.0). Phylogenetic analysis based on the 16S rRNA sequence showed that this strain was related to two species within the genus Halobellus (Hbs.), with 98.4% and 95.8% similarity to Hbs. salinus CSW2.24.4 ^T and Hbs. clavatus TNN18^T, respectively. The major polar lipids of the strain MBLA160^T were phosphatidylglycerol, phosphatidylglycerol sulfate, and phosphatidylglycerol phosphate methyl ester. The genome size, G?+?C content, and N50 value of MBLA0160^T were 3.49 Mb, 66.5 mol%, and 620,127 bp, respectively. According to predicted functional proteins of strain MBLA0160^T, the highest category was amino acid transport and metabolism. Genome rapid annotation showed that amino acid and derivatives was the most subsystem feature counts. Pan-genomic analysis showed that strain MBLA0160^T had 97 annotated unique KEGG, which were mainly included metabolism and environmental information processing. Ortholog average nucleotide identities (OrthoANI) and in silico DNA-DNA hybridization (isDDH) values between the strain MBLA0160^T and other strains of the genus Halobellus were under 84,4% and 28.1%, respectively. The genome of strain MBLA0160^T also contain the biosynthetic gene cluster for C₅₀ carotenoid as secondary metabolite. Based on the phylogenetic, phenotypic, chemotaxonomic properties, and comparative genomic analyses, strain MBLA0160^T is considered to represent a novel species of the genus Halobellus, for which the name Halobellus ruber sp. nov. is proposed. The type strain is MBLA0160^T (=?KCTC 4291 ^T?=?JCM 34172 ^T).

     

Chromohalobacter moromii sp. nov., a moderately halophilic bacterium isolated from lupine-based moromi fermentation

Three moderately halophilic strains, TMW 2.2308^T, TMW 2.2299 and TMW 2.2304, were isolated from a lupine-based moromi fermentation. Initial identification based on their low molecular sub-proteome using mass spectrometry showed relation to the genus Halomonas, however, low score values indicated novelty. The comparison of 16S rRNA gene sequences placed these strains within the genus Chromohalobacter with C. japonicus CECT 7219^T (99.67% 16S rRNA sequence similarity to strain TMW 2.2308^T), C. canadensis DSM 6769^T (99.54%) and C. beijerinckii LMG 2148^T (99.32%) being their closest relatives. However, average nucleotide highest identity values of TMW 2.2308^T to C. beijerinckii LMG 2148^T of 93.12% and 92.88% to C. japonicus CECT 7219^T demonstrate that it represents a novel species within the genus Chromohalobacter with additional strains TMW 2.2299 (96.91%) and TMW 2.2304 (96.98%). The isolated strains were non-spore-forming, motile and able to grow at temperatures from 5 to 45 °C with an optimum at 37 °C. Growth of TMW 2.2308^T occurs at 5 to 25% (w/v) NaCl with optimum growth between 10 and 12.5%. The genome of TMW 2.2308^T has a size of 3.47 Mb and a G + C content of 61.0 mol%. The polyphasic evidence lead to the classification of TMW 2.2308^T, TMW 2.2299 and TMW 2.2304 as members of a novel species of the genus Chromohalobacter. We propose a novel species as Chromohalobacter moromii sp. nov., with TMW 2.2308^T (=DSM 113153^T =CECT 30422^T) as the type strain.     

Haloarchaeobius litoreus sp. nov., isolated from a marine solar saltern

Two extremely halophilic archaeal strains GX1^T and GX60 were isolated from the Gangxi marine solar saltern, China. Cells from the two strains were observed to be rod-shaped and stained Gram-negative, with red-pigmented colonies. Strains GX1^T and GX60 were found to be able to grow at 25–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.6 M), at pH 5.5–9.5 (optimum pH 7.0) and neither strain required Mg²⁺ for growth. The cells lysed in distilled water and the minimal NaCl concentration to prevent cell-lysis was found to be 8 % (w/v). The major polar lipids of the two strains were identified as phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and three glycolipids chromatographically identical to those of Haloarchaeobius iranensis IBRC-M 10013^T. 16S rRNA gene analysis revealed that each strain had two dissimilar 16S rRNA genes and both strains were phylogenetically related to Hab. iranensis IBRC-M 10013^T (94.9–98.9 % nucleotide identity). The rpoB′ gene similarity between strains GX1^T and GX60, and between these strains and Hab. iranensis IBRC-M 10013^T were found to be 99.6, 96.0 and 95.8 %, respectively. The DNA G + C content of strain GX1^T and GX60 were determined to be 67.7 and 67.8 mol %, respectively. The DNA–DNA hybridization value of strains GX1^T and GX60 was 86 % and the two strains showed low DNA–DNA relatedness with Hab. iranensis IBRC-M 10013^T (38 and 32 %). It was concluded that strain GX1^T (= CGMCC 1.10390^T = JCM 17114^T) and strain GX60 (= CGMCC 1.10389 = JCM 17120) represent a new species of Haloarchaeobius, for which the name Haloarchaeobius litoreus sp. nov. is proposed.     

Halorubrum salinum sp. nov., isolated from a marine solar saltern

The halophilic archaeal strain GX71^T was isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells of the strain were pleomorphic and lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. Strain GX71^T was able to grow at 25–45 °C (optimum 30 °C), in the presence of 1.7–4.8 M NaCl (optimum 2.6 M NaCl), with 0.005–0.7 M MgCl₂ (optimum 0.05 M MgCl₂) and at pH 5.5–9.5 (optimum pH 7.0–7.5). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 10 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-3) and an unidentified lipid was also detected. The 16S rRNA gene sequence of strain GX71^T showed 94.0–97.0 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB′ gene sequence of strain GX71^T was 87.3–93.4 % similarity to current members of the genus Halorubrum. The DNA G+C content of GX71^T was 67.1 mol%. Strain GX71^T showed low DNA–DNA relatedness with Halorubrum lipolyticum CGMCC 1.5332^T, Halorubrum saccharovorum CGMCC 1.2147^T, Halorubrum kocurii CGMCC 1.7018^T and Halorubrum arcis CGMCC 1.5343^T, the most closely related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX71^T represents a novel species of the genus Halorubrum, for which the name Halorubrum salinum sp. nov. is proposed. The type strain is GX71^T (= CGMCC 1.10458^T = JCM 17093^T).     

Halobacterium rubrum sp. nov., isolated from a marine solar saltern

Halophilic archaeal strain TGN-42-S1^T was isolated from the Tanggu marine solar saltern, China. Cells from strain TGN-42-S1^T were observed to be pleomorphic rods, stained Gram-negative, and formed red-pigmented colonies on solid media. Strain TGN-42-S1^T was found to be able to grow at 20–50 °C (optimum 35–37 °C), at 1.7–4.8 M NaCl (optimum 3.1 M), at 0–1.0 M MgCl₂ (optimum 0.1 M), and at pH 5.0–9.0 (optimum pH 7.0–7.5). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell-lysis was found to be 10 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, galactosyl mannosyl glucosyl diether (TGD-1), sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), sulfated galactosyl mannosyl galactofuranosyl glucosyl diether (S-TeGD), and three unidentified glycolipids which were chromatographically identical to those of the Halobacterium species. The 16S rRNA gene and rpoB′ gene of strain TGN-42-S1^T were phylogenetically related to the corresponding genes of Halobacterium jilantaiense CGMCC 1.5337^T (98.8 and 93.5 % nucleotide identity, respectively), Halobacterium salinarum CGMCC 1.1958^T (98.4 and 91.9 %), and Halobacterium noricense JCM 15102^T (96.9 and 91.1 %). The DNA G + C content of strain TGN-42-S1^T was determined to be 69.2 mol %. Strain TGN-42-S1^T showed low DNA–DNA relatedness with Hbt. jilantaiense CGMCC 1.5337^T and Hbt. salinarum CGMCC 1.1958^T, the most closely related members of the genus Halobacterium. The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain TGN-42-S1^T (=CGMCC 1.12575^T =JCM 19908^T) represents a new species of Halobacterium, for which the name Halobacterium rubrum sp. nov. is proposed.     

Prokaryotic diversity of a Tunisian multipond solar saltern

16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied. A. Sghir and E. Ammar have equally contributed to this work.