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.

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.

Halobaculum rubrum sp. nov., an extremely halophilic archaeon isolated from a salt lake.

A halophilic archaeal strain, designated C46T, was isolated from an inland salt lake in Qinghai Province, PR China. Results of phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C46T belongs to the genus Halobaculum, and the closest phylogenetic relative is Halobaculum gomorrense DSM 9297T with 97.7 % similarity. Despite this, strain C46T was more related to Halobaculum saliterrae WSA2T than other members of the genus Halobaculum based on genome comparison and analysis, and the average nucleotide identity, in silico DNA-DNA hybridization, amino acid identity and percentage of conserved protein values between the two strains were 89.1, 53.3, 89.2 and 75.6 %, respectively, which are lower than the cutoff values proposed for species delimitation. The physiological, biochemical, genetic and genomic characteristics of strain C46T were different from those of its closest phylogenetic neighbours, which indicated that this strain represents a novel species of the genus Halobaculum, for which the name Halobaculum rubrum sp. nov. is proposed. The type strain is C46T (=CGMCC 1.13737T=JCM 32959T).

Halosimplex halophilum sp. nov. and Halosimplex salinum sp. nov., isolated from saline soil and a salt mine.

A polyphasic study was undertaken to determine the taxonomic position of two halophilic archaeal strains, TH32T and YPL4T, isolated from saline soil and a salt mine in PR China, respectively. Strains TH32T and YPL4T both have two dissimilar 16S rRNA genes. The two strains exhibited sequence similarities of 91.5-95.5 % for 16S rRNA genes and 90.9 % for the rpoB' gene. Sequence similarities of 16S rRNA genes and the rpoB' gene between the two strains and the current four members of Halosimplex were 90.6-97.4 % and 91.4-93.5 %, respectively. Phylogenetic analysis revealed that the two strains formed different branches separating them from the current Halosimplex members. Several phenotypic characteristics differentiate strains TH32T and YPL4T from current Halosimplex members. The polar lipids of the two strains are phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and four glycolipids. Two of the glycolipids are chromatographically identical to disulfated mannosyl glucosyl diether and sulfated mannosyl glucosyl diether, respectively, and the remaining two glycolipids are unidentified. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values between the two strains and the current members of Halosimplex (ANI 80.4-89.2 % and in silico DDH 24.0-41.8 %) were much lower than the threshold values proposed as a species boundary, suggesting that the two strains represent novel species of Halosimplex. The values between the two strains (ANI 81.3 % and in silico DDH 24.9 %) were also much lower than the recommended threshold values, which revealed that the two strains represent two genomically different species of Halosimplex. These results showed that strains TH32T (=CGMCC 1.15190T=JCM 30840T) and YPL4T (=CGMCC 1.15329T=JCM 31108T) represent two novel species of Halosimplex, for which the names Halosimplex halophilum sp. nov. and Halosimplex salinum sp. nov. are proposed.

Natronorubrum halalkaliphilum sp. nov., a haloalkaliphilic archaeon isolated from soda lake in Inner Mongolia Autonomous Region, China.

A haloalkaliphilic strain JWXQ-INN-674T was isolated from the water sample of a soda lake in Inner Mongolia Autonomous Region, China. Cells of the strain were coccoid, motile, and strictly aerobic. The strain was able to grow in presence of 2.6-5.4 M NaCl (optimum concentration is 3.4 M) at 30-50 °C (optimum temperature is 42 °C) and pH 7-9.5 (optimum pH is 9.0). The 16S rRNA gene sequence of strain JWXQ-INN-674T showed 95.3-96.6% similarity to members of the genus Natronorubrum of the family Natrialbaceae. The whole genome sequencing of strain JWXQ-INN-674T revealed a genome size of 4.56 M bp and a DNA G + C content of 62.5 mol%. Genome relatedness of strain JWXQ-INN-674T and other species in the genus Natronorubrum was analyzed by average nucleotide identity and digital DNA-DNA hybridization with the values of 76.8-90.6 and 23.1-39.3%, respectively. The strain possessed the polar lipids phosphatidylglycerol and methylated phosphatidylglycerol phosphate lipid. No glycolipids were detected. Based on phylogenetic analysis, phenotypic characteristics, chemotaxonomic properties and genome relatedness, the isolate was proposed as the type strain of a novel species of genus Natronorubrum, Natronorubrum halalkaliphilum sp. nov. (type strain JWXQ-INN-674T = CGMCC 1.17283T = JCM 34245T).

Genomic analysis of facultatively oligotrophic haloarchaea of the genera Halarchaeum, Halorubrum, and Halolamina, isolated from solar salt.

Extremely halophilic archaea (haloarchaea) belonging to the phylum Euryarchaeota have been found in high-salinity environments. In this study, Halarchaeum sp. CBA1220, Halorubrum sp. CBA1229, and Halolamina sp. CBA1230, which are facultatively oligotrophic haloarchaea, were isolated from solar salt by culture under oligotrophic culture conditions. The complete genomes of strains CBA1220, CBA1229, and CBA1230 were sequenced and were found to contain 3,175,875, 3,582,278, and 3,465,332 bp, with a G + C content of 68.25, 67.66, and 66.75 mol %, respectively. In total, 60, 36, and 33 carbohydrate-active enzyme genes were determined in the respective strains. The strains harbored various genes encoding stress-tolerance proteins, including universal stress proteins, cold-shock proteins, and rubrerythrin and rubrerythrin-related proteins. The genome data produced in this study will facilitate further research to improve our understanding of other halophilic strains and promote their industrial application.

Salinadaptatus halalkaliphilus gen. nov., sp. nov., a haloalkaliphilic archaeon isolated from salt pond in Inner Mongolia Autonomous Region, China.

A haloalkaliphilic strain XQ-INN 246T was isolated from the sediment of a salt pond in Inner Mongolia Autonomous Region, China. Cells of the strain were rods, motile and strictly aerobic. The strain was able to grow in the presence of 2.6-5.3 M NaCl (optimum concentration is 4.4 M) at 30-50 °C (optimum temperature is 42 °C) and pH 7.0-10.0 (optimum pH is 8.0-8.5). The whole genome sequencing of strain XQ-INN 246T revealed a genome size of 4.52 Mbp and a DNA G+C content of 62.06 mol%. Phylogenetic tree based on 16S rRNA gene sequences and concatenated amino acid sequences of 122 single-copy conserved proteins revealed a robust lineage of the strain XQ-INN 246T with members of related genera of the family Natrialbaceae. The strain possessed the polar lipids of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Based on phylogenetic analysis, phenotypic characteristics, chemotaxonomic properties and genome relatedness, the isolate was proposed as the type strain of a novel species of a new genus within the family Natrialbaceae, for which the name Salinadaptatus halalkaliphilus gen. nov., sp. nov. is proposed. The type strain is XQ-INN 246T (=CGMCC 1.16692T=JCM 33751T).

Natronomonas halophila sp. nov. and Natronomonas salina sp. nov., two novel halophilic archaea.

Two halophilic archaeal strains, C90T and YPL13T, were isolated from a salt lake and a salt mine in PR China. The two strains were found to form two clusters (97.5 and 89.5 % similarity between them, respectively) separating them from the three current members of the genus Natronomonas (95.4-97.0 % and 86.6-89.3 % similarity, respectively) on the basis of the 16S rRNA and rpoB' gene sequence similarities and phylogenetic analysis. Diverse phenotypic characteristics differentiate strains C90T and YPL13T from current Natronomonas members. The polar lipids of strain C90T were phosphatidic acid, phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulphate, two unidentified glycolipids, a major glycolipid and a minor glycolipid, while those of strain YPL13T were PG, PGP-Me, two unidentified phospholipids and a glycolipid. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between the two strains were 79.8 and 27.1 %, respectively, which were much lower than the threshold values proposed as a species boundaries (ANI 95-96 % and isDDH 70 %), which revealed that the two strains represent two novel species; these values (ANI 76.6-80.0 % and isDDH 21.6-27.0 %) of the strains examined in this study and the current members of Natronomonas are much lower than the recommended threshold values, suggesting that strains C90T and YPL13T represent two genomically different species of Natronomonas. These results showed that strains C90T (=CGMCC 1.13738T=JCM 32961T) and YPL13T (=CGMCC 1.13884T=JCM 31111T) represent two novel species of Natronomonas, for which the names Natronomonas halophila sp. nov. and Natronomonas salina sp. nov. are proposed.

MALDI-TOF analysis of a novel extremophile peptide purified from Halarchaeum acidiphilum ASDL78 with antiarchaeal and antibacterial activities.

In hypersaline environments, halophilic archaea synthesize antimicrobial substances called halocins. There is a promise to make new drugs for antibiotic-resistant strains. Here, we report the antibacterial activity of a new haloarchaea selected from Lut Desert, Iran. A total of 38 isolated halophilic bacteria and archaea were screened for the antagonistic activity test of each strain against other bacterial and archaeal strains. Finally, a strain, recognized as Halarchaeum acidiphilum, with a fast grown strain and high antagonistic potential against different strains was identified by morphological, physiological, and molecular characteristics. The halocin was produced in a semisolid submerge medium and partially purified by heat treatments and molecular weight ultrafiltration cutoff (3, 50, and 10 kDa). It was a cell-free, heat-resistant (85°C for 2 h) protein with a molecular mass near to 20 kDa produced at the endpoint of logarithmic growth. The molecular weight of halocin was 17 kDa, and indicated no apparent homology with known halocins, suggesting that this might be a new halocin. Therefore, a new strain belonging to Halarchaeum genus was isolated and characterized here that produced an antimicrobial and anti-haloarchaea halocin.

Halobacterium bonnevillei sp. nov., Halobaculum saliterrae sp. nov. and Halovenus carboxidivorans sp. nov., three novel carbon monoxide-oxidizing Halobacteria from saline crusts and soils.

Three novel carbon monoxide-oxidizing Halobacteria were isolated from Bonneville Salt Flats (Utah, USA) salt crusts and nearby saline soils. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains PCN9T, WSA2T and WSH3T belong to the genera Halobacterium, Halobaculum and Halovenus, respectively. Strains PCN9T, WSA2T and WSH3T grew optimally at 40 °C (PCN9T) or 50 °C (WSA2T, WSH3T). NaCl optima were 3 M (PCN9T, WSA2T) or 4 M NaCl (WSH3T). Carbon monoxide was oxidized by all isolates, each of which contained a molybdenum-dependent CO dehydrogenase. G+C contents for the three respective isolates were 66.75, 67.62, and 63.97 mol% as derived from genome analyses. The closest phylogenetic relatives for PCN9T, WSA2T and WSH3T were Halobacterium noricense A1T, Halobaculum roseum D90T and Halovenus aranensis EB27T with 98.71, 98.19 and 95.95 % 16S rRNA gene sequence similarities, respectively. Genome comparisons of PCN9T with Halobacterium noricense A1T yielded an average nucleotide identity (ANI) of 82.0% and a digital DNA-DNA hybridization (dDDH) value of 25.7 %; comparisons of WSA2T with Halobaculum roseum D90T yielded ANI and dDDH values of 86.34 and 31.1 %, respectively. The ANI value for a comparison of WSH3T with Halovenus aranensis EB27T was 75.2 %. Physiological, biochemical, genetic and genomic characteristics of PCN9T, WSA2T and WSH3T differentiated them from their closest phylogenetic neighbours and indicated that they represent novel species for which the names Halobaculum bonnevillei, Halobaculum saliterrae and Halovenus carboxidivorans are proposed, respectively. The type strains are PCN9T (=JCM 32472=LMG 31022=ATCC TSD-126), WSA2T (=JCM 32473=ATCC TSD-127) and WSH3T (=JCM 32474=ATCC TSD-128).

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

Two extremely halophilic archaea strains, CBA1112T and CBA1113, were isolated from solar salt in Korea. The genome sizes and G+C content of CBA1112T and CBA1113 were 3.77 and 3.53Mb, and 66.0 and 66.5mol%, respectively. Phylogenetic analysis based on closely related taxa and environmental Haloplanus sequences indicated that both CBA1112T 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 CBA1112T and CBA1113 are described as novel species of the genus Haloplanus. Thus, we propose the name Haloplanus rubicundus sp. nov. The type strain is CBA1112T (=KCCM 43224T=JCM 30475T).

Reclassification of the genus Natronolimnobius: proposal of two new genera, Natronolimnohabitans gen. nov. to accommodate Natronolimnobius innermongolicus and Natrarchaeobaculum gen. nov. to accommodate Natronolimnobius aegyptiacus and Natronolimnobius sulfurireducens.

The genus Natronolimnobius, currently including four species, is a member of the order Natrialbales, class Halobacteria, and consists of obligately alkaliphilic and extremely halophilic members found exclusively in highly alkaline hypersaline soda lakes. The species were classified into this genus mostly based on phylogenetic analysis of the 16S rRNA gene. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein markers clearly indicates a polyphyletic origin of the species included into this genus, thus warranting its reclassification into three separate genera. We therefore propose to transfer Nlb. innermongolicus (type strain N-1311) to a new genus Natronolimnohabitans as Nlh. innermongolicus comb. nov. and to transfer Nlb. aegyptiacus (type strain JW/NM-HA 15) and Nlb. sulfurireducens (type strain AArc1) to a new genus Natrarchaeobaculum as Nbl. aegyptiacum comb. nov. and Nbl. sulfurireducens comb. nov. The phylogenomic differentiation of these four species is also supported by the ANI/AAI distances and unique phenotypes. The most important physiological differences includes a previously unreported ability for cellulose and xylan utilization in Nlb. baerhuensis, thermophily in Nbl. aegyptiacus and anaerobic sulfur respiration in Nbl. sulfurireducens. We further present an emended description of Natronolimnobius baerhuensis.

Halocatena pleomorpha gen. nov. sp. nov., an extremely halophilic archaeon of family Halobacteriaceae isolated from saltpan soil.

A novel archaeal strain designated as SPP-AMP-1T was isolated from saltpan soil, using the serial dilution method on a halophilic archaeal medium supplemented with ampicillin. Cells were both rod-shaped and pleomorphic in nature, non-motile, unable to produce acid from a variety of sugars or grow anaerobically with different substrates (l-arginine) and electron acceptors (DMSO, nitrate). Optimal growth was observed at 42 °C, 3.4-4.2 M NaCl and pH 7.2. Cells did not lyse in distilled water and grew in the absence of Mg2+ ions. Phylogenetic analysis based on the sequences of 16S rRNA gene, amino acid sequence of ß'-subunit of RNA polymerase and 400 conserved proteins retrieved from the whole genome assemblies showed that strain SPP-AMP-1T was distantly related to any existing genera within the family Halobacteriaceae. MK-8 was the only quinone detected. Polar lipid analysis showed a unique combination of diethyl derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, glycosyl-mannosyl-glucosyl diether and sulphated glycosyl-mannosyl-glucosyl diether as the major lipids. The G+C content of genomic DNA is 57.7 mol%. The phenotypic, phylogenetic and genomic data supported the concept of the novel genus status of strain SPP-AMP-1T in the family Halobacteriaceae for which the name Halocatena pleomorpha gen. nov., sp. nov., is proposed; the type strain is SPP-AMP-1T (=JCM 31368T=KCTC 4276T=MTCC 12579T).

Halorussus halophilus sp. nov., A Novel Halophilic Archaeon Isolated from a Marine Solar Saltern.

The halophilic archaeal strain ZS-3T (= CGMCC 1.12866T = JCM 30239T) was isolated from a sediment sample of Zhoushan marine solar saltern, P. R. China. Phylogenetic analyses based on 16S rRNA, rpoB' genes and the concatenation of 738 protein sequences reveal that strain ZS-3T was related to members of the genus Halorussus. The OrthoANI and in silico DDH values between strain ZS-3T and the current Halorussus members are much lower than the threshold values proposed as the species boundary (ANI 95-96% and in silico DDH 70%), suggesting that strain ZS-3T represents a novel species of Halorussus (Halorussus halophilus sp. nov.). Diverse phenotypic characteristics differentiate strain ZS-3T from current Halorussus members. Since the strain expressed diverse hydrolyzing enzyme activity, its complete genome was sequenced. The genome of strain ZS-3T was found to be 4,450,731 bp with total GC content of 61.51%, and comprises one chromosome and three plasmids. A total of 4694 protein coding genes, 43 tRNA genes and 6 rRNA genes were predicted. A CRISPR-Cas system was also detected. The genome encodes sixteen putative glycoside hydrolases, nine extracellular proteases, seventeen aminopeptidases, seven carboxypeptidases, one esterase and one nitrite reductase. The exploration of the hydrolase genes may expand our understanding of adapted mechanism of halophilic archaea surviving optimally in hypersaline environments where containing organic matter. Meanwhile, various hydrolyzing enzymes may extend this microorganism for further applications in salt-based fermentation.

Natronorubrum halophilum sp. nov. isolated from two inland salt lakes.

Two halophilic archaeal strains, SHR37T and NEN6, were isolated from salt lakes located in the Tibet and Xinjiang regions of China. The two strains were found to form a single cluster (99.9% and 99.3% similarity, respectively) separating them from the six current members of Natronorubrum (94.7-96.9% and 86.1-90.8% similarity, respectively) on the basis of the 16S rRNA and rpoB' gene sequence similarities and phylogenetic analysis. Diverse phenotypic characteristics differentiate strains SHR37T and NEN6 from current Natronorubrum members. Their polar lipids are C20C20 and C20C25glycerol diether derivatives of PG, PGP-Me, and a major gycolipid chromatographically identical to disulfated mannosyl glucosyl diether (S2-DGD). Four minor unidentified gycolipids are also present. The OrthoANI and in silico DDH values of the two strains were 97.3% and 76.1%, respectively, which were much higher than the threshold values proposed as a species boundary (ANI 95-96% and in silico DDH 70%), which revealed that the two strains represent one species; the two values (ANI 79.0-81.9% and in silico DDH 23.5-25.7%) of the strains examined in this study and the current members of Natronorubrum are much lower than the recommended threshold values, suggesting that strains SHR37T and NEN6 represent a genomically different species of Natronorubrum. These results showed that strains SHR37T (= CGMCC 1.15233T = JCM 30845T) and NEN6 (= CGMCC 1.17161) represent a novel species of Natronorubrum, for which the name Natronorubrum halophilum sp. nov. is proposed.

Natrialba swarupiae sp. nov., a halophilic archaeon isolated from a hypersaline lake in India.

A haloarchaeal strain (ESP3B_9T) was isolated from the salt pan of Sambhar salt lake, Rajasthan, India. Cells were coccoid, non-motile, Gram-stain-negative and formed reddish-pink pigmented colonies. The strain was aerobic, able to grow at 35-55 °C (optimum, 40 °C), in 20-35 % (25 %) NaCl and pH 8-10 (pH 9). Mg2+ not required for growth. The cells were lysed in distilled water and the minimum NaCl concentration that prevented cell lysis was 5 % w/v. The 16S rRNA gene sequence similarities between strain ESP3B_9T and Natrialba hulunbeirensis JCM 10989T and Natrialba magadii ATCC 43099T were 96.53 and 96.25 % respectively. The similarities of the RNA polymerase subunit B gene between strain ESP3B_9T and N. hulunbeirensis JCM 10989T and N. magadii ATCC 43099T were 84.47 and 84.9 % respectively. Genome sequencing revealed a genome size of 4.20 Mbp with DNA G+C content of 62.5 mol%. The major polar lipids were phosphotidylglycerol and phosphatidylglycerol phosphate methyl esters with minor amounts of unidentified lipids. The results of polyphasic analysis determined that strain ESP3B_9T represents a novel species of the genus Natrialba, for which the name Natrialba swarupiae sp. nov. is proposed. The type strain is ESP3B_9T (MCC 3419T=JCM 33002T=KCTC 4279T=CGMCC 1.16737T).

Halostella pelagica sp. nov. and Halostella litorea sp. nov., isolated from salted brown alga Laminaria.

Three rod-shaped halophilic archaeal strains, DL-M4T, LYG-109 and DLLS-108T, were isolated from the salted brown alga Laminaria produced in different marine areas of PR China. Cells of strains were motile, formed red-pigmented colonies on agar and lysed in distilled water. The three strains grew optimally with 2.6 M NaCl, with 0.05-0.3 M MgCl2, at 37 °C and at pH 7.0-7.5. The results of phylogenetic analyses based on the 16S rRNA and rpoB' genes differentiated these strains into two clusters belonging to the genus Halostella, which currently contains Halostella salina CBA1114T and Halostella limicola LT12T. Strains DL-M4T and LYG-109 formed a single cluster separate from the current two members of Halostella (94.4-95.7 and 90.0-90.9 % similarities, respectively) while strain DLLS-108T had Hsl. salina CBA1114T as its nearest neighbour (97.7-97.8 and 95.9 % similarities, respectively) and was separated from Hsl. limicola LT12T (94.4-95.8 and 93.4 % similarities, respectively). These clusters represented two distinct novel species as indicated by phenotypic characteristics, polar lipid compositions and whole-genome comparisons. Diverse phenotypic characteristics, morphology and growth characteristics, nutrition and miscellaneous biochemical tests differentiate strains DL-M4T, LYG-109, DLLS-108T from Hsl. limicola LT12T and Hsl. salina CBA1114T. Strains DL-M4T and LYG-109 contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three unidentified glycolipids, while strain DLLS-108T contained these polar lipids and two unidentified phospholipids. The major respiratory quinones detected in the three isolates were menaquinone MK-8 and MK-8(H2). The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between the isolated strains and the current two members of Halostella were found to be 79.3-86.6 (ANI) and 22.9-49.8 % (isDDH). All these results showed that the three isolates represent two novel species of the genus Halostella for which the names Halostella pelagica sp. nov. [type strain dl-M4T (=CGMCC 1.13603T=JCM 32954T)] and Halostella litorea sp. nov. [type strain DLLS-108T(=CGMCC 1.13610T=JCM 32955T)] are proposed.

MBSP1: a biosurfactant protein derived from a metagenomic library with activity in oil degradation.

Microorganisms represent the most abundant biomass on the planet; however, because of several cultivation technique limitations, most of this genetic patrimony has been inaccessible. Due to the advent of metagenomic methodologies, such limitations have been overcome. Prevailing over these limitations enabled the genetic pool of non-cultivable microorganisms to be exploited for improvements in the development of biotechnological products. By utilising a metagenomic approach, we identified a new gene related to biosurfactant production and hydrocarbon degradation. Environmental DNA was extracted from soil samples collected on the banks of the Jundiaí River (Natal, Brazil), and a metagenomic library was constructed. Functional screening identified the clone 3C6, which was positive for the biosurfactant protein and revealed an open reading frame (ORF) with high similarity to sequences encoding a hypothetical protein from species of the family Halobacteriaceae. This protein was purified and exhibited biosurfactant activity. Due to these properties, this protein was named metagenomic biosurfactant protein 1 (MBSP1). In addition, E. coli RosettaTM (DE3) strain cells transformed with the MBSP1 clone showed an increase in aliphatic hydrocarbon degradation. In this study, we described a single gene encoding a protein with marked tensoactive properties that can be produced in a host cell, such as Escherichia coli, without substrate dependence. Furthermore, MBSP1 has been demonstrated as the first protein with these characteristics described in the Archaea or Bacteria domains.

Halomicrococcus hydrotolerans gen. nov., sp. nov., an extremely halophilic archaeon isolated from a subterranean salt deposit.

A halophilic archaeon, strain H22T, was isolated from a subterranean salt deposit sampled at Yunnan salt mine, PR China. Colonies of strain H22T were light pink-pigmented. Cells were coccus, non-motile, Gram-stain-negative, and did not lyse in distilled water. The strain was aerobic and grew at 20-55 °C (optimum, 37 °C), in the presence of 10-30 % (w/v) NaCl (20 %) and at pH 6.5-9.0 (pH 7.0). Mg2+ was required for growth (optimum, 0.005 M). Major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and sulfated mannosyl-glucosyl-glycerol diether-1. Sequence similarity search based on the multiple 16S rRNA genes (rrnA, rrnB and rrnC) of strain H22T revealed that it was most closely related to species of the genera Haloarchaeobius, Haladaptatus, Halorussus and Halorubellus with relative low sequence similarities (91.9-93.7 %). The strain, however, shared highest rpoB' gene sequence identities with Halorussus rarus TBN4T (90.8 % rpoB' gene sequence similarity). Phylogenetic trees based on 16S rRNA and rpoB' gene sequences revealed a robust lineage of the strain H22T with members of related genera of the family Halobacteriaceae. The DNA G+C content of strain H22T was 62.9 mol%. Genome-based analysis of average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) between strains H22T and its closest relative were equal or lower than 77.7 and 22.4 %, respectively, which were far below the threshold for delineation of a new species. Based on ANI values, in silico DDH, and distinct morphological and physiological differences from the previously described taxa, we suggest that strain H22T represents a novel species of a new genus within the family Halobacteriaceae, for which the name Halomicrococcus hydrotolerans gen. nov., sp. nov. is proposed. The type strain is H22T (=CGMCC 1.16291T=NBRC 113231T).

Halorientalis pallida sp. nov., an extremely halophilic archaeon isolated from a marine saltern.

An extremely halophilic archaeon, strain F13-25T, was isolated from a marine saltern located in Isla Cristina, Huelva, on the south-west coast of Spain. The novel strain had pink-pigmented, non-motile, coccoid cells. Optimal growth was achieved at 25 % (w/v) NaCl, pH 7.5 and 37 °C. Strain F13-25T possessed two heterogeneous 16S rRNA genes (rrnA and rrnB) most closely related to Halorientalis persicus D108T (97.6-99.2 % sequence similarity) and Halorientalis regularis TNN28T (95.9-98.8 %). On the basis of the results of rpoB' gene sequence analysis, strain F13-25T was also closely related to Halorientalis persicus IBRC-M 10043T (89.9 %) and Halorientalis regularis TNN28T (92.3 %). Relatedness values, computed using the Genome-to-Genome Distance Calculator, between strain F13-25T and Halorientalis persicus IBRC-M 10043T and Halorientalis regularis IBRC-M 10760T were 34.6 and 36.2 %, respectively. Average nucleotide identity values based on orthoANI, ANIb and ANIm of strain F13-25T and Halorientalis persicus IBRC-M 10043T and Halorientalisregularis IBRC-M 10760T were 88.0 and 88.8, 87.1 and 87.6 %, and 89.2 and 89.6 %, respectively. All values were far below the threshold accepted for prokaryotic species delineation. The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and one glycolipid chromatographically identical to sulfated diglycosyl diether. The DNA G+C content was 65.7 mol% (genome). The results of phylogenetic, phenotypic and chemotaxonomic analyses indicated that strain F13-25T represents a novel species of the genus Halorientalis, for which the name Halorientalis pallida sp. nov., with type strain F13-25T (=CECT 9384T=IBRC-M 11176T), is proposed.