Natrinema halophilum sp. nov., Natrinema salinisoli sp. nov., Natrinema amylolyticum sp. nov. and Haloterrigena alkaliphila sp. nov., four extremely halophilic archaea isolated from salt mine, saline soil and salt lake.

Four halophilic archaeal strains, YPL8T, SLN56T, LT61T and KZCA68T, were isolated from a salt mine, saline soil and a salt lake located in different regions of China. Sequence similarities of 16S rRNA and rpoB' genes among strains YPL8T, SLN56T, LT61T and the current members of Natrinema were 94.1-98.2 % and 89.3-95.1 %, respectively, while these values among strain KZCA68T and the current members of Haloterrigena were 97.2-97.4 % and 91.7-91.9 %, respectively. The average nucleotide identity, in silico DNA-DNA hybridization and average amino acid identity values among these four strains and their closely related species were all lower than the threshold values for species boundary. All four strains were unable to hydrolyse casein, gelatin, or Tween 80. Strain YPL8T contained phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), sulfated mannosyl glucosyl diether (S-DGD-1), disulfated mannosyl glucosyl diether (S2-DGD) and sulfated mannosyl glucosyl diether-phosphatidic acid (S-DGD-PA). Strain SLN56T contained PA, PG, phosphatidylglycerol sulphate (PGS), PGP-Me, S-DGD-1, S2-DGD and S-DGD-PA. Strain LT61T contained PA, PG, PGS, PGP-Me, S-DGD-1 and S2-DGD. The phospholipids of strain KZCA68T were PA, PG and PGP-Me. These results showed that strains YPL8T (=CGMCC 1.13883T=JCM 31181T), SLN56T (=CGMCC 1.14945T=JCM 30832T) and LT61T (=CGMCC 1.14942T=JCM 30668T) represent novel species of the genus Natrinema, for which the names, Natrinema halophilum sp. nov., Natrinema salinisoli sp. nov. and Natrinema amylolyticum sp. nov. are proposed. Strain KZCA68T (=CGMCC 1.17211T=JCM 34158T) represents a novel species of Haloterrigena, for which the name Haloterrigena alkaliphila sp. nov. is proposed.

A novel halolysin without C-terminal extension from an extremely halophilic archaeon.

Halolysins are extracellular proteases secreted by halophilic archaea for nutritional purposes. They bear great application potentials in various industries. Yet the diversity of halolysins remains underexplored. In this study, a halolysin from the extremely halophilic archaeon Haladaptatus sp. DYF46 (HlyHap) was identified to be a novel type of halolysin without C-terminal extension (CTE). Addition of the CTE of a halolysin from Halococcus salifodinae to HlyHap did not significantly affect its extracellular proteolytic activity. Mature HlyHap was generated from recombinant HlyHap precursor by high-affinity column refolding. HlyHap displayed optimal activity at 0.25-0.50 M NaCl, 45 °C and pH 8.5-9.0. Interestingly, HlyHap preferred a low salinity and was stable in a broad range of salinity, albeit from an extremely halophilic archaeon. Ca2+ and Mg2+ significantly promoted HlyHap activity. HlyHap activity was stable with organic solvents and detergents. The Km and Vmax values of HlyHap against azocasein were 0.018 mM and 7,179 U/mg, and those against succinyl-Ala-Ala-Pro-Phe-pNA were 0.32 mM and 3×106 µmol/min/µg, respectively. The unusual traits of HlyHap, a novel type of halolysin without CTE, may endow it with strong potential for various industrial uses, such as biocatalysis in fluctuating salinities and aqueous-organic solvent. KEY POINTS: • This is the first report of a novel type of halolysin without C-terminal extension • HlyHap was obtained by heterologous expression and high-affinity column refolding • HlyHap exhibited good salinity tolerance.

Genome-based taxonomy of genera Halomicrobium and Halosiccatus, and description of Halomicrobium salinisoli sp. nov.

The genera Halosiccatus and Halomicrobium are the most closely related genera within the family Haloarculaceae (class Halobacteria). All species of these two genera are closely related to each other in phylogenetic analyses based on their 16S rRNA gene sequences, and also using the sequences of four housekeeping genes. The genus Halosiccatus was proposed based on inferred phylogeny using only one of the three distinct 16S rRNA genes detected in strain DC8T, while Halomicrobium zhouii, one of three species of Halomicrobium, was omitted from the reference species used in these analyses. The related 16S rRNA gene sequence similarities of type strains of Halomicrobium katesii and Halomicrobium mukohataei were as high as 99.5%-99.7%, much higher than the threshold values proposed as species boundaries. These issues could have resulted in taxonomic inaccuracies in the genera Halosiccatus and Halomicrobium, and a thorough study was undertaken to clarify the status of all species in both genera. Based on phylogenetic and phylogenomic analyses, the current four species of the two genera form a single clade with high bootstrap confidence, indicating that the genus Halosiccatus should be merged with Halomicrobium. Halomicrobium katesii Kharroub et al. 2008 is proposed as a later heterotypic synonym of Halomicrobium mukohataei (Ihara et al. 1997) Oren et al. 2002. An additional species is also described (strains LT50T and TH30), and was isolated from different Gobi saline soil samples of Tarim Basin, Xinjiang, China. Phenotypic, chemotaxonomic, genomic and phylogenetic properties indicated that strains LT50T (=CGMCC 1.15187T = JCM 30837T) and TH30 (=CGMCC 1.15189 = JCM 30839) represent a novel species of the genus Halomicrobium, for which the name Halomicrobium salinisoli sp. nov. is proposed.

Haloprofundus salilacus sp. nov., Haloprofundus halobius sp. nov. and Haloprofundus salinisoli sp. nov.: three extremely halophilic archaea isolated from salt lake and saline soil.

Three halophilic archaeal strains, Gai1-5T, SEDH52T and SQT7-1T were isolated from Gaize salt lake and Xiadi salt lake in Tibet, and saline soil from Xinjiang, respectively. Phylogenetic analysis based on 16S rRNA gene and rpoB' gene sequences showed that these three strains formed different branches separating them from Haloprofundus halophilus NK23T (97.7-98.3% similarities for 16S rRNA gene and 94.7-94.8% similarities for rpoB' gene, respectively) and Haloprofundus marisrubri SB9T (94.7-96.4% similarities for 16S rRNA gene and 92.3-93.2% similarities for rpoB' gene, respectively). Several phenotypic characteristics distinguish the strains Gai1-5 T, SEDH52T and SQT7-1T from Haloprofundus halophilus NK23T and Haloprofundus marisrubri SB9T. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values among the three strains and current Haloprofundus members were in the range of 83.3-88.3% and 27.2-35.7%, respectively, far below the species boundary threshold values. The major polar lipids of three strains were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylglycerol sulphate (PGS), phosphatidylglycerol phosphate methyl ester (PGP-Me), sulfated mannosyl glucosyl diether (S-DGD-1), mannosyl glucosyl diether-phosphatidic acid (DGD-PA) and sulfated mannosyl glucosyl diether-phosphatidic acid (S-DGD-PA). These results showed that strains Gai1-5T (= CGMCC 1.16079T = JCM 33561T), SQT7-1T (= CGMCC 1.16063T = JCM 33553 T) and SEDH52T (= CGMCC 1.17434T) represented three novel species in the genus Haloprofundus, for which the names Haloprofundus salilacus sp. nov., Haloprofundus salinisoli sp. nov., and Haloprofundus halobius sp. nov. are proposed.