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.

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.

Salinigranum halophilum sp. nov., isolated from marine solar salterns.

Three halophilic archaeal strains, YJ-53T, ZS-5 and DYF38, were isolated from marine solar salterns located in different provinces of China. The three strains formed a single cluster (99.7-99.8 and 97.9-99.2 % similarities, respectively) that was separate from the current two members of Salinigranum (96.7-98.0 and 89.8-92.9 % similarities, respectively) on the basis of 16S rRNA and rpoB' gene sequence comparisons and phylogenetic analysis. Diverse phenotypic characteristics differentiated strains YJ-53T, ZS-5 and DYF38 from Salinigranum rubrum GX10T and Salinigranum salinum YJ-50-S2T. The major polar lipids of isolated strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and two major glycolipids chromatographically identical to mannosyl glucosyl diether and sulfated mannosyl glucosyl diether, detected in the current members of Salinigranum. The OrthoANI and in silico DNA-DNA hybridization (DDH) values between the three strains were in the range of 97.7-98.4 % and 80.3-86.1 %, respectively, much higher than the threshold values proposed as species boundaries (average nucleotide identity 95-96 % and in silico DDH 70 %), revealing that the three strains represent one species. Results of comparative OrthoANI and in silico DDH analyses of the strains described in this study with validly described members of the genus Salinigranum supported that strains YJ-53T (=CGMCC 1.12860T=JCM 30238T), ZS-5 (=CGMCC 1.12867=JCM 30240) and DYF38 (=CGMCC 1.13779=JCM 33557) represent a novel species of the genus Salinigranum, for which the name Salinigranum halophilum sp. nov. is proposed.

Salinirubellus salinus gen. nov., sp. nov., isolated from a marine solar saltern.

A halophilic archaeal strain, designated ZS-35-S2T, was isolated from Zhoushan marine solar saltern in Zhejiang Province, China. Cells were pleomorphic, Gram-stain-negative and formed red-pigmented colonies on agar plates. The cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 8 % (w/v). Strain ZS-35-S2T was able to grow at 25-50 °C (optimum, 37 °C), with 1.4-4.8 M NaCl (optimum, 2.1 M), with 0-1.0 M MgCl2 (optimum, 0.1 M) and at pH 5.0-9.5 (optimum, pH 7.5). Phylogenetic tree reconstructions based on 16S rRNA genes and rpoB' genes revealed that strain ZS-35-S2T was distinct from the related genera Halomarina, Natronomonas, Halorientalis, Salinirubrum and Halobaculum of the order Halobacteriales. The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three unidentified glycolipids. The two major glycolipids were chromatographically identical to S-DGD-1 and DGD-1, respectively. The DNA G+C content of strain ZS-35-S2T is 67.0 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain ZS-35-S2T (=CGMCC 1.12551T=JCM 30036T) represents a novel species of a new genus within the order Halobacteriales, for which the name Salinirubellus salinus gen. nov., sp. nov. is proposed.