Halosubterraneus shenae gen. nov., sp. nov., an extremely halophilic archaeon isolated from salt mine

Two halophilic archaeal strains, AD34^T and PAK95, were isolated from a salt mine in Yunnan and rock salt in Jiangsu, two provinces in China that are 2,000 km apart, respectively. Cells of strain AD34^T were spherical while those of PAK95 were rod-shaped. Strains AD34^T and PAK95 were Gram-stain-negative, aerobic and able to grow at 1.4-4.8 M NaCl (with optimum at 3.1 M NaCl), pH 5.0-9.0 (optimum, pH 7.5) and 20-60 °C (optimum, 37 °C and 40 °C, respectively). Their 16S rRNA gene sequences showed the closest similarity to Haloparvum sedimenti DYS4^T, with similarities of 95.11% (AD34^T) and 95.46% (PAK95). The rpoB' gene sequences of strains AD34^T and PAK95 showed the highest similarity to species of Haloparvum alkalitolerans MK62-1^T with 91.40% (PAK95) and 91.53% (AD34^T) identity. Comprehensive phylogenetic analyses revealed that strains AD34^T and PAK95 form a robust, monophyletic clade with 100% bootstrap support, clearly distinct from related genera, particularly from the genus Haloparvum. From whole-genome sequencing, the DNA G+C content of AD34^T and PAK95 is 66.5 and 66.4 mol%, respectively, while that of DYS4^T and MK62-1^T is 68.3 and 69.1 mol%, respectively. The average nucleotide identity between AD34^T, PAK95 and the family Haloferacaceae ranged from 66.5 to 79.2%. The digital DNA-DNA hybridization values ranged from 19.6 to 32.8%, while the average amino acid identity (AAI) values were between 58.0 and 72.0%. Strains AD34^T and PAK95 may represent a novel species of a novel genus within the family Haloferacaceae, as judged by the AAI cutoff value (≤72.1%) proposed for differentiating genera within this family. The major polar lipids of strain AD34^T were phosphatidylglycerol (PG), PG phosphate methyl ester, PG sulphate and sulphated mannosyl glucosyl diether. Based on the phenotypic and phylogenetic analyses, it is proposed that strains AD34^T (=MCCC 4K00176^T=KCTC 4324^T) and PAK95 (=MCCC 4K00231=CGMCC 1.62791=KCTC 4376) represent a novel genus and species within the family Haloferacaceae, for which the name Halosubterraneus shenae gen. nov., sp. nov. is proposed. The type strain is AD34^T.