Heng-Lin Cui

Intraspecific polymorphism of 16S rRNA genes in two halophilic archaeal genera, Haloarcula and Halomicrobium

All members of the genera Haloarcula and Halomicrobium whose names have been validly published were surveyed for 16S rRNA gene polymorphism, and the transcription of the genes from two species was investigated during growth at different NaCl concentrations. The species of Haloarcula and Halomicrobium harbour at least two different 16S rRNA gene copies, and 18 new sequences of 16S rRNA genes were obtained. The type I and type II 16S rRNA genes of Haloarcula are divergent at 4.8–5.6% of their nucleotide positions. The type III and type IV 16S rRNA genes from Halomicrobium mukohataei JCM 9738T are 9.0% divergent, which represents the highest intraspecific divergent 16S rRNA genes so far seen. Phylogenetic analysis based on 16S rRNA genes indicated that all type I 16S rRNA genes were clustered, and the same was true for the type II 16S rRNA genes of Haloarcula species. The two clusters, respectively generated from type I and type II 16S rRNA genes, were sharply separated and their divergences (4.8–5.6%) are in the range of various divergence usually found between genera in the order Halobacteriales (about 5–10%). Results from reverse transcription-PCR showed that the type I and type II copies of Har. amylolytica BD-3T and type III and type IV copies of Hmc. mukohataei JCM 9738T were all transcribed to 16S rRNA molecules under different salt concentrations (15–28% NaCl).

Halobellus clavatus gen. nov., sp. nov. and Halorientalis regularis gen. nov., sp. nov., two new members of the family Halobacteriaceae

Four halophilic archaeal strains, designated TNN18(T), TBN12, TNN28(T) and TBN19, were isolated from brines sampled from two artificial marine solar salterns in eastern China. Strains TNN18(T) and TNN28(T) were isolated from the Tainan marine solar saltern, whereas strains TBN12 and TBN19 were from the Taibei marine solar saltern. Colonies of the four strains were red-pigmented and their cells were pleomorphic, motile, Gram-reaction-negative rods. Strains TNN18(T) and TBN12 were able to grow at 25-50 °C (optimum 37 °C), in 10-3  % (w/v) NaCl (optimum 15 %), with 0-1.0 M MgCl(2) (optimum 0.05 M) and at pH 5.5-9.0 (optimum pH 7.0-7.5), while strains TNN28(T) and TBN19 were able to grow at 20-50 °C (optimum 37 °C), in 15-30 % (w/v) NaCl (optimum 18-20 %), in 0.005-1.0 M MgCl(2) (optimum 0.01-0.3 M) and at pH 6.0-9.0 (optimum pH 7.0-7.5). Cells of these strains lyse in distilled water; minimal NaCl concentrations to prevent cell-lysis are 10 % (w/v) for strains TNN18(T) and TBN12 and 12 % (w/v) for strains TNN28(T) and TBN19. The major polar lipids of strains TNN18(T) and TBN12 were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfate (PGS) and one major glycolipid (GL1), which was chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1). Minor amounts of other lipids (GL0, GL2, GL3 and GL4) were also detectable. The polar lipid profiles of strains TNN28(T) and TBN19 contained PG, PGP-Me, GL1, which was chromatographically identical to S-DGD-1, and three to four minor unidentified glycolipids (GL2-GL5). Phylogenetic analyses revealed that strains TNN18(T) and TBN12 formed a distinct clade with strains of the closest related species, Haloquadratum walsbyi (91.5-91.8 % 16S rRNA gene sequence similarity) and strains TNN28(T) and TBN19 formed a distinct clade with strains of the species Halosimplex carlsbadense (89.9-93.3 % similarity) and two members of the genus Halorhabdus (92.5-93.3 % similarity). The DNA G+C contents of strains TNN18(T), TBN12, TNN28(T) and TBN19 were 61.5, 62.4, 61.9 and 61.5 mol%, respectively. DNA-DNA hybridization values between strains TNN18(T) and TBN12, and strains TNN28(T) and TBN19 were 82.9 % and 88.2 %, respectively. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the four strains represent two novel species of two new genera within the family Halobacteriaceae, for which the names Halobellus clavatus gen. nov., sp. nov. (type strain TNN18(T ) = CGMCC 1.10118(T ) = JCM 16424(T)) and Halorientalis regularis gen. nov., sp. nov. (type strain TNN28(T ) = CGMCC 1.10123(T ) = JCM 16425(T)) are proposed.