A considerable number of species of the Halobacteriaceae possess multiple copies of the 16S rRNA gene that exhibit more than 5 % divergence, complicating phylogenetic interpretations. Two additional problems have been pointed out: (i) the genera Haloterrigena and Natrinema show a very close relationship, with some species being shown to overlap in phylogenetic trees reconstructed by the neighbour-joining method, and (ii) alkaliphilic and neutrophilic species of the genus Natrialba form definitely separate clusters in neighbour-joining trees, suggesting that these two clusters could be separated into two genera. In an attempt to solve these problems, the RNA polymerase B9 subunit has been used as an additional target molecule for phylogenetic analysis, using partial sequences of 1305 bp. In this work, a primer set was designed that consistently amplified the full-length RNA polymerase B9 subunit gene (rpoB9) (1827-1842 bp) from 85 strains in 27 genera of the Halobacteriaceae. Differences in sequence length were found within the first 15 to 31 nt, and their downstream sequences (1812 bp) were aligned unambiguously without any gaps or deletions. Phylogenetic trees reconstructed from nucleotide sequences and deduced amino acid sequences by the maximum-likelihood method demonstrated that multiple species/strains in most genera individually formed cohesive clusters. Two discrepancies were observed: (i) the two species of Natronolimnobius were placed in definitely different positions, in that Natronolimnobius innermongolicus was placed in the Haloterrigena/Natrinema cluster, while Natronolimnobius baerhuensis was closely related to Halostagnicola larsenii, and (ii) Natronorubrum tibetense was segregated from the three other Natronorubrum species in the protein tree, while all four species formed a cluster in the gene tree, although supported by a bootstrap value of less than 50 %. The six Haloterrigena species/strains and the five species of Natrinema formed a large cluster in both trees, with Halopiger xanaduensis and Nln. innermongolicus located in the cluster in the protein tree and Nln. innermongolicus in the gene tree. Hpg. xanaduensis broke into the cluster of the genus Halobiforma, instead of the Haloterrigena/Natrinema cluster, in the gene tree. The six Natrialba species formed a tight cluster with two subclusters, of neutrophilic species and alkaliphilic species, in both trees. Overall, our data strongly suggest that (i) Nln. innermongolicus is a member of Haloterrigena/Natrinema, (ii) Nrr. tibetense might represent a new genus and (iii) the two genera Haloterrigena and Natrinema might constitute a single genus. As more and more novel species and genera are proposed in the family Halobacteriaceae, the full sequence of the rpoB9 gene may provide a supplementary tool for determining the phylogenetic position of new isolates.Abbreviations: ML, maximum-likelihood; NJ, neighbour-joining.The GenBank/EMBL/DDBJ accession numbers for the rpoB9 gene sequences determined in this study are AB477139-AB477222 and AB478421 a...

A halophilic archaeon, Haloarcula sp. strain S-1, produced extracellular organic solvent-tolerant alpha-amylase. Molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This amylase exhibited maximal activity at 50 degrees C in buffer containing 4.3 M NaCl, pH 7.0. Moreover, the enzyme was active and stable in various organic solvents (benzene, toluene, and chloroform, etc.). Activity was not detected at low ionic strengths, but it was detected in the presence of chloroform at low salt concentrations. On the other hand, no activity was detected in the presence of ethyl alcohol and acetone.

A novel halophilic archaeon, strain MH1-52-1T, was isolated from solar salt imported from Australia. Cells were pleomorphic, non-motile and Gram-negative. Strain MH1-52-1T required at least 3.0 M NaCl and 1 mM Mg2+ for growth. Strain MH1-52-1T was able to grow at pH 4.0–6.0 (optimum, pH 4.4–4.5) and 15–45 °C (optimum, 37 °C). The diether phospholipids phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, derived from both C20C20 and C20C25 archaeol, were present. Four unidentified glycolipids were also detected. The 16S rRNA gene sequence showed the highest similarity to that of Halobacterium noricense A1T (91.7 %); there were lower levels of similarity to other members of the family Halobacteriaceae. The G+C content of its DNA was 61.4 mol%. Based on our phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolate should be classified as a representative of a new genus and species, for which the name Halarchaeum acidiphilum gen. nov., sp. nov. is proposed. The type strain of Halarchaeum acidiphilum is MH1-52-1T (=JCM 16109T =DSM 22442T =CECT 7534T).

Strain YSM-123T was isolated from commercial salt made from Japanese seawater in Niigata prefecture. Optimal NaCl and Mg2+ concentrations for growth were 4.0–4.5 M and 5 mM, respectively. The isolate was a mesophilic and slightly alkaliphilic haloarchaeon, whose optimal growth temperature and pH were 37 °C and pH 8.0–9.0. Phylogenetic analysis based on 16S rRNA gene sequence analysis suggested that strain YSM-123T is a member of the phylogenetic group defined by the family Halobacteriaceae, but there were low similarities to type strains of other genera of this family (≤90 %); for example, Halococcus (similarity <89 %), Halostagnicola (<89 %), Natronolimnobius (<89 %), Halobiforma (<90 %), Haloterrigena (<90 %), Halovivax (<90 %), Natrialba (<90 %), Natronobacterium (<90 %) and Natronococcus (<90 %). The G+C content of the DNA was 63 mol%. Polar lipid analysis revealed the presence of phosphatidylglycerol, phosphatidylglycerophosphate methyl ester, disulfated diglycosyl diether and an unknown glycolipid. On the basis of the data presented, we propose that strain YSM-123T should be placed in a new genus and species, Natronoarchaeum mannanilyticum gen. nov., sp. nov. The type strain of Natronoarchaeum mannanilyticum is strain YSM-123T (=JCM 16328T =CECT 7565T).

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and 'the rest' of the present family Halobacteriaceae.