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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 B' 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 B' subunit gene (rpoB') (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 rpoB' gene may provide a supplementary tool for determining the phylogenetic position of new isolates.

Further refinement of the phylogeny of the Halobacteriaceae based on the full-length RNA polymerase subunit B′ (rpoB′) gene