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

Abstract

A considerable number of species of theHalobacteriaceaepossess 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 generaHaloterrigenaandNatrinemashow 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 genusNatrialbaform 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 theHalobacteriaceae. 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 ofNatronolimnobiuswere placed in definitely different positions, in thatNatronolimnobius innermongolicuswas placed in theHaloterrigena/Natrinemacluster, whileNatronolimnobius baerhuensiswas closely related toHalostagnicola larsenii, and (ii)Natronorubrum tibetensewas segregated from the three otherNatronorubrumspecies 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 sixHaloterrigenaspecies/strains and the five species ofNatrinemaformed a large cluster in both trees, withHalopiger xanaduensisandNln. innermongolicuslocated in the cluster in the protein tree andNln. innermongolicusin the gene tree.Hpg. xanaduensisbroke into the cluster of the genusHalobiforma, instead of theHaloterrigena/Natrinemacluster, in the gene tree. The sixNatrialbaspecies formed a tight cluster with two subclusters, of neutrophilic species and alkaliphilic species, in both trees. Overall, our data strongly suggest that (i)Nln. innermongolicusis a member ofHaloterrigena/Natrinema, (ii)Nrr. tibetensemight represent a new genus and (iii) the two generaHaloterrigenaandNatrinemamight constitute a single genus. As more and more novel species and genera are proposed in the familyHalobacteriaceae, the full sequence of therpoB′ gene may provide a supplementary tool for determining the phylogenetic position of new isolates.