A phylogenetic tree of the Mycoplasma mycoides cluster was inferred from a set of concatenated sequences from five housekeeping genes (fusA, glpQ, gyrB, lepA and rpoB). The relevance of this phylogeny was reinforced by detailed analysis of the congruence of the phylogenies derived from each of the five individual gene sequences. Two subclusters were distinguished. The M. mycoides subcluster comprised M. mycoides subsp. mycoides biotypes Small Colony (SC) and Large Colony (LC) and M. mycoides subsp. capri. The latter two groups could not be clearly separated, which supports previous proposals that they be united into a single taxonomic entity. The Mycoplasma capricolum subcluster included M. capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae and Mycoplasma sp. bovine group 7 of Leach, a group of strains that remains unassigned. This group constituted a distinct branch within this cluster, supporting its classification as a subspecies of M. capricolum. Mycoplasma cottewii and Mycoplasma yeatsii clustered in a group that was distinct from Mycoplasma putrefaciens and they were all clearly separated from the M. mycoides cluster. In conclusion, this approach has allowed us to assign phylogenetic positions to all members of the M. mycoides cluster and related species and has proved the need to adjust the existing taxonomy. Furthermore, this method may be used as a reference technique to assign an unequivocal position to any particular strain related to this cluster and may lead to the development of new techniques for rapid species identification.
In order to clarify the current phylogeny of the haloarchaea, particularly the closely related genera that have been difficult to sort out using 16S rRNA gene sequences, the DNA-dependent RNA polymerase subunit B′ gene (rpoB′) was used as a complementary molecular marker. Partial sequences of the gene were determined from 16 strains of the family Halobacteriaceae. Comparisons of phylogenetic trees inferred from the gene and protein sequences as well as from corresponding 16S rRNA gene sequences suggested that species of the genera Natrialba, Natronococcus, Halobiforma, Natronobacterium, Natronorubrum, Natrinema/Haloterrigena and Natronolimnobius formed a monophyletic group in all trees. In the RpoB′ protein tree, the alkaliphilic species Natrialba chahannaoensis, Natrialba hulunbeirensis and Natrialba magadii formed a tight group, while the neutrophilic species Natrialba asiatica formed a separate group with species of the genera Natronorubrum and Natronolimnobius. Species of the genus Natronorubrum were split into two groups in both the rpoB′ gene and protein trees. The most important advantage of the use of the rpoB′ gene over the 16S rRNA gene is that sequences of the former are highly conserved amongst species of the family Halobacteriaceae. All sequences determined so far can be aligned unambiguously without any gaps. On the other hand, gaps are necessary at 49 positions in the inner part of the alignment of 16S rRNA gene sequences. The rpoB′ gene and protein sequences can be used as an excellent alternative molecular marker in phylogenetic analysis of the Halobacteriaceae.
The occurrence of genes encoding nitrogenase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was investigated in the members of the family Ectothiorhodospiraceae. This family forms a separate phylogenetic lineage within the Gammaproteobacteria according to 16S rRNA gene sequence analysis and mostly includes photo- and chemoautotrophic halophilic and haloalkaliphilic bacteria. The cbbL gene encoding the large subunit of ‘green-like’ form I RubisCO was found in all strains, except the type strains of Alkalispirillum mobile and Arhodomonas aquaeolei. The nifH gene encoding nitrogenase reductase was present in all investigated species of the phototrophic genera Ectothiorhodospira, Halorhodospira and Thiorhodospira, but not of the genus Ectothiorhodosinus. Unexpectedly, nifH fragments were also obtained for the chemotrophic species Thioalkalispira microaerophila and Alkalilimnicola halodurans, for which diazotrophic potential has not previously been assumed. The cbbL-, nifH- and 16S rRNA gene-based trees were not highly congruent in their branching patterns since, in the ‘RubisCO’ and ‘nitrogenase’ trees, representatives of the Ectothiorhodospiraceae are divided in a number of broadly distributed clusters and branches. However, the data obtained may be regarded as evidence of the monophyletic origin of the cbbL and nifH genes in most species within the family Ectothiorhodospiraceae and mainly corresponded to the current taxonomic structure of this family. The cbbL phylogeny of the chemolithoautotrophic sulfur-oxidizers Thioalkalivibrio nitratireducens and Thioalkalivibrio paradoxus and the nitrifier Nitrococcus mobilis deviated significantly from the 16S-rRNA gene-based phylogeny. These species clustered with one of the duplicated cbbL genes of the purple sulfur bacterium Allochromatium vinosum, a member of the family Chromatiaceae.
The mitochondrial cytochrome-c oxidase subunit 1 (cox1) gene has been proposed as a DNA barcode to identify animal species. To test the applicability of the cox1 gene in identifying ciliates, 75 isolates of the genus Tetrahymena and three non-Tetrahymena ciliates that are close relatives of Tetrahymena, Colpidium campylum, Colpidium colpoda and Glaucoma chattoni, were selected. All tetrahymenines of unproblematic species could be identified to the species level using 689 bp of the cox1 sequence, with about 11 % interspecific sequence divergence. Intraspecific isolates of Tetrahymena borealis, Tetrahymena lwoffi, Tetrahymena patula and Tetrahymena thermophila could be identified by their cox1 sequences, showing <0.65 % intraspecific sequence divergence. In addition, isolates of these species were clustered together on a cox1 neighbour-joining (NJ) tree. However, strains identified as Tetrahymena pyriformis and Tetrahymena tropicalis showed high intraspecific sequence divergence values of 5.01 and 9.07 %, respectively, and did not cluster together on a cox1 NJ tree. This may indicate the presence of cryptic species. The mean interspecific sequence divergence of Tetrahymena was about 11 times greater than the mean intraspecific sequence divergence, and this increased to 58 times when all isolates of species with high intraspecific sequence divergence were excluded. This result is similar to DNA barcoding studies on animals, indicating that congeneric sequence divergences are an order of magnitude greater than conspecific sequence divergences. Our analysis also demonstrated low sequence divergences of <1.0 % between some isolates of T. pyriformis and Tetrahymena setosa on the one hand and some isolates of Tetrahymena furgasoni and T. lwoffi on the other, suggesting that the latter species in each pair is a junior synonym of the former. Overall, our study demonstrates the feasibility of using the mitochondrial cox1 gene as a taxonomic marker for ‘barcoding’ and identifying Tetrahymena species and some other ciliated protists.