Figures 1 and 2 provide rough comparisons of the relationships between these organisms within this gene cluster by analyzing gene order, size, and similarity. A more rigorous measure of the relationships between these organisms is provided by constructing phylogenetic trees that directly measure genetic relationships based upon multiple sequence alignments, direct sequence comparisons, and evolutionary reconstruction. While a number of different methods are available for calculating phylogenetic relationships, the distances between the organisms compared here are significant enough that similar results are obtained if different methods are used (data not shown). Therefore we depict the results of one phylogenetic measure in comparing representative coding regions for each of the three operons (phylogenetic reconstructions for the genes not shown were all similar to those presented in this figure).
These four panels depict the phylogenetic relationships between these organisms for the 16S ribosomal RNA nucleotide sequences, and for operons S10, spc, and alpha. All multiple sequence alignments were constructed using the GCG program PileUp. 16S rRNA sequences were aligned at the nucleotide level, while all of the other sequences were aligned at the protein level. Theses gapped alignments based on protein sequence were then reconstructed for the nucleotide sequences. The nucleotide alignments were used for all phylogenetic reconstructions. The phylogeny of 16S rRNA sequences are shown for comparison purposes.
Phylogenetic relationships were determined using the GCG programs Distances and GrowTree, along with ClustalW. These programs construct phylogenetic relationships by calculating distance matrices based on the nucleotide alignments using the Kimura 2-parameter statistic to correct for multiple substitutions. Tree topology is then calculated using the Neighbor-joining method. ClustalW was used for bootstrap analysis to provide confidence measures on the reliability of each node within the tree. Trees are displayed as non-rooted with their branch lengths proportional to distance. The numbers along the branches provide the bootstrap values (percentage of 1,000 runs) for the corresponding tree node. Scale is not maintained between the comparisons for each protein.
These phylogenetic reconstructions support the rough comparisons previously shown in which Mg and Mp are the most closely related organisms, and then Uu the next most closely related sequence to this group. Mc and Bs are seen to be approximately equidistant between the Mg, Mp, Uu group, and a group consisting of Ec and Hi. Mc lies a bit closer to the other mycoplasma species than does Bs, but the distance is not great. These relationships are maintained for all proteins across this cluster of genes.
