For the first time a single experimental approach, 16 S ribosomal RNA sequence characterization, has been used to develop an overview of phylogenetic relationships in the bacterial world. The technique permits the tracing of relationships back to the common ancestor of all extant life. This first glimpse of bacterial phylogeny reveals a world whose roots appear to span more than 3 billion years. A deep phylogenetic split exists among the bacteria, which necessitates their division into two major lines of descent, the archaebacteria and the true bacteria (or eubacteria). It is a general finding that the most ancient bacterial phenotypes are anaerobic, and that aerobic phenotypes have arisen a number of times. Photosynthetic phenotypes are also extremely ancient. Many nonphotosynthetic groups appear to have arisen from photosynthetic ancestry, which is reason to question the generally held belief that the first bacteria were anaerobic heterotrophs. The two ultimate lines of bacterial descent are no more closely related to one another than either is to the cytoplasmic aspect of the eukaryotic cell. However, in that the eukaryotic cell is a phylogenetic chimera, it itself cannot be seen as a line of descent comparable to the two bacterial lines—although some of its individual parts can be so viewed. In this way, the chloroplast and perhaps the mitochondrion are each eubacterial, and at least one ribosomal protein is archaebacterial. A third line of descent that is neither eubacterial nor archaebacterial is represented in the 18 S ribosomal RNA.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. The borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated from the shrew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema ilini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.

The phylogenetic relationships between the mycoplasmas and bacteria have been established from a comparative analysis of their 16S rRNA oligonucleotide citalogs. The genera Mycoplasma, Spiroplasma, and Atholeplasma arose b degenerative evolution, as a deep branch of the subline of costridial ancestry that led to Bacillus and Lactobacillus.Thermoplasma has no specific relationship to the other mycoplasmas; it belongs with the archaebacteria.Mycoplasma is the general name for a group of prokaryotes that do not have cell walls; each cell is bounded by a single lipoprotein membrane (for reviews, see refs. 1 and 2). The various isolates have been placed in the class Mollicutes, order Mycoplasmatales and classified into the genera Mycoplasma (containing about 50 currently recognized species), Acholeplasma (6 species), Ureaplasma (1 species), and Spiroplasma (1 species). In addition, the class Mollicutes contains two genera of uncertain taxonomic position: Anaeroplasma (two species) are obligate anaerobes and Thermoplasma (one species) is an acidophilic thermophile. The taxonomy and characteristics of these genera have been reviewed recently by Tully (3). All mycoplasmas, except Acholeplasma and some Anaeroplasma isolates, require sterol for growth. The genome size of the Mycoplasma and Ureaplasma is about 0.5 X 109 daltons, that of the Acholeplasma, Spiroplasma, and Thermoplasma about 1.0 X 109 daltons. The G.C content of the cell DNA is: Mycoplasma, 23-41%; Acholeplasma, 29-35%; Ureaplasma, 28%; Spiroplasma, 26%; Anaeroplasma, 29-34%; and Thermoplasma, 46% (2).A review of the biochemical and biophysical studies of the mycoplasmas concluded that, in general, their biology is qualitatively similar to that of other prokaryotes, the only differences being quantitative ones due to the limited size of the mycoplasma cell and genome (1). In particular, mycoplasma ribosomal and transfer RNAs contain many kinds of modified bases, but in smaller amounts than reported for other prokaryotes (1, 4, 5).The origin of and relationships among mycoplasmas have been controversial for many years. Their small genome sizes and general simplicity led some workers to suggest that they might be descendants of a primitive type of organism that preceded the typical bacteria in the evolutionary progression (6). However, mycoplasmas may merely be degenerate forms. If so, there is still the question of whether mycoplasmas constitute a separate and major branch of the bacteria, as the name Mollicutes connotes, or whether they represent a diverse collection of wall-less forms derived from many different branches of bacteria.In the studies presented here, we have examined the question of mycoplasma genealogy through comparative analyses of their 16S rRNA sequences. This technique, rRNA cataloging, has been used successfully in determining the phylogenetic structure of a variety of bacterial groups (7,8). We find that the mycoplasmas are not a phylogenetically coherent group in the sense that all derive from a common ancestor, itself a mycopl...

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