Sequence of the 16S Ribosomal RNA from Halobacterium volcanii, an Archaebacterium.

The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

Secondary structure of 16S ribosomal RNA.

A secondary structure model for 16S ribosomal RNA which is based on available chemical, enzymatic, and comparative sequence data shows good agreement between constraints dictated by the model and a wide variety of experimental observations. The four major structural domains created by the base-pairing scheme correspond closely to RNA fragments isolated after nuclease digestion in the presence of bound ribosomal proteins. Functionally important sites appear to be located in unpaired regions and are phylogenetically highly conserved.

A common origin of rickettsiae and certain plant pathogens.

On the basis of ribosomal RNA sequence comparisons, the rickettsia Rochalimaea quintana has been found to be a member of subgroup 2 of the alpha subdivision of the so-called purple bacteria, which is one of about ten major eubacterial divisions. Within subgroup alpha-2, R. quintana is specifically related to the agrobacteria and rhizobacteria, organisms that also have close associations with eukaryotic cells. This genealogical grouping of the rickettsiae with certain plant pathogens and intracellular symbionts suggests a possible evolution of the rickettsiae from plant-associated bacteria.

Gram-positive bacteria: possible photosynthetic ancestry.

A 16S ribosomal RNA gene has been sequenced from Heliobacterium chlorum, the recently discovered photosynthetic bacterium that contains a novel form of chlorophyll. Comparisons with other 16S ribosomal RNA sequences show that the organism belongs to the Gram-positive bacteria (one of ten eubacterial "phyla")--more precisely to the so-called low G + C (G, guanine; C, cytosine) subdivision thereof. This brings to five the number of such phyla that contain photosynthetic species, the other four being the purple bacteria and relatives, the green sulfur bacteria, the green nonsulfur bacteria, and the cyanobacteria. The finding suggests that Gram-positive bacteria may be of photosynthetic ancestry, and it strengthens the case for a common photosynthetic ancestry for all eubacteria.

A euryarchaeal lysyl-tRNA synthetase: resemblance to class I synthetases.

The sequencing of euryarchaeal genomes has suggested that the essential protein lysyl-transfer RNA (tRNA) synthetase (LysRS) is absent from such organisms. However, a single 62-kilodalton protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes this protein was cloned. The predicted amino acid sequence of M. maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the crenarchaeote Sulfolobus solfataricus. The presence of amino acid motifs characteristic of the Rossmann dinucleotide-binding domain identifies M. maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases. These data question the concept that the classification of aminoacyl-tRNA synthetases does not vary throughout living systems.

Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the "gemini group. " (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.

Whither microbiology? Phylogenetic trees.

The direct cloning and sequencing of genes from uncultured microorganisms in a hot spring suggests that the diversity of life on Earth may be far in excess of that estimated from culturable species.

A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics.

The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.

The ribosomal gene spacer region in archaebacteria.

Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so. The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.

A partial phylogenetic analysis of the "flavobacter-bacteroides" phylum: basis for taxonomic restructuring.

On the basis of small subunit rRNA sequence analyses five major subgroups within the flavobacteria-bacteroides phylum have been defined. These are tentatively designated the cytophaga subgroup (comprising largely Cytophaga species), the flavobacter subgroup (comprising the true flavobacteria and the polyphyletic genus Weeksella), the bacteroides subgroup (comprising the bacteroides and certain cytophaga-like bacteria), the sphingobacter subgroup (which contains the known sphingolipid-producing members of the phylum), and the saprospira subgroup (comprising particular species of Flexibacter, Flavobacterium, Haliscomenobacter, and, of course, the genus Saprospira). These groupings are given not only by evolutionary distance analysis, but can be defined and distinguished on the basis of a simple small subunit rRNA signatures.

Archaebacterial phylogeny: perspectives on the urkingdoms.

Comparisons of complete 16S ribosomal RNA sequences have been used to confirm, refine and extend earlier concepts of archaebacterial phylogeny. The archaebacteria fall naturally into two major branches or divisions, I--the sulfur-dependent thermophilic archaebacteria, and II--the methanogenic archaebacteria and their relatives. Division I comprises a relatively closely related and phenotypically homogeneous collection of thermophilic sulfur-dependent species--encompassing the genera Sulfolobus, Thermoproteus, Pyrodictium and Desulfurococcus. The organisms of Division II, however, form a less compact grouping phylogenetically, and are also more diverse in phenotype. All three of the (major) methanogen groups are found in Division II, as are the extreme halophiles and two types of thermoacidophiles, Thermoplasma acidophilum and Thermococcus celer. This last species branches sufficiently deeply in the Division II line that it might be considered to represent a separate, third Division. However, both the extreme halophiles and Tp. acidophilum branch within the cluster of methanogens. The extreme halophiles are specifically related to the Methanomicrobiales, to the exclusion of both the Methanococcales and the Methanobacteriales. Tp. acidophilum is peripherally related to the halophile-Methanomicrobiales group. By 16S rRNA sequence measure the archaebacteria constitute a phylogenetically coherent grouping (clade), which excludes both the eubacteria and the eukaryotes--a conclusion that is supported by other sequence evidence as well. Alternative proposals for archaebacterial phylogeny, not based upon sequence evidence, are discussed and evaluated. In particular, proposals to rename (reclassify) various subgroups of the archaebacteria as new kingdoms are found wanting, for both their lack of proper experimental support and the taxonomic confusion they introduce.

A Phylogenetic Analysis of Lactobacilli, Pediococcus pentosaceus and Leuconostoc mesenteroides.

Lactobacillus acidophilus, L. helveticus, L. lactis, L. brevis, L. plantarum, L. ruminis, L. casei, L. bifermentans, L. viridescens, L. fermentum, Pediococcus pentosaceus and Leuconostoc mesenteroides have been characterized by oligonucleotide cataloguing of their 16 S ribosomal RNAs. All Lactobacillus species investigated together with P. pentosaceus and L. mesenteroides form a phylogenetically coherent and ancient group. L. acidophilus and L. helveticus is the only closely related pair of species; all other species are only remotely related among each other, indicating that they are well-defined species. Lactobacillus, together with Streptococcus and Bacillus, constitute a super-cluster within the 'clostridium' sub-branch of Gram-positive eubacteria showing a low guanine plus cytosine content of their deoxyribonucleic acid.

Archaeal phylogeny: reexamination of the phylogenetic position of Archaeoglobus fulgidus in light of certain composition-induced artifacts.

A major and too little recognized source of artifact in phylogenetic analysis of molecular sequence data is compositional difference among sequences. The problem becomes particularly acute when alignments contain ribosomal RNAs from both mesophilic and thermophilic species. Among prokaryotes the latter are considerably higher in G + C content than the former, which often results in artificial clustering of thermophilic lineages and their being placed artificially deep in phylogenetic trees. In this communication we review archaeal phylogeny in the light of this consideration, focusing in particular on the phylogenetic position of the sulfate reducing species Archaeoglobus fulgidus, using both 16S rRNA and 23S rRNA sequences. The analysis shows clearly that the previously reported deep branching of the A. fulgidus lineage (very near the base of the euryarchaeal side of the archaeal tree) is incorrect, and that the lineage actually groups with a previously recognized unit that comprises the Methanomicrobiales and extreme halophiles.

Methanopyrus kandleri: an archaeal methanogen unrelated to all other known methanogens.

Analysis of its 16S rRNA sequence shows that the newly discovered hyperthermophilic methanogen, Methanopryus kandleri, is phylogenetically unrelated to any other known methanogen. The organism represents a separate lineage originating near the root of the archaeal tree. Although the 16S rRNA sequence of Mp. kandleri resembles euryarchaeal 16S rRNAs more than it does crenarchaeal, it shows more crenarchaeal signature features than any known euryarchaeal rRNA. Attempts to place it in relation to the root of the archaeal tree show that the Mp. kandleri lineage likely arises from the euryarchaeal branch of the tree. While the existence of so deeply branching a methanogenic lineage brings into question the thesis that methanogenesis evolved from an earlier metabolism similar to that seen in Thermococcus, it at the same time reinforces the notion that the aboriginal [correction of aborginal] archaeon was a thermophile.

The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales.

We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei. This is the first such sequence from a member of the Methanomicrobiales. Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles. Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so. Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.

Were the original eubacteria thermophiles?

Thermotoga maritima is one of the more unusual eubacteria: It is highly thermophilic, growing at temperatures higher than any other eubacterium; its cell wall appears to have a unique structure and its lipids a unique composition; and the organism is surrounded by a loose-fitting sheath of unknown function. Its phenotypic uniqueness is matched by its phylogenetic position; Thermotoga maritima represents the deepest known branching in the eubacterial line of descent, as measured by ribosomal RNA sequence comparisons. T. maritima also represents the most slowly evolving of eubacterial lineages. The fact that the two deepest branchings in the eubacterial line of descent (the other, the green non-sulfur bacteria and relatives, i.e. Chloroflexus, Thermomicrobium, etc.) are both basically thermophilic and slowly evolving, strongly suggests that all eubacteria have ultimately arisen from a thermophilic ancestor.

Rooting the archaebacterial tree: the pivotal role of Thermococcus celer in archaebacterial evolution.

The sequence of the 16S ribosomal RNA gene from the archaebacterium Thermococcus celer shows the organism to be related to the methanogenic archaebacteria rather than to its phenotypic counterparts, the extremely thermophilic archaebacteria. This conclusion turns on the position of the root of the archaebacterial phylogenetic tree, however. The problems encountered in rooting this tree are analyzed in detail. Under conditions that suppress evolutionary noise both the parsimony and evolutionary distance methods yield a root location (using a number of eubacterial or eukaryotic outgroup sequences) that is consistent with that determined by an "internal rooting" method, based upon an (approximate) determination of relative evolutionary rates.