Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.

Ribosomal DNA: molecular evolution and phylogenetic inference.

The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases

Polyphasic taxonomy, a consensus approach to bacterial systematics.

A new hierarchic classification structure for the taxa between the taxonomic levels of genus and class is Proposed for the actinomycete line of descent as defined by analysis of small subunit (16S) rRNA and genes coding for this molecule (rDNA). While the traditional circumscription of a genus of the actinomycete subphylum is by and large in accord with the 16S rRNA/rDNA-based phylogenetic clustering of these organisms. most of the higher taxa proposed in the past do not take into account the phylogenetic clustering of genera. The rich chemical, morphological and physiological diversity of phylogenetically closely related genera makes the description of families and higher taxa so broad that they become meaningless for the description of the enclosed taxa. Here we present a classification system in which phylogenetically neighboring taxa at the genus level are clustered into families, suborders, orders, subclasses, and a class irrespective of those phenotypec characteristics on which the delineation of taxa has been based in the past. Rather than being based on a listing of a wide array of chemotaxonomic, morphological, and physiological properties, the delineation is based solely on 16S rDNA/rRNA sequence-based phylogenetic clustering and the presence of taxon-specific 16S rDNA RNA signature nucleotides.

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Proposal for a New Hierarchic Classification System, Actinobacteria classis nov.

16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.

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How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.

16S rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. marinum, M. chelonae, M. smegmatis, M. terrae, M. gordonae, M. scrofulaceum, M. szulgai, M. intracellulare, M. nonchromogenicum, M. xenopi, M. malmoense, M. simiae, M. flavescens, M. fortuitum, and M. paratuberculosis were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotochromogenic, and nonchromogenic mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kansasii when this kind of analysis was used.

Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium.

The compilation of 5S rRNA and 5S rRNA gene nucleotide sequences as of 30 September 1996, contains a total of 1661 primary structures of 5S rRNAs or their genes, which is an increase of 928 new sequence entries over the last compilation. It covers sequences from 54 archaea, 449 eubacteria, 34 plastids, nine mitochondria and 430 eukaryotes. The databank uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information. The taxonomic classification of organisms was totally updated. Now the database is also available via anonymous FTP or WWW.

Compilation of 5S rRNA and 5S rRNA gene sequences

Large insertions and deletions in the variable regions of eukaryotic 16S-like rRNA relative to the archaebacterial structure have been defined as a marker for rapidly evolving taxa. Deletions in the rRNA occur in the diplomonad Giardia and the microsporidian Vairimorpha, whereas insertions occur in Euglenozoa (Euglena and the kinetoplastids), Acanthamoeba, Naegleria, Physarum, Dictyostelium, the apicomplexan Plasmodium, the ciliate Euplotes, and some metazoa. Except Acanthamoeba and Euplotes, all of these protists were previously placed at the base of the eukaryote phylogeny. A re-analysis of the 16S-like rRNA and 5S rRNA data with the neighborliness method revealed a close relationship of Apicomplexa to the dinoflagellate-ciliate clade, most probably closer to the dinoflagellates. Morphological evidence that supports this grouping is the layer of sacs underneath the plasma membrane in all three taxa and the identical structure of trichocysts in the apicomplexan Spiromonas and dinoflagellates. The remaining rapidly evolving organisms might still be misplaced in the 16S-like rRNA trees.

The troublesome parasites--molecular and morphological evidence that Apicomplexa belong to the dinoflagellate-ciliate clade.

The 16S rRNAs from nine rapidly growing Mycobacterium species were partially sequenced by using the dideoxynucleotide-terminated, primer extension method with cDNA generated by reverse transcriptase. The sequences were aligned with 47 16S rRNA or DNA sequences that represented 30 previously described and 5 undescribed species of the genus Mycobacterium, and a dendrogram was constructed by using equally weighted distance values. Our results confirmed the phylogenetic separation of the rapidly and slowly growing mycobacteria and showed that the majority of the slowly growing members of the genus represent the most recently evolved organisms. The 24 strains which represented 21 rapidly growing species constituted several sublines, which were defined by the following taxa: (i) Mycobacterium neoaurum and M. diernhoferi, (ii) M. gadium, (iii) the M. chubuense cluster, (iv) the M. fortuitum cluster, (v) M. kommossense, (vi) M. sphagni, (vii) M. fallax and M. chitae, (viii) M. aurum and M. vaccae, (ix) the M. flavescens cluster, and (x) M. chelonae subsp. abscessus. Our phylogenetic analysis confirmed the validity of the phenotypically defined species mentioned above, but our conclusions disagree with most of the conclusions about intrageneric relationships derived from numerical phenetic analyses.

Phylogeny of rapidly growing members of the genus Mycobacterium.

1. Summary Comparative 16S rRNA sequencing was used to infer the phylogenetic relationship among different serovars of the Mycobacterium avium-M. intracellulare complex as well as to define signature nucleotides characteristic for different serovars. In general, the groups defined by rRNA sequencing reflect the classification obtained with sensitin tests and pathogenicity examinations in chickens. Unique 16S rRNA sequence patterns could be defined for (1) M. avium , (2) M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11, (3) M. intracellulare serovars 12, 13, 14, 15, 17, 19 and 20, (4) M. intracellulare serovar 7 and (5) M. intracellulare serovar 18. Phylogenetically, groups 1 and 2 on one hand and groups 3, 4 and 5 on the other hand each share a common ancestor. M. paratuberculosis was indistinguishable from M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11 by this kind of analysis.

Jörn Wolters

Papers

Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium.

Compilation of 5S rRNA and 5S rRNA gene sequences

The troublesome parasites--molecular and morphological evidence that Apicomplexa belong to the dinoflagellate-ciliate clade.

Phylogeny of rapidly growing members of the genus Mycobacterium.

Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level.