Phylogeny Based on Secondary article 16S rRNA/DNA Article Contents . Introduction Erko Stackebrandt, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, . Semantic : a Basis for Phylogenetic Braunschweig, Germany Studies . Sequence Determination, Sequence Alignment and Determination of Sequence Similarities Modern systematics of is based on comparative analysis of the evolutionarily . Recognition of the Higher Taxa of Prokaryotes conservative genes coding for 16S ribosomal RNA. Dendrograms of phylogenetic . Polyphasic Approach to Bacterial Systematics relatedness show the order in which evolved in time, thus providing a basis for . The Taxonomic Rank ‘’ in Bacteriology their classification.

Introduction are the historical record of , and the determina- In contrast to more highly evolved , in which tion of their primary structure provides a powerful means complex morphologies visibly reflect their evolutionary by which evolutionary relationships can be measured. In history, the microscopic and ultrastructural features of essence, two organisms possessing a given stretch of microorganisms cannot be used to deduce the way in which semantides which differ in only a few changes (mutations, the prokaryotes and the morphologically simple eukar- nucleotide order or amino acid positions) are more closely yotic forms evolved. Before 1960 taxonomists were unable related to each other than those organisms in which a to appreciate the complexity of microbial systematics and higher number of changes have accumulated. Thus these to recognize that groups based on superficial properties molecules can be considered as chronometers. As different alone did not necessarily reflect those which arose due to genes are subjected to different rates of changes (same evolutionary processes. However, at this same time (when frequency of mutation but different level of manifestation most microbiologists resigned themselves to the belief that of changes), each cell possesses a variety of chronometers the true course of phylogeny could never be from which different evolutionary events can be deter- unravelled) concepts were developed that turned out to mined. Slow running clocks will reflect early evolutionary change our view about the inter-relatedness of living events such as the separation of the main lines of descent, organisms. while fast running clocks will reflect more recent events, such as the more precise separation of genera and species. Comparative analysis of one particular homologous semantide from many organisms will allow the determina- Semantic Macromolecules: a Basis for tion of the order in which this gene or evolved during evolution, thus unravelling its family tree. Phylogenetic Studies Episemantic molecules, which are synthesized under the control of tertiary semantides, are adenosine triphosphate Biological molecules can be classified into three categories (ATP), carotenoids and chemotaxonomic markers. These – semantides, episemantides and asemantides – according molecules are not phylogenetic markers per se but the past to the information they carry (Zuckerkandl and Pauling, has shown that groups of organisms that form a 1965). At the highest level is the semantides, which are phylogenetic cluster can, in most cases, be recognized by information-carrying molecules. From the evolutionary the chemical composition of markers such as peptidogly- origin of the first cell to contemporary cells, information on can, , fatty acids, isoprenoid quinones, polyamines reproduction, behaviour, survival, maintenance, etc. have and mycolic acids. been laid down in blueprints and passed in semantides Asemantic molecules are molecules that are not pro- from one generation to the other. duced by the organisms themselves and therefore do not Three semantides have been defined according to their express any of the information that this contains. role in the cell. Following the biological dogma of For example, molecules such as exogenously supplied information flow, DNA is the primary semantide, RNA vitamins, phosphate ions, , , etc. cannot be is the secondary semantide, and are the tertiary used in the reconstruction of evolutionary events. semantides. As changes in the primary structure of DNA occur by an ongoing process of random mutation and selection, the composition of this molecule is constantly changing and this information is passed through messen- ger RNA to the proteins. The sequences of these molecules

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16S rRNA and the gene coding for it are ribosomal proteins are assembled to form the two reliable homologous molecules ribosomal subunits of which each ribosome is composed. The small 30S ribosomal subunit contains the 16S rRNA The most useful molecular chronometers used today for and 21 proteins, while the larger 50S subunit contains the the determination of phylogenetic relationships are the 16S 23S rRNA, the 5S rRNA and 32 proteins. Fully assembled ribosomal (r)RNA genes (rDNA) and their gene products, ribosomes are part of the process, in which the the 16S rRNAs (Figure 1) (Woese, 1987). In most organisms genetic information, channelled through the ribosomes via these genes occur in multiple copies per cell, but very slow- transcribed messenger RNA is translated into polypeptides growing species may have only a single copy. The 16S and proteins. It can be deduced from the universality of the rRNA gene is part of an rrn operon, located at the 5’ biological code that such a process, although terminus, followed by the larger 23S rDNA gene and the primitive in early evolution, had already been functioning small 5S rRNA gene. These genes are separated by spacers, during the early stages of life. Thus, the 16S rRNA genes, as some of which contain genes for transfer RNA. During well as the genes coding for other components of the translation, the pre-rRNA is folded under the influence of ribosomes, are presumably derived from a common the ribosomal proteins into tertiary and quaternary ancestor and are homologous molecules. structures. This is the basis for the maturation process, in Several criteria have been identified which make the 16S which the 5’ and 3’ flanking region of the rRNA genes are rRNA and their genes the most widely studied phyloge- digested by specific . The mature RNA and the netic markers: (1) the function of ribosomes has not

1100r 1114f

519f 907r 536f 27f 926f

1392r

5’ 357f 342r

3’

Figure 1 Secondary structure of a 16S rRNA molecule based on the E. coli structure (Maidak et al., 1994; available in the public domain Ribosomal Database Project). Highly variable regions are red; highly conservative stretches are green. Binding sites of primers used in PCR amplification of the rRNA gene are blue, with the direction of amplification indicated by arrows. The other nucleotides are black.

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Phylogeny Based on 16S rRNA/DNA changed for about 3.8 billion years, (2) the 16S rDNA sequence analysis is most conveniently carried out as a genes are universally present among all cellular life forms, linear PCR cycle sequencing reaction. The sequences are (3) the size of 1540 nucleotides makes them easy to analyse, then aligned, which means that homologous nucleotides (4) the primary structure is an alternating sequence of derived from a common position within the ancestral invariant, more or less conserved to highly variable sequence are arranged in columns, and consequently are regions, and (5) lateral gene transfer has not (yet) been recognized as being identical or different (Stackebrandt observed among organisms. Other homologous molecules and Rainey, 1995). have been sequenced, e.g. the 23S rDNA, the 5S rRNA, Phylogenetic relationships can be assessed by pairwise genes coding for enzymes, and ribosomal proteins, but the similarities. One hundred per cent similarity found database is not nearly as extensive for these molecules as between a pair of 16S rDNA sequences using different 16S rDNA, for which about 8000 sequences have been methods indicates very high relatedness, if not identity of deposited. the investigated organisms. The lower the value the more unrelated the compared organisms. If, however, the number of organisms is too large, the respective similarity matrix cannot be interpreted meaningfully. In this case Sequence Determination, Sequence phylogenetic relationships can be visualized graphically by using algorithms that transform the similarity values into Alignment and Determination of dissimilarity values to compensate for superimposed Sequence Similarities (multiple) substitutions. These phylogenetic distances form the basis for phylogenetic trees or dendrograms. Progress in the elucidation of phylogenetic relationships The most widely applied treeing methods are distance parallels the development of sequencing methods. Com- methods but other approaches, such as maximum parsi- parative sequence analysis of 16S was introduced by Carl mony and maximum likelihood methods are frequently Woese and collaborators about 20 years ago (Woese and used as well. Tree topologies are best tested by comparing Fox, 1977). The demanding and expensive sequence the evolution of different phylogenetic markers with a analysis of T1-generated 16S rRNA oligonucleotides similar degree of sequence conservatism. The results of (16S rRNA cataloguing), applied by only a few scientists, comparative analyses of other conserved molecules re- led to the discovery of the kingdom Archaebacteria (now sponsible for central functions such as elongation factors ) as a third main line of evolutionary descent and (associated with ribosomes and necessary for the elonga- to the first main outline of the evolution of prokaryotes. tion of the growing ), b subunit of ATPase (used to Ten years later, with the introduction of reverse tran- catalyse the breakdown of ATP to ADP 1 Pi) and RNA scriptase sequence analysis, sequence analysis of amplified polymerases (necessary for the growth of mRNA strands copies of rRNA developed into a routine method. Today, during transcription), do in general, support the branching rRNA genes are amplified by the polymerase chain pattern of organisms within major lines of descent as reaction (PCR) and rDNA sequencing is used worldwide inferred by 16S rDNA analysis (Figure 2). in assessing the phylogenetic position of novel strains and microbial communities. The main reason for the routine application of this method is the presence of a set of conservative nucleotide Recognition of the Higher Taxa of stretches, which are scattered over the rDNA genes, Prokaryotes serving as target sites of oligonucleotide primers (usually 14–20 bases in length) (Figure 1); these primers are needed Prokaryotes do not constitute a coherent phylogenetic for amplification and subsequent sequence analysis. Thus, group of organisms but form two lineages, the Archae- a set of not more than 10 primers is sufficient to analyse a (now Archaea) and the Eubacteria (now Bacteria), wide spectrum of phylogenetically diverse organisms. each of them being as unrelated to the 18S rRNA – Sequence analysis can be performed on both purified representing the nucleus component of the eukaryotes – as nucleic acid preparations and crude extracts of bacterial they were related among each other (Figure 3) (Woese and cells. Fox, 1977). Considering that the root of the tree is placed Today, the amplified DNA fragments are sequenced within the eubacterial lineage, the eukaryotes and Archae- directly by applying the chain termination method, in bacteria appear to be specifically related. The distinctness which the statistical introduction of a nucleotide analogue, of the three major rRNA lines of descent was later such as dideoxynucleotides, allows them to compete with confirmed by analyses of the genes coding for proteins conventional nucleotides and cause base-specific termina- involved in the regulation of translation and in energy- tion of the elongation products. This results in populations yielding processes. The concept of these new primary of single-stranded (ss)DNA fragments of different lengths kingdoms contrasted dramatically with the traditional, sharing a common 5’ end (the primer). Automated DNA more widely accepted five-kingdom classification, describ-

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ABthis method is still too slow to provide information on the Mycoplasma large set of organisms required for meaningful analysis. Pectinatus Presently, besides the comparison of a homologous gene, it Peptococcus is the presence of certain phenotypic traits that confirms Enterococcus Lactobacillus the separateness of the two prokaryotic domains. Char- Lactobacillus Enterococcus Bacillus firmus acters shared between members of two of the three highest Bacillus megaterium taxa are of no use for placing strains in a phylogenetically Corynebacterium correct taxon, but certain characters have been identified Propionigenium that are domain specific and hence are of diagnostic value. Synechococcus Among others, these are the chemical linkages and Anabaena compositions of amino acids and sugars in the Pirellula Wolinella (peptidoglycan (Bacteria) versus pseudomurein, proteina- Stigmatella cous and polysaccharide walls (Archaea)), ester-linked Rhodospirillum (Bacteria) versus ether-linked (Archaea) lipids, the mod- Escherichia ification pattern of tRNAs, and the resistance to antibiotics (Danson et al., 1992). Bacteroides Both of the two prokaryotic domains contain a number Flavobacterium Cytophaga of more or less well-separated sublines of descent, formerly Chlorobium named the phyla or divisions. With the introduction of the Herpetosiphon taxon domain, within the Archaea the phyla were Thermotoga described as kingdoms. Most of them are evolutionarily more ancient than the presently described eukaryotic Figure 2 Phylogenetic dendrograms based on ATP synthase b subunit kingdoms but it can be assumed that the number of amino acid (A) and 16S rDNA nucleotide (B) sequences, showing the kingdoms within the domain Eukarya will increase general agreement in branching patterns obtained from analyses of different evolutionary conserved macromolecules. The trees were significantly once the lower eukaryotes have been investi- reconstructed by neighbour-joining analysis. Organisms belonging to the gated by extensive phylogenetic analysis. phyla of Gram-positive bacteria, cyanobacteria, and Bacteroides/Cytophaga are displayed in blue, green, red and pink, The kingdoms of the Archaea respectively. The bar indicates 10% estimated changes. ATP synthase data modified from Ludwig et al. (1993). Three main lines, defined as kingdoms, have so far been defined within the domain Archaea: the , ing four eukaryotic kingdoms (animals, plants, fungi and the Crenarchaeota and the Korarchaeota. The first king- protozoa) and a single prokaryotic kingdom (bacteria). dom contains the physiologically defined methanogenic However, in phylogenetic, molecular and cellular terms the organisms. Related to them are nonmethanogenic taxa eukaryotic kingdoms are virtually identical and the five- such as the wall-less thermoplasmas, the extreme halophi- kingdom concept does not recognize the fundamental lic and alkaliphilic archaea and the peculiar thermophilic differences that distinguish the two groups of prokaryotes. fulgidus, an organism capable of forming methane and reducing sulfate. Also included is the thermophilic species celer and the symbiotic The Domain concept that serve as hydrogen sinks in many protozoa. The second kingdom contains the hyperthermo- The three-kingdom classification was subsequently re- philic archaea, some of which grow optimally at tempera- placed by the Domain concept (Woese et al., 1990). This tures up to 1058C and most of which require elemental taxon has been created at the highest taxonomic rank to for optimal growth. This taxon is broadened highlight the importance of the tripartite division of the phylogenetically by an enormous number of yet uncul- living world. The suffix ‘bacteria’ was omitted in the names tured bacteria from a geothermal hot spring in Yellow- of the two prokaryotic domains to reflect their exclusive stone National Park. The third kingdom contains only a evolutionary relationship. Instead the terms Archaea, few uncultured hot spring organisms that, as derived from Bacteria and Eukarya have been proposed. the 16S rDNA sequence, may represent evolutionary Sequence comparisons provide neutral genotypic mea- primitive life forms (Barns et al., 1996). surements but they do not reveal differences in . If, however, molecular analyses point towards the ex- istence of major evolutionary groups, one would expect to The phyla within the domain Bacteria find additional profound differences at the molecular level The phylogenetic structure of the domain Bacteria is much that are expressed at the phenotypic level. The most more complex than that of the Archaea and a formal straightforward approach in the determination of differ- kingdom structure has not yet been proposed. The reason ences at the DNA level is analysis of whole , but for the delay can be explained by the large number of phyla,

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Domain Archaea Kingdom Methanococcus Kingdom Euryarchaeota Sulfolobus Crenarchaeota Thermococcus

Methanobacterium Pyrodictium Thermofilum

Methanosarcina pJP78 Cyanobacteria Thermotoga Kingdom Korarchaeota Gram-positives Fibrobacter Aquifex Proteobacteria Thermus Bacteroides Chlorobium Domain Bacteria Microsporidia Giardia Higher evolved Eukarya Domain Eukarya 5%

Figure 3 The phylogenetic tripartation of living organisms based on the analysis of the gene coding for small subunit rRNA (16S and 18S rDNA). Within the prokaryotic domains most of the main lines of descent, called kingdoms in the domain Archaea, and phyla in the domain Bacteria, are indicated. The origin of the evolutionary lineages appears to be located close to the branching point of the lineage of the Bacteria (indicated by a circle). The bar represents 5 nucleotide substitutions per 100 nucleotides. some of which are separated by such small internode more confusing within the class Proteobacteria, where a distances that the precise order of their emergence in hierarchical structure between the ranks of subclasses and evolution cannot be deduced (Olsen et al., 1994). The families is still missing. These organisms lack the rich relationships within the individual lines are often un- spectrum of chemotaxonomic properties found among the expected and support for these groupings from the sharing Gram-positive bacteria and only broad taxonomic groups of properties other than similarities in conserved genes is can be circumscribed by the structure of pigments, rare. polyamine patterns, ubiquinone types, fatty acid composi- A few phyla, i.e. those embracing Gram-positive tion, chemical composition of A and the core region of bacteria, the Proteobacteria, the Bacteroides-Cytophaga the lipopolysaccharides. Chemotaxonomy is only slowly taxon, the spirochetes and the cyanobacteria, embrace the winning ground on the taxons in which morphological and majority of described species. Consequently, these phyla physiological properties have traditionally played the show a complex phylogenetic structure. Of the species- major role in defining genera. poorer phyla, some are traditional taxa, such as Chloro- flexus, Chlorobium and Chlamydia and their respective relatives, but most of the taxa that have been identified to represent new and phylogenetically ancient phyla have Polyphasic Approach to Bacterial been isolated during the past 20 years, such as the orders Aquifecales, Thermotogales, Planctomycetales, Verruco- Systematics microbiales, and the genera Fibrobacter, Acidobacterium, Microbiologists should be aware that the available Deinococcus and Thermus and their relatives. phylogenetic branching pattern reflects the actual situation The most convincing 16S rDNA branching pattern is in nature quite incompletely. Phylogenetic reconstructions that of the Gram-positive bacteria. The presence of two are based on inferred homologies from only a few major subdivisions correlates nicely with the distribution molecules and thus can be considered, at best, an of the DNA G+C content of their members (i.e. the low approximation. The gradually emerging 16S rRNA tree G+C ‘clostridial’ subline and the high G+C ‘actinobac- is probably best considered as presenting a hypothesis teria’ subline). The class Actinobacteria is the first example about relationships which should be tested by other data. of a fully hierarchical, phylogeny-based classification In order to maintain stability in systematics the description system above the level within the domain Bacteria. of the two most important levels in , the species Here, a rich pattern of chemotaxonomic properties has and the genus, should be based on a combination of facilitated the circumscription of genera. The situation is properties; this strategy is referred to as the ‘polyphasic

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5 Phylogeny Based on 16S rRNA/DNA approach to taxonomy’. In this approach the phylogenetic merical phenetic taxonomy) to identify phenotypically branching pattern serves as an aid to recognize clusters of homogeneous clusters which, by convention, are then phylogenetically related strains but the delineation of called ‘species’. The higher the overall resemblance among phylogenetically neighbouring clusters is predominantly strains of a species and the more discriminating these made on the basis of morphology, biochemical properties properties from other species, the more convincing the and episematic molecules. These include the chemical validity of the description. structure of certain cell constituents, such as fatty acids, With the development of DNA:DNA reassociation lipids, peptidoglycan, polyamines, isoprenoid quinones, assays, in which genomic similarity was measured, a new but also selected nucleotides of the 16S rDNA and other quality of assessment of relatedness was introduced. gene sequences. Nucleic acid pairing assays have the advantage that they Above the genus level (family! order! class! king- theoretically sample a very large number of genomic dom (phylum)) the availability of phenotypic properties characters, i.e. the reassociation between short DNA for the circumscription of taxa is poor and descriptions are fragments. Bias is introduced by the presence of large based mainly on sequence information. The tree mirrors and differences in size of the pairing the presence of certain categories, e.g. many of the deeply partner DNA and different methods may give higher or rooting phyla within the domain Bacteria, which are lower values for the same strains. All methods are subject emerging constantly, no matter which molecule and to significant experimental error, typically in the region of a method used. However, the isolated position of these few per cent, but these deviations will not change the groups may disappear as more organisms are investigated. general picture of genomic relatedness. The greatest The main advantage of the phylogenetic system lies in its problem, however, is that it is not possible to build a stability; only the rank (either vertically or horizontally) cumulative database for hybridization values. within a hierarchical structure, but not its place within a The current definition of a prokaryotic species is a higher taxon will change. cluster of highly related strains as revealed by the quantitative assessment of similarity amongst the primary structure of their DNA; the strains must also be phenotypically similar. A guideline of 70% or greater The Taxonomic Rank ‘Species’ in DNA:DNA reassociation with high thermal stability of Bacteriology heterologous duplexes has been recommended as the boundary value for strains in the same species. A value of The biological species concept (BSC), which was originally 70% DNA similarity as determined by hybridization formulated for ‘higher’ eukaryotes, describes groups of corresponds to about 96% sequence identity as determined interbreeding natural populations which are reproduc- by sequence analysis. This level is based upon the empirical tively isolated from other such groups. Gene flow does observation that strains of bacteria which are highly occur between prokaryotes but it occurs by different related phenotypically often share at least this amount of mechanisms and has different effects compared to those in DNA:DNA reassociation. sexual eukaryotes. Prokaryotes do not produce gametes The level of DNA:DNA reassociation between strains of and do not undergo meiosis. Consequently, any concept established taxonomic species can be greater than 70% but such as the BSC which requires these characteristics (i.e. is seldom much lower. For example, the two species reproduction linked to sex) cannot be applied. Gene Neisseria gonorrhoea and N. meningitidis share over 74% transfer among individuals of different bacteria varies in DNA:DNA reassociation but are kept as separate species frequency and the rate of recombination may vary among because they can be phenotypically distinguished and loci encoding proteins of different types. Prokaryotic because they cause different important diseases. Another species may evolve in a defined ecological niche but their well known case is the genera pair Escherichia and Shigella, enormous ability to move around in the environment and whose species cannot be distinguished genomically, except to explore new niches makes them prone to new kinds of for a few genes that cause their different pathogenicities. competition, stress, exchange of genetic material, and Strains of a species that share less than 70% DNA hence to rapid changes of the genotype and phenotype at similarity are not reclassified as new species when the the strain level. new taxon lacks any phenotypic properties that would These characteristics mean that prokaryotic taxono- allow taxonomists to recognize the entity as a new species. mists face the problem of seeing how bacterial population Where does rDNA sequence similarity fit into this genetics can provide a consistent BSC for prokaryotes. In discussion? Among highly related strains the correlation order to define bacterial species, bacterial taxonomists between DNA:DNA similarity and rDNA similarity is not have traditionally used approaches based on overall linear, in many examples there is actually very little resemblance (i.e. phenetic relationship). Methods have correlation. A compilation of data obtained with the same been used to analyse a group of strains for many pairs of randomly selected strains have shown that despite unweighted characters, applying statistical analysis (nu- higher than 99% sequence similarity the corresponding

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DNA:DNA similarity was lower than 40% (Stackebrandt Maidak BL, Larsen N, McCaughey MJ et al. (1994) The Ribosomal and Goebel, 1994). This lack of correspondence is not Database Project. Nucleic Acids Research 22: 3485–3487. surprising when it is considered that the overall nucleotide Olsen GJ, Woese CR and Overbeek R (1994) The winds of (evolutionary) similarity of the genome is compared, and that this changes: breathing new life into microbiology. Journal of Bacteriology 178: 1–6. represents a mosaic of slowly to rapidly evolving genes Stackebrandt E and Goebel BM (1994) A place for DNA–DNA with single genes usually slowly evolving. It can be assumed reassociation and 16S rRNA sequence analysis in the present species that evolutionary changes occurring at the DNA level will definition in bacteriology. International Journal of Systematic only be mirrored by changes at the rRNA gene level after Bacteriology 44: 846–849. millions of years. Calculations for the 16S rDNA of Stackebrandt E and Rainey FA (1995) Partial and complete 16S rDNA prokaryotic symbionts, for which the age of the host and sequences, their use in generation of 16S rDNA phylogenetic trees and thus the approximate invasion of the host by the symbiont their implications in molecular ecological studies. In: Akkermans ADL, van Elsas JD and de Bruijn FJ (eds) Molecular Microbial is known, reveal that 40–50 million years are required Ecology Manual. Amsterdam: Kluwer Academic Publishers. before the 16S rDNA primary structure is altered by 1% Woese CR (1987) Bacterial evolution. Microbiological Reviews 51: 221– ( 15 nucleotides). During such a period, prokaryotic 271. strains may alter significantly with respect to their Woese CR and Fox GE (1977) Phylogenetic structure of the prokaryotic physiological and biochemical properties. Experience has domain: the primary kingdoms. Proceedings of the National Academy shown that the vast majority of intraspecies 16S rDNA of Sciences of the USA 74: 5088–5090. sequence similarities are 100%. Woese CR, Kandler O and Wheelis ML (1990) Towards a natural system of organisms. Proposal for the domains Archaea, Bacteria, and Another phenomenon that prohibits the sole use of 16S Eucarya. Proceedings of the National Academy of Sciences of the USA rDNA sequence similarity to define species is the finding 87: 4576–4579. that this gene evolves at a different rate in different groups Zuckerkandl E and Pauling L (1965) Molecules as documents of of prokaryotes. The time that elapsed for changing the 16S evolutionary history. Journal of Theoretical Biology 8: 357–366. rDNA primary structure of two genus A species by 2% may be significantly different to the time that is needed for a 2% change in the homologous molecule of two genus B species. It is therefore expected that these two genera also Further Reading differ in the extent of their overall taxonomic properties. One cannot expect a single gene, the 16S rDNA gene, to Atlas RM (1997) Microbial systematics, evolution, phylogeny and solve all problems in prokaryotic systematics and taxon- classification. In: Atlas RM (ed.) Principles of Microbiology, 2nd edn, pp. 888–951. Chicago: Wm.C. Brown Publishers. omy. This molecule has had (and still has) a greater impact Balows A, Tru¨per HG, Dworkin M, Harder W and Schleifer K-H (eds) on our understanding of the history of living forms on the (1991) The Prokaryotes. A Handbook on the Biology of Bacteria: planet Earth than any other molecule analysed. rRNA and Ecophysiology, Isolation, Identification, Applications, 2nd edn. 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New York: Springer-Verlag. the USA 93: 9188–9193. Vandamme P, Pot B, Gillis M, De Vos P, Kersters K and Swings J (1996) Danson MJ, Hough DW and Lunt GG (1992) The Archaebacteria: Polyphasic taxonomy, a consensus approach to bacterial systematics. Biochemistry and Biotechnology. Biochemical Society Symposium 58. Microbiological Reviews 60: 407–438. London: Portland Press. Woese CR, Gutell R, Gupta R and Noller H (1983) Detailed analysis of Ludwig W, Neumaier J, Klugbauer N et al. (1993) Phylogenetic the higher order structure of 16S like ribosomal ribonucleic acids. relationships of Bacteria based on comparative sequence analysis of Microbiological Reviews 47: 621–669. elongation factor TU and ATP-synthase b-subunit genes. Antonie van Zillig W (1991) Comparative biochemistry of Bacteria and Archaea. Leeuwenhoek 64: 285–305. Current Opinions in Genetics and Development 1: 544–551.

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