Taxonomy: Definition, Objectives and Characteristics

In this article we will discuss about:- 1. Definitions of Taxonomy 2. Objectives of Taxonomy 3. Characteristics.

Definitions of Taxonomy:

Taxonomy is the science dealing with classification. In biology or in any other field, systematic classification is necessary to bring an order in the apparent chaos, so that the different types in a heterogeneous assemblage can be arranged into groups having common properties.

In biology, taxonomy aims at grouping organisms on the basis of mutual similarities into units called taxa (singular taxon). The taxonomic unit, taxon, may have different levels depending on the extent of similarities among the organisms included in it. Each level or rank has a different designation and these ranks form a hierarchical arrangement.

For example, organisms having the highest similarity are placed in one taxonomic unit, called a species. A group of several species is then assembled into the next higher unit, called a genus (pl. genera). A group of genera is then collected to form the next higher taxon, called a family. Similarly, a group of families form an order, several orders form a class and several classes, a kingdom. In recent times, particularly in bacterial taxonomy, a rank above kingdom level has been created, called a domain.

The basic taxonomic unit of biological organisms is a species. In case of bacteria, a species is defined as a collection of strains which resemble each other in many characteristics and differ significantly from other collections of strain. A strain is defined as the descendants of a single colony of a pure culture.

A colony ideally is formed from a single cell or spore growing on an agar medium. So, a bacterial species, according to the above definition, comprises a number of strains which are closely similar, differing in one or a few characteristics. Thus, the concept of a bacterial species is different from the species concept applicable to higher organisms, like plants and animals.

In the latter, a species represents a group of distinctly identifiable organisms which are sexually isolated from the members of other species. This means that exchange of genetic materials by the normal process of sexual interaction is possible only between two individuals belonging to the same species. Inter-specific genetic exchanges are rare.

Bacteria lack, generally, a sexual mechanism as found in higher organisms and, therefore, the definition of a bacterial species is not so rigid, rather it leaves considerable amount of freedom to the taxonomist to create “new” species on the basis of even one or a few differences in characteristics. The higher taxonomic ranks, like genus, family, order also suffer from the same uncertainties due to the inherent laxity in their definitions.

Objectives of Taxonomy:

Taxonomy aims at fulfilling three main objectives:

1. Firstly, taxonomy aims at classifying organisms into taxa on the basis of similarities in phenotypic (phenetic) characteristics i.e. the characteristics which are expressed in an organism and can be examined visually or can be tested by other means.

As each phenotypic characteristic is controlled by one or a group of genes, two individuals which possess similar phenotypic characteristic must have similar genes. Large number of similar phenotypic characteristic, therefore, reveals a genetic closeness between organisms.

Genetic closeness is also often linked with phylogenetic relatedness, because organisms having many common genes among them must have originated from a common stock during evolution. For instance, if two organisms, A and B, are phylogenetically closer than to another organism, C, it means that A and B have branched off from a common stock in more recent times than C which branched off earlier. Obviously, A and B will have more common genes between them than they will have with C.

2. The second objective of taxonomy is to assign each taxon a name. This naming of a taxon is known as nomenclature. Assigning a name to an organism is necessary for identifying it without confusion throughout the scientific world. Therefore, nomenclature needs to be made following certain internationally accepted rules. Scientific naming of bacteria is governed by the International code of nomenclature of bacteria (The Revised code of 1975). An updated edition of the Revised code has been published in 1992.

Like other organisms, bacterial names have two parts, — a genus name and a species name. Both names must be in Latinized form. This type of naming of biological organisms called, binomial nomenclature, was first introduced by Carl Linnaeus in 1753. The generic name is a Latinized noun starting with a capital letter and the specific epithet is generally a Latinized adjective qualifying the genus name.

The species name is written in small hand. As an international convention, the generic and specific names are italicized. To avoid confusion, a scientific name of an organism also requires the citation of the author’s name who first proposed the name. In assigning a name to a newly discovered bacterium, the author or authors must strictly observe the rules laid down by the International Code.

A new species is indicated by the abbreviation sp. nov. and a new genus by gen. nov. The authors describing a new bacterium are also required to deposit a culture of the organism in an authorized culture collection where it is maintained as the type culture of the particular organism for future reference. Each entry is provided with a separate index number by the collection authority.

Besides scientific names, bacteria are often known by their trivial or informal names. Some trivial names are often widely used. For example, Koch’s bacillus is the common name of Mycobacterium tuberculosis, pneumococci of Streptococcus pneumoniae, meningococci of Neisseria meningitidis etc.

3. The third objective of taxonomy is to serve as an instrument for identification of bacteria. A newly isolated organism can be assorted to its nearest allies or can be identified as a new hitherto unknown taxon. This makes taxonomy a dynamic branch of biology, because discovery of new organisms constantly demands changes in the existing classification.

Also, adoption of new techniques for classifying organisms often necessitates changes, sometimes thorough changes in the existing framework. For example, the developments in the molecular biological techniques, like DNA hybridization, have made a great impact on the taxonomy of bacteria. In fact, a new branch of taxonomy called molecular taxonomy has come into existence. The determination of homology of ribosomal RNA’s is another development which has revolutionized bacterial taxonomy.

Characteristics of Taxonomy:

(i) Classical Taxonomy:

Classification of any group of organism uses selected stable characteristics which vary among the taxa. These are known as taxonomic characteristics. Classically, the bacteria have been classified on the basis of similarities in phenotypic characteristics, like morphological features, response to Gram stain, cultural characteristics, physiological biochemical properties, pathogenicity, antibiotic sensitivity, serological relationships etc. Taxonomically important morphological, cultural and physiological- biochemical characteristics are shown in Table 3.1, 2 and 3 respectively.

(ii) Molecular Taxonomy:

Approach to bacterial taxonomy has undergone drastic changes since the development of molecular biology in the second half of the twentieth century. The concept that macromolecules, like proteins and nucleic acids, could be used as an indicator of evolution of living organisms was first suggested by Zuckerkandl and Pauling in 1965.

They described these macromolecules as “molecular chronometers”, because the sequences of monomers in them have changed slowly and randomly and the number of changes in a particular macromolecule has increased linearly with geological time scale.

A comparison of the sequence of monomers of a particular macromolecule from two organisms should, therefore, give a measure of their phylogenetic relationship. If the sequences differ considerably, it indicates that the two organisms are phylogenetically distant.

This new approach has given rise to the molecular taxonomy. Although initially amino acid sequencing of proteins was used as a parameter for determination of phylogenetic relations, nucleic acids soon replaced proteins. Among the characteristics of nucleic acids, DNA base composition, DNA homology, DNA sequencing, r-RNA sequence analysis etc. have been used for solving taxonomic problems.

The principles of some of the methods are briefly described:

(a) DNA base composition:

The first characteristic that was applied in solving taxonomic problems was the base composition of DNA. A unique feature of DNA is that the ratio of (G + C): (A + T) is more or less constant for a biological species. The ratio is conventionally expressed as G + C moles %. Organisms which are closely related, like the strains of a given species have close values of G + C moles %. In bacteria; this value varies from about 25% to 80%.

The values of some representative bacterial genera are shown in Table 3.4:

Several methods are available for experimental determination of DNA base ratio. Of these, two methods commonly employed are those by determination of melting temperature of DNA and buoyant density. The principles are described briefly.

A characteristic feature of double-stranded DNA helix is that at a high temperature the helical structure collapses producing two single strands due to dissolution of the H-bonds. This is known as melting and the temperature at which melting occurs is a character of a particular species of DNA.

Because there are three H-bonds between G and C, and two between A and T, a DNA molecule having more of G + C melts at a higher temperature. Another important characteristic of DNA is that molten or denatured DNA shows an increase in optical density at 260 nm, a phenomenon known as hyperchromicity.

This means that as the double stranded DNA is dissociated into single-stranded state, its optical density at 260 nm (OD₂₆₀) gradually increases and reaches a maximum when all the DNA present in a sample becomes denatured. This can be measured in an instrument called UV spectrophotometer having an arrangement for gradually raising the temperature of a DNA solution.

By plotting % increase of OD₂6o against temperature, a curve is obtained as shown in Fig. 3.4. The temperature at which 50% of maximum increase is reached is taken as the melting temperature (T_m) of that particular species of DNA. From the melting temperature, the G + C moles % can be calculated from the relation, T_m = 69.3 + 0.41(G + C) %.

Although G + C content of DNA is a useful taxonomic character, it alone may not indicate a close relation, because two quite unrelated organisms may have by chance close G + C content. But for two organisms resembling each other in most other phenotypic characters, a close G + C content of DNA can be taken as a reliable indication of their phylogenetic relatedness.

By analysis of G + C content of DNA of large number of bacteria, it has been found that strains within a species have more or less identical values and its variation in different species within a genus usually does not exceed by more than 10%.

It should be remembered that G + C content gives only the overall composition of DNA and gives no information about the sequence of bases in the DNA molecule. It is this sequence in a DNA segment that constitutes the specificity of a gene. So, G + C content of DNA does not give any information regarding the similarity of genes of two organisms.

Another technique of determination of G + C moles % of DNA utilizes a different property of double-stranded (ds-) DNA. The buoyant density of ds-DNA increase linearly with its G + C content. In an equilibrium density gradient, homogeneous nucleic acid accumulates as a symmetrical band, the width of which is inversely proportional to the square root of its molecular weight.

Smaller molecules having a lower molecular weight tend to diffuse more rapidly than larger molecules and, hence, have wider bands. Also, DNA samples differing in G + C content form separate bonds, because of their difference in density.

In equilibrium density gradient centrifugation using caesium chloride (CsCl), the samples of DNA, previously purified by removing proteins and RNA, are mixed thoroughly with a 6M solution of the caesium salt in a PVC (polyvinyl chloride) centrifuge tube and the mixture is subjected to high speed ultracentrifugation for a sufficiently long time to allow the formation of a density gradient.

The highest density is at the bottom of the tube and it gradually decreases upwards. The different components of the DNA sample collect in distinct bands at levels where the density of a particular component equals that of the gradient.

The fractions can be collected by puncturing the PVC tube at the bottom and their density determined. From the buoyant density of the DNA bands, their G + C moles % can be calculated from the relation, p (buoyant density) = 1.660 + 0.98 (G + C) %.

(b) Nucleic acid hybridization:

A more reliable information about the similarity of the genomes of two organisms can be obtained by DNA-DNA hybridization, because formation of a heteroduplex between two single stranded DNA molecules derived from two organisms depends on the degree of complementarity of the two single strands.

A double-stranded DNA can be dissociated into single strands by application of heat. An interesting feature of single-stranded DNA is that on cooling, the strands tend to re-associate to form double-helix structure automatically. This process, known as annealing, occurs optimally when the temperature is brought to about 25°C below the melting temperature in a solution of high ionic concentration, such as 0.3M NaCl which reduces electrostatic repulsion between the DNA strands.

Various methods have been developed for quantitative determination of heteroduplex formation. One of the most commonly employed technique involves binding of comparatively long DNA molecules of one organism to nitrocellulose filter and allowing the bound DNA molecules to hybridize with comparatively short DNA molecules of the other organism.

For differentiating between the two species of DNA, one of them — usually the second one — is made radioactive by labeling with either ³²P or ³H. Radioactive DNA is obtained by growing an organism in a medium containing a radioactive salt e.g. ³²P labelled phosphate. DNA becomes labelled and is then isolated and purified for use in hybridization.

For DNA-DNA hybridization, the longer non-radioactive single stranded DNA molecules are first allowed to bind to a nitrocellulose filter, unbound DNA is removed by washing and the filter with bound DNA is incubated with the radioactive smaller single-stranded DNA under optimal conditions of annealing.

During incubation the smaller radioactive molecules hybridize with the longer DNA molecules depending on their homology in the base sequences. Then the filter is washed to remove the unbound radioactive DNA molecules and the radioactivity of the filter is measured.

Evidently, the radioactivity retained is due to hybridization of the non-radioactive DNA with radioactive DNA and it gives a quantitative measure of the degree of complementarity of the two species of DNA i.e. homology between the two DNAs. The procedure is schematically shown in Fig. 3.5.

It has been experimentally determined that heteroduplex formation between two different single stranded DNA molecules is prevented if their non-complementarity exceeds 10-20%. From the taxonomic point of view, if the DNAs of two organisms have at least 70% homology and their T_m– values do not have more than 5% difference, the two organisms can be considered to belong to the same species, otherwise they can be considered as different species.

DNA homology studies have been conducted on more than 10,000 bacteria belonging to about 2,000 species and several hundred genera. It has proved to be a powerful tool in solving many problems of bacterial taxonomy, particularly at species level.

It should be noted that for determining relationships among distantly related organisms, DNA-DNA hybridization, cannot give any positive information, because DNAs of such organisms do not possess enough base-pair complementarity to allow heteroduplex formation. Only information that may be obtained is that the organisms concerned are not related to each other. From taxonomic point of view, such information is not of much value.

(c) Ribosomal RNA homology:

An important discovery made in 1965 revealed that in all living organisms, the DNA segments transcribing ribosomal RNA (r-cistrons or r-DNA) have changed more slowly in course of evolution than the rest of the genome. In other words, the r-cistrons are more conserved in comparison to the genes encoding proteins.

This provided an instrument for comparing the phylogenetic relationships between distantly related organisms through determination of base sequences of r-RNA or r-DNA. Among the different r-RNAs, the 16S r-RNA of prokaryotic organisms and the analogous 18S r-RNA of eukaryotes have been found to be most suitable for comparison of their sequences in taxonomic studies.

The method used in the beginning for determination of r-RNA homology was oligonucleotide cataloging. Purified r-RNA was cleaved into oligonucleotides by specific enzymes, like bacteriophage T1 RNase, separated by two-dimensional electrophoresis, further hydrolysed into smaller segments and again electrophoresed to determine their nucleotide sequences.

The sequence of one unique oligonucleotide of each organism was stored in computer. Sequences of different organisms were compared to determine their similarity. Ribosomal RNA of most of the major taxonomic group has been found to possess one or more unique sequences which are known as their oligonucleotide signature. Such signature sequences have been determined for most of the major taxonomic groups of bacteria.

One of the major impacts of r-RNA studies on taxonomy is the recognition of three major domains — the Archaea, the Eucarya including all eukaryotes, and the Bacteria. It has been claimed by Woese, Kandler and Wheelis (1990) that the three major evolutionary lines diverged from a common ancestral form.

Advances in the molecular biological techniques have now made it possible to determine nucleotide sequences of r-DNA for preparing phylogenetic trees with the help of computers. Such trees are built up by comparing the sequences of two molecules by alignment. The number of mismatches in the sequence is counted and used to calculate the evolutionary distance. The similarity between the two molecules is expressed as similarity coefficient.

A group of closely related organisms e.g. species of the same genus, will generally have a narrow range of similarity coefficients. Conversely, a wider range of similarity coefficients indicates that the organisms have branched off from each other in more remote past.

On this basis a dendrogram or a phylogenetic tree can be prepared (Fig. 3.6):

Determination of r-RNA/r-DNA homology has been made with thousands of bacterial organisms for revelation of their taxonomic relationships. Such studies were made till 1997-1998 with pure cultures of bacteria, but, since then, techniques have been developed to recover r-RNA genes directly from natural habitats and have eliminated the necessity of bringing the bacteria in pure culture in the laboratory.

This has come to be known as community analysis of 16S r-RNA from natural microbial community. One of the surprising findings of such studies is that the number of microorganisms that is not yet brought under cultivation far exceeds the number of those that has been cultured so far. This means that the diversity of the microbial world is far more extensive that it is known till now.