In this article we will discuss about:- 1. Meaning of Bacterial Taxonomy 2. Importance of Bacterial Taxonomy 3. Ranks or Levels 4. Characteristics used.
Meaning of Bacterial Taxonomy:
The science of classification of bacteria is called bacterial taxonomy. Bacterial taxonomy (G: taxis = arrangement or order, nomos = law or nemein = to distribute or govern), in a broader sense, consists of three separate but interrelated disciplines: classification, nomenclature, and identification.
Classification refers to the arrangements of bacteria into groups or taxa (sing, taxon) on the basis of their mutual similarity or evolutionary relatedness.
Nomenclature is the discipline concerned with the assignment of names to taxonomic groups as per published rules. Identification represents the practical side of taxonomy, which is the process of determining that a particular isolate belongs to a recognized taxon. It is to mention here that the term Bacterial systematics often is used for bacterial taxonomy.
But, systematics bears broader sense than taxonomy and is defined by many as the scientific study of organisms with the ultimate object of characterizing and arranging them in an orderly fashion. Systematics therefore encompasses disciplines such as morphology, ecology, epidemiology, biochemistry, molecular biology, and physiology of bacteria.
Importance of Bacterial Taxonomy:
Bacterial taxonomy, however, is important due to following reasons:
1. Bacterial taxonomy senses to be a library catalogue that helps easily access large number of books. Taxonomy therefore helps classifying and arranging bewildering diversity of bacteria into groups or taxa on the basis of their mutual similarity or evolutionary relatedness.
2. The science of bacteriology is not possible without taxonomy because the latter places bacteria in meaningful, useful groups with precise names so that bacteriologists can work with them and communicate efficiently.
3. Bacterial taxonomy helps bacteriologists to make predictions and frame hypotheses for further research based on knowledge of identical bacteria. For convenience, the bacteriologist can predict that a bacterium in question would be possessing similar characteristics to its relative bacterium whose characteristics are already known.
4. Contribution of bacterial taxonomy in accurately identifying bacteria is of practical significance. For convenience, bacterial taxonomy contributes particularly in the area of clinical microbiology. Treatment of bacterial disease often become exceptionally difficult if the pathogen is not properly identified.
Ranks or Levels of Bacterial Taxonomy:
In bacterial taxonomy, a bacterium is placed within a small but homogenous group in a rank or level. Groups of this rank or level unite creating a group of higher rank or level. In bacterial taxonomy, the most commonly used ranks or levels in their ascending order are: species, genera, families, orders, classes, phyla, and domain (Table 3.1).
Species is the basic taxonomic group in bacterial taxonomy. Groups of species are then collected into genera (sing, genus). Groups of genera are collected into families (sing, family), families into orders, orders into classes, classes into phyla (sing, phylum), and phyla into domain (the highest rank or level). Groups of bacteria at each rank or level have names with endings or suffixes characteristic to that rank or level.
Characteristics Used In Bacterial Taxonomy:
1. Classical Characteristics (Classical Taxonomy):
Several phenotypic characteristics (e.g., morphological, physiological and metabolic, ecological) and genetic analysis have been used in bacterial (microbial) taxonomy for many years.
These characteristics are assessed and the data are used to group bacteria up to the taxonomic ladder from species to domain. Classical characteristics are quite useful in routine identification of bacteria and also provide clues for phylogenic relationships amongst them as well as with other organisms.
Various morphological features, e.g., cell shape, cell size, colonial morphology, arrangement of flagella, cell motility mechanism, ultra structural characteristics, staining behaviour, endospore formation, spore morphology and location, and colour are normally used to classify and identify microorganisms.
Morphological characteristics play important role in microbial classification and identification due to following reasons:
(i) They are easily studied and analysed especially in eukaryotic microorganisms and comparatively complex prokaryotes.
(ii) They normally do not vary greatly with environmental changes as they are resulted in by the expression of many genes and, therefore, are usually genetically stable.
(iii) Morphological similarity amongst microorganisms often is a good indication of phylogenetic relatedness.
Some taxonomically useful morphological characteristics and their variations are shown in Table 3.2.
Physiological and Metabolic Characteristics:
Some physiological and metabolic characteristics are very useful in classifying and identifying microorganisms because they are directly related to the nature and activity of microbial enzymes and transport proteins.
Some most important physiological and metabolic characteristics used in microbial taxonomy are nutritional types, cell wall components, carbon and nitrogen sources, energy metabolism, osmotic tolerance, oxygen relationships, temperature relationships, salt requirements and tolerance, secondary metabolites, storage inclusions, etc.
Some taxonomically useful physiological and metabolic characteristics and their variations are given in Table 3.3.
Ecological characteristics, i.e., the characteristics of relationship of microorganisms to their environment significantly contribute in microbial taxonomy. It is because even very closely related microorganisms may vary considerably with respect to their ecological characteristics.
For convenience, microorganisms inhabiting freshwater, marine, terrestrial, and human body environments differ from one another and from those living in different environments.
However, some of the most important ecological characteristics used in microbial taxonomy are – life cycle patterns, the nature of symbiotic relationship, pathogenic nature, and variations in the requirements for temperature, pH, oxygen, and osmotic concentrations.
Genetic analysis is mostly used in the classification of eukaryotic microorganisms because the species is defined in these organisms in terms of sexual reproduction which occurs in them. This analysis is sometimes employed in the classification of prokaryotic microorganisms particularly those that use the processes of conjugation and transformation for gene exchange.
For example, members of genus Escherichia may conjugate with the members of genera Shigella and Salmonella but not with those of genera Enterobacter and Proteus. This shows that the members of first three genera are more closely related to one another than to Enterobacter and Proteus.
Studies of transformation with genera like Bacillus, Haemophilus Micrococcus, Rhizobium, etc. reveal that transformation takes place between different bacterial species but only rarely between genera.
This provides evidence of a close relationship between species since transformation tails to occur unless the genomes are very much similar. Bacterial plasmids that carry genes coding tor phenotypic traits undoubtedly contribute in microbial taxonomy as they occur in most genera.
2. Molecular Characteristics:
Some recent molecular approaches such as genomic DNA GC ratios, nucleic acid hybridization, nucleic acid sequencing, ribotyping, and comparison of proteins have become increasingly important and are used routinely for determining the characteristics of microorganisms to be used in microbial taxonomy.
Genomic DNA GC ratios (G + C content):
Genomic DNA GC ratio (G + C) is the first, and possibly the simplest, molecular approach to be used in microbial taxonomy. The GC ratio is defined as the percentage of guanine plus cytosine in an organism’s DNA.
The GC ratio the base sequence and varies with sequence changes as follows:
The GC ratio of DNA from animals and higher plants averages around 40% and ranges between 30 and 50%. Contrary to it, the GC ratio of both eukaryotic and prokaryotic microorganisms varies greatly; prokaryotic GC ratio is the most variable, ranging from around 20% to almost 80%. Despite such a wide range of variation, the GC ratio of strains within a particular species is constant.
Genomic DNA GC ratios of a wide variety of microorganisms have been determined, and knowledge of this ratio can be useful in microbial taxonomy, depending on the situation. For convenience, two microorganisms can possess identical GC ratios and yet turn out to be quite unrelated both taxonomically and phylogenetically because a variety of base sequences is possible with DNA of a single base composition.
In this case, the identical GC ratios are of no use with view point of microbial taxonomy. In contrast, if two microorganisms’ GC ratio differs by greater than about 10%, they will share few DNA sequences in common and are therefore unlikely to be closely related.
GC ratio data are valuable in microbial taxonomy in at least two following ways:
(i) They can confirm a scheme of classification of microorganisms developed using other data. If microorganisms in the same taxon vary greatly in their GC ratios, the taxon deserves to be divided.
(ii) GC ratio appears to be helpful in characterizing bacterial genera since the variation within a genus is usually less than 10% even though the content may vary greatly between genera.
For convenience, Staphylococcus and Micrococcus are the genera of gram-positive cocci having many features in common but differing in their GC ratio by more than 10%. The former has a GC ratio of 30-38%, whereas the latter of 64-75%.
Nucleic acid hybridization (Genomic hybridization):
Nucleic acid hybridization or genomic hybridization measures the degree of similarity between two genomes (nucleic acids) and is useful for differentiating two bacteria (microorganisms). DNA-DNA hybridization is useful to study only closely related bacteria, whereas DNA-RNA hybridization helps comparing distantly related bacteria.
1. DNA-DNA hybridization:
Double-stranded DNA isolated from one bacterium is dissociated into single strands at appropriate temperature which are made radioactive with 32P, 3H, or 14C.
Similarly, double-stranded DNA isolated from other bacterium is dissociated into single strands which are not made radioactive. The non-radioactive single- stranded DNA molecules are first allowed to bind to a nitrocellulose filter and unbound strands are removed by washing.
Now the filter with bound strands of DNA is incubated with the radioactive single-stranded DNA under optimal conditions of annealing. Annealing is an interesting feature of single-stranded DNA in which the strands, on cooling, tend to re-associate to form double-helix structure automatically.
Annealing occurs optimally when the temperature is brought to about 25°C below the melting temperature (Tm) in a solution of high ionic concentration.
However, during incubation the radioactive single strands of DNA hybridize with non-radioactive single strands of DNA depending on their homology in the base sequence. Then the filler is washed to remove unbound radioactive DNA molecules and the radioactivity of the hybridized radioactive DNA molecules is measured.
Following this, the amount of radioactivity in the hybridized radioactive DNA molecules is compared with the control which is taken as 100%, and this comparison 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. 3.1.
Though there is no fixed convention as to how much hybridization between two DNAs is necessary to assign two bacteria to the same taxonomic rank, 70% or greater degree of complementarity of the two DNAs is recommended for considering the two bacteria belonging to the same species.
By contrast, degree of at least 25% is required to argue that the two bacteria should reside in the same genus. Degree of complementarity to the range of 10% or less denotes that the two bacteria are more distantly related taxonomically.
DNA-DNA hybridization is a sensitive method for revealing subtle differences in the genes of two bacteria (other microbes also) and is therefore useful for differentiating closely related bacteria.
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.
2. DNA-RNA hybridization:
DNA-RNA hybridization helps compare, unlike DNA-DNA hybridization, distantly related bacteria (microorganisms) using radioactive ribosomal RNA (rRNA) or transfer RNA (tRNA).
It becomes possible because the DNA segments (genes) transcribing rRNA and tRNA represent only a small portion of the total DNA genome and have not evolved as rapidly as most other genes encoding proteins (i.e., they are more conserved in comparison to genes encoding proteins).
Among the different rRNAs, the 16S rRNA of prokaryotes and the analogous 18S rRNA of eukaryotic organisms have been found to be most suitable for comparison of their sequences is taxonomic studies. One of the major impacts of rRNA studies on taxonomy, for convenience, is the recognition of three major domains—the Archaea, the Bacteria, and the Eukarya by Woese and Colleagues in 1990.
DNA-RNA hybridization technique is similar to that employed for DNA-DNA hybridization. The filter- bound non-radioactive ssDNA is incubated with radioactive rRNA, washed, and counted.
An even more accurate measurement of complementarity is obtained by finding the temperature required to dissociate and remove half the radioactive rRNA from the filter; the higher this temperature, the stronger the DNA-rRNA complex and the more similar the base sequences.
However, DNA-RNA hybridization has been done with thousands of bacteria for relevation of their taxonomic relationships. Such studies were made with pure cultures of bacteria till 1997-98, but since then, techniques have been developed to recover rRNA genes directly from natural habitats. This has come to be called as community analysis of rRNA from natural bacterial community.
Nucleic acid sequencing:
Nucleic acid (DNA and RNA) sequencing is another molecular characteristic that helps directly compare the genomic structures. Most attention has been given to the sequencing of 5S and 16S rRNAs isolated from the 50S and 30S subunits of 70S prokaryotic ribosome, respectively.
As mentioned in DNA-RNA hybridization, rRNAs are almost ideal for the studies of bacterial (microbial) evolution and relatedness because:
(i) They are essential to ribosomes found in all bacteria,
(ii) Their functions are same in all ribosomes, and
(iii) Their structure changes very slowly with time, i.e., they are more conserved.
The procedure of rRNA sequencing involves the following steps:
(i) rRNA is isolated from the ribosome and purified,
(ii) Reverse transcriptase enzyme is used to make complementary DNA (cDNA) using primers that are complementary to conserved rRNA sequences,
(iii) The cDNA is amplified using polymerase chain reaction (PCR) and finally,
(iv) The cDNA is sequenced and the rRNA sequence deduced from the results.
Shotgun sequencing and other genome sequencing techniques have led to the characterization of many prokaryotic genomes (approximately 100) in a very short time and many more are in the process of being sequenced. Direct comparison of complete genome sequences undoubtedly will become an important tool in determining the classificatory categories of prokaryotes.
Ribotyping is a technique which measures the unique pattern that is generated when DNA from a bacterium (all other organisms also) is digested by a restriction enzyme and the fragments are separated and probed with a rRNA probe.
The technique does not involve nucleic acid sequencing. Ribotyping has proven useful for bacterial identification and has found many applications in clinical diagnostics and for the microbial analyses of food, water, and beverages.
Ribotyping is a rapid and specific method for bacterial identification; it is so specific that it has been given the nickname ‘molecular fingerprinting’ because a unique series of bands appears for virtually any bacterium (any organism).
In ribotyping, first the DNA is isolated from a colony or liquid culture of the bacterium to be identified. Using polymerase chain reaction (PCR), genes of DNA for rRNA (preferably 16S rRNA) and related molecules are amplified, treated with one or more restriction enzymes, separated by electrophoresis, and then probed with rRNA genes.
The pattern generated from the fragments of DNA on the gel is then digitized and a computer used to make comparison of this pattern with patterns from other bacteria available from a database.
Comparison of proteins:
The amino acid sequences of proteins are direct reflections of mRNA sequences and therefore closely related to the structures of the genes coding for their synthesis. In the light of this, the comparisons of proteins from different bacteria prove very useful taxonomically.
Although there are many methods to compare proteins, the most direct approach is to determine the amino acid sequences of proteins with the same function.
When the sequences of proteins of the same functions in two bacteria are similar, the bacteria possessing them are considered to be closely related. However, the sequences of cytochromes and other electron transport proteins, histones, heat-sock proteins, and a variety of enzymes have been used in taxonomic studies.