Molecular Characteristics Used in Microbial Taxonomy

 

Molecular Characteristics Used in Microbial Taxonomy

Taxonomy [Greek taxis, arrangement or order, and nomos, law, or nemein, to distribute or govern] is defined as the science of biological classification. It consists of three separate but interrelated parts:  classification, nomenclature, and identification.

Classification is the arrangement of organisms into groups or taxa (s., taxon) based on mutual similarity or evolutionary relatedness.

Nomenclature is the branch of taxonomy concerned with the assignment of names to taxonomic groups in agreement with published rules.

Identification is the process of determining that a particular isolate or organism belongs to a recognized taxon.

Classification Systems: There are two general way of constructing classification systems. Organisms can be grouped together based on overall similarity to form a phenetic system or they can be grouped based on probable evolutionary relationships to produce a phylogenetic or phyletic system. Computers may be used to analyze data for the production of phenetic classifications and this process is called numerical taxonomy.

Major Characteristics Used in Taxonomy

Many characteristics are used in classifying and identifying microorganisms. These characteristics are divided into two groups: classical and molecular.

Examples of Classical Characteristics are Morphological Characteristics, Physiological and Metabolic Characteristics, Ecological Characteristics, Genetic Analysis, etc. 

Molecular Characteristics: Molecular Characteristics are some of the most powerful approaches to taxonomy.  It includes the study of proteins and nucleic acids. Because proteins and nucleic acids are either direct gene products or the genes themselves, their comparisons yield considerable information about true relatedness between organisms.

Nucleic Acid Base Composition: Microbial genomes can be directly compared to analyze taxonomic similarity in many ways. The first and the simplest technique is the determination of DNA base composition. DNA contains four purine and pyrimidine bases: adenine (A), guanine (G), cytosine (C), and thymine (T). In double-stranded DNA, A pairs with T, and G pairs with C.

The (G + C)/ (A+T) ratio or G + C content or the percent of G + C in DNA, reflects the base sequence and varies with sequence changes as follows:

Mol% G + C =        G + C         X 100

  G + C + A + T

The base composition of DNA can be determined in several ways. The G + C content can be ascertained after hydrolysis of DNA and analysis of its bases with high-performance liquid chromatography (HPLC). 

When solutions of DNA are exposed to extremes of pH or heat or to solutes such as urea or amides, the double helical structure of DNA undergoes a transition into a randomly single-stranded form known as denatured DNA. During denaturation the interactions between successive base pairs are interrupted. When DNA denatures, significant changes occur in a number of its physical properties, such as an increase in buoyant density, decrease in viscosity and an increase in the UV absorption at 260 nm. This last effect is known as the hyperchromic effect and provides a convenient method for monitoring the denaturation of DNA.

The G + C content often is determined from the melting temperature (Tm) of DNA. In double-stranded DNA three hydrogen bonds join GC base pairs, and two bonds connect AT base pairs. As a result, DNA with a greater G + C content will have more hydrogen bonds, and its strands are more strongly held together.  So the strands having high G + C content will separate only at higher temperatures.  It will have a higher melting point. DNA melting can be measured using a spectrophotometer because the absorbance of UV light (260 nm) by DNA increases during strand separation. When a DNA sample is slowly heated, the absorbance increases as hydrogen bonds are broken and reaches a plateau when the entire DNA has become single stranded. The midpoint of the rising curve gives the melting temperature or Tm, which is a direct measure of the G + C content.

Since DNA with higher G + C content have high density, the percent G + C can also be obtained by centrifuging DNA in a CsCl density gradient.

The G + C content of DNA of animals and higher plants averages around 40% and ranges between 30 and 50%. The DNA of both eucaryotic and procaryotic microorganisms varies greatly in G + C content.  Procaryotic G + C content is the most variable, ranging from around 25 to 80%. But the G + C content of strains within a particular species is constant. If two organisms differ in their G + C content by more than about 10%, their genomes have quite different base sequences. Since very different base sequences can be constructed from the same proportions of AT and GC base pairs, the organism are also to be compared for similarity in their   phenotypic characters.

Nucleic Acid Hybridization: The similarity between genomes can be compared more directly by use of nucleic acid hybridization studies. Single stranded DNA (ssDNA) is formed by heating or by keeping double stranded DNA (dsDNA) at high pH.  If a mixture of single stranded DNA is held at a temperature about 25°C below the Tm, strands with complementary base sequences will reassociate to form stable dsDNA, whereas non-complementary strands will remain single.  Incubation at 10 to 15°C below the Tm permits hybrid formation only with almost identical strands.

In one of the more widely used hybridization techniques, nitrocellulose filters with bound nonradioactive DNA strands are incubated at the appropriate temperature with single-stranded DNA fragments made radioactive with ³²P, ³H, or ¹⁴C. After radioactive fragments are allowed to hybridize with the membrane-bound ss-DNA, the membrane is washed to remove any nonhybridized ssDNA and its radioactivity remaining on the filter is measured. The quantity of radioactivity bound to the filter reflects the amount of hybridization and thus the similarity of the DNA sequences. The degree of similarity or homology is expressed as the percent of experimental DNA radioactivity retained on the filter compared with the percent of homologous DNA radioactivity bound under the same conditions.

Two strains whose DNAs show at least 70% relatedness under optimal hybridization conditions and less than a 5% difference in Tm often are considered members of the same species.

DNA-DNA hybridization is used to study only closely related microorganisms. More distantly related organisms are compared by carrying out DNA-RNA hybridization experiments using radioactive ribosomal or transfer RNA (rRNA or tRNA). Distant relationships can be detected because rRNA and tRNA genes represent only a small portion of the total DNA genome and have not evolved as rapidly as most other microbial genes. The technique is similar to that employed for DNA-DNA hybridization.

Nucleic Acid Sequencing: Despite the usefulness of G + C content determination and nucleic acid hybridization studies, genome structures can be directly compared only by sequencing DNA or RNA. RNA sequencing has been used more extensively in microbial taxonomy.

rRNA sequences are important indices of genentic relatedness among prokaryotes since

·         Small differences in rRNA sequence can be used to determine evolutionary relatedness between organisms.

·         Their functional role is the same in all ribosomes. Furthermore, their structure changes very slowly with time, presumably because of their constant and critical role.

·         Because rRNA contains variable and stable sequences, both closely related and very distantly related microorganisms can be compared.

Among the three rRNA molecules (5S, 16S and 23S), 16S (contains 1500 nucleotides) is mostly used.  The small size of 5S rRNA (125 nucleotides) limits the amount of information that can be obtained from it, while the large size of 23S rRNA (2900 nucleotides) makes the sequencing difficult. 

In this technique, complete rRNAs are sequenced. First, RNA is isolated and purified. Then, reverse transcriptase is used to make complementary DNA (cDNA) using primers that are complementary to rRNA sequences. Next, the polymerase chain reaction amplifies the cDNA. Finally, the cDNA is sequenced and the rRNA sequence deduced from the results.

Ribosomal RNAs can also be characterized in terms of partial sequences by the oligonucleotide cataloging method. Purified, radioactive 16S rRNA is treated with the enzyme T1 ribonuclease, which cleaves it into fragments. The fragments are separated, and all fragments composed of at least six nucleotides are sequenced. The sequences of corresponding 16S rRNA fragments from different procaryotes are then aligned and compared using a computer, and association coefficients (Sab values) are calculated.

DNA Fingerprinting: DNA from two organisms is treated with the same restriction enzyme and the restriction fragments produced are separated by electrophoresis.  The pattern is known as DNA fingerprints.  Comparison of the number and sizes of restriction fragments produced from the DNA of the organisms provide information about their genetic similarities and differences.  The more close the organisms are, the more similar the pattern of the DNA fingerprint would be.  Restriction Fragment Length Polymorphism (RFLP) is one technique used in DNA fingerprinting.  RFLP methodology involves cutting DNA with restriction enzymes, then separating the DNA fragments by agarose gel electrophoresis and determining the number of fragments and relative sizes.

Ribotyping:  The fingerprinting of genomic DNA restriction fragments that contain all or part of the genes coding for the 16S and 23S rRNA. Ribotyping is a method that can identify and classify bacteria based upon differences in rRNA. In this method, DNA is extracted from a colony of bacteria and then treated with restriction enzymes into fragments. The DNA fragments is then transferred to a membrane and probed with a region of the rRNA to interpret the rRNA genes. The pattern is recorded, digitized and stored in a database. Databases for Listeria (80 pattern types), Salmonella (97 pattern types), Escherichia (65 pattern types) and Staphylococcus (252 pattern types) have been established. Ribotyping generates a highly reproducible and precise fingerprint that can be used to classify bacteria from the genus and the species level.

Fluorescent in situ hybridization (FISH) is a powerful technique for detecting RNAor DNA sequences in cells, tissues, and tumors.

Fluorescent in situ hybridization is a technique in which single-stranded nucleic acids (DNA or RNA) are allowed to form hybrids with suitably similar, complementary sequences. From the extent of hybridization, the degree of sequence similarity can be determined, and specific sequences can be detected and located on a given chromosome. FISH uses Fluorescent probes that bind to complementary parts of the chromosome.  Flourescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes. FISH can be used to compare the genomes of two biological species, to deduce evolutionary relationships. Bacterial FISH probes are often primers for the 16s rRNA region. Microorganisms are fixed or kept in place in a slide; fluorescent labeled probe enters and reacts with target ribosome in cell and could be visualized using a fluorescence microscope.

FISH also has a large number of applications in molecular biology and medical science, including gene mapping, diagnosis of chromosomal abnormalities, diagnosis of infectious viral and bacterial diseases, tumor  diagnosis, and studies of cellular structure and function.

Southern blotting: This is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. The method is named after its inventor, the British biologist Edwin Southern.

1. Restriction endonucleases are used to cut DNA strands into fragments.
2. The DNA fragments are electrophoresed on agarose gel to separate them based on size.
3. A sheet of nitrocellulose (or nylon) membrane is placed on the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If some of the DNA fragments are larger than 15 kb, prior to blotting, the gel may be treated with acid or alkali which breaking it into smaller pieces allowing efficient transfer from the gel to membrane.
4. The membrane is then baked in an oven at 80°C for 2 hours (nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.
5. The membrane is then exposed to a hybridization probe (a single DNA or RNA fragment with a specific complementary sequence). The probe DNA is labelled (usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye) so that it can be detected.

6. After hybridization, excess probe is washed from the membrane and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or by fluorescence in case of fluorescent probe or by development of color on the membrane if a chromogenic detection method is used.

The northern blot is used to study the expression patterns of a specific type of RNA molecule as relative comparison among a set of different samples of RNA. In this process RNA is separated based on size by electrophoresis and is then transferred to a membrane that is then probed with a labeled probe of a sequence of interest.

In western blotting, proteins are separated based on size using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography.

Eastern blotting: Eastern blotting technique is to detect post-translational modification of proteins.  Proteins are electrophoresed and blotted on to the PVDF or nitrocellulose membrane. Transferred proteins are analyzed for post-translational modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification.

DNA chip (genome chip or gene array): A DNA chip is made with thousands of nucleotide sequences attached to a chip in a grid pattern.  The attached nucleotide sequences act as probes to detect whether the given test sample contains complementary RNA or DNA.  Probe-target hybridization is usually detected and quantified by detection of fluorescence or chemiluminescence-labeled targets.   The probes are attached to a solid surface by a covalent bond and the solid surface can be glass or a silicon chip.

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. For this reason, comparisons of proteins from different microorganisms are very useful taxonomically.

There are several ways to compare proteins.

Protein sequencing or determination of the amino acid sequences of proteins. If the sequences of proteins with the same function are similar, the organisms possessing them are probably closely related. Examples are cytochromes and other electron transport proteins, histones, heat-shock proteins, transcription and translation proteins, a variety of metabolic enzymes, etc.

Because protein sequencing is slow and expensive, more indirect methods of comparing proteins are mostly employed. The electrophoretic mobility of proteins and immunologic techniques using antibodies to compare proteins from different microorganisms are examples. The physical, kinetic, and regulatory properties of enzymes are also employed.

Serotyping: Serotyping refers to serological procedures used to differentiate strains (serovars or serotypes) of microorganisms that differ in the antigenic composition. It is possible to identify a microorganism serologically by testing for cell wall antigens using specific antibodies. For example, there are 84 strains of Streptococcus pneumoniae, each differing in the nature of its capsular material. These differences can be detected by capsular swelling (termed the Quellung reaction) if antisera or antibody specific for the capsular types are used.  Slide agglutination test, ELISA, western blotting, etc are also used for serological testing. 

Bacteriophage Typing: Bacteriophages (phages) are viruses that attack members of a particular bacterial species, or strains within a species. Bacteriophage (phage) typing is based on the specificity of phage surface receptors for cell surface receptors. Only those bacteriophages that can attach to these surface receptors can infect bacteria and cause lysis. On a petri dish culture, lytic bacteriophages cause plaques or clear areas on lawns of sensitive bacteria. These plaques represent infection by the virus. 

In bacteriophage typing the bacterium to be tested is inoculated onto a petri plate to form a solid layer or lawn of cells. The plate is then marked into squares (15 to 20 mm per side), and each square is inoculated with a drop of suspension from the different phages available for typing. After the plate is incubated for 24 hours, it is observed for plaques. The phage type is reported as a specific genus and species followed by the types that can infect the bacterium. For example, the series 10/16/24 indicates that this bacterium is sensitive to phages 10, 16, and 24, and belongs to a collection of strains, called a phagovar, that have this particular phage sensitivity.

Enzyme-Linked Immunosorbent Assay (ELISA) or enzyme immunoassay (EIA): This is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. ELISA involves specific antigen-antibody interaction. The sample with an unknown amount of antigen is immobilized on a solid support (on a microtiter plate) either non-specifically (adsorption) or specifically (by another antibody specific to the same antigen). After the antigen is immobilized, the detection antibody is added which can form a complex with the antigen. This detection antibody may be linked to an enzyme, or can be detected by a secondary antibody that is linked to enzyme. On addition of the chromogenic substrate to the enzyme a visible colour signal is produced, which indicates the presence and quantity of antigen in the sample.