GENERAL MICROBIOLOGY - T.P. Pyrog - 2004

7. PROKARYOTE SYSTEMATICS

7.6. MODERN TRENDS IN BACTERIAL SYSTEMATICS

7.6.2. Bacterial Genosystematics

A fundamentally new approach that differs from phenotypic systematics is bacterial genosystematics. The degree of genetic divergence among organisms can be assessed using the following indicators: DNA GC content; DNA—DNA and DNA—RNA Hybridization; protein Amino acid sequences; and Gene nucleotide sequences. Let us examine the application of each of these parameters in bacterial systematics.

DNA GC Content. Initially, only the overall base composition of DNA was used to compare bacterial genomes. The GC content of DNA is of significant taxonomic value. In Bacteria, the molar GC content ranges from 22 to 75%. This value remains constant for a given Organism. However, the GC percentage does not account for the linear arrangement of NUCLEOTIDES in DNA, meaning that organisms with identical GC contents are not necessarily identical.

DNA—DNA and DNA—RNA Hybridization. The Essence of this method is that the DNA double helix is denatured by heating, and the separated strands are stabilized. Upon cooling, DNA renaturation becomes possible. However, only a complementary (matching) strand can pair with a single-stranded DNA. The amount of renatured double-stranded DNA serves as a measure of similarity between the organisms being compared. The sequences forming double-stranded complexes are not necessarily complementary at all nucleotides. The proportion of non-complementary nucleotide pairs can be determined by the rate at which DNA strands in the duplex separate as the Temperature increases. For this purpose, T_m (melting temperature) is determined, which is the temperature at which 50% of the double-stranded DNA dissociates. ΔT_m is calculated using the formula

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A ΔT_m of 1º C corresponds to approximately 1% of non-complementary nucleotide pairs. Similar reassociation can be performed between DNA and RNA molecules, as Double helices also form between single-stranded DNA and complementary RNA strands. DNA Homology experiments are most useful at the species Classification level.

Practical guidelines for using the DNA—DNA hybridization method in bacterial systematics were outlined in the decisions of the Committee on Reconciliation of Approaches to Bacterial Taxonomy, published as early as late 1987. Specifically, they state that a species should encompass strains with a DNA—DNA similarity level of 70% or higher, and a ΔT_m of 5 °C or lower. Phenotypic characteristics must be consistent with this definition and may deviate from the phylogenetic species concept only as an exception.

In the early 1970s, DNA—DNA and DNA—RNA hybridization helped refine the taxonomic placement of species in various groups, such as Pseudomonas, anaerobic bacteria, and others. For instance, it was demonstrated that species belonging to the genus Pseudomonas form five genetically isolated homologous groups. Bacteroides fragilis comprises five subspecies that proved to be distinct groups based on DNA homology. The genus Salmonella contains only a single DNA-homologous group, and the proposal to recognize only a single Salmonella species has gained widespread support.

Protein Amino Acid Sequences. The comparison of protein amino acid sequences relies on the reflection of the organism's primary gene sequence within them, serving as a kind of DNA "fingerprint." In practical research, Electrophoresis is used for Comparative Protein Analysis. The electrophoretic Determination of protein profiles is based on the premise that closely related organisms feature identical cellular Proteins. However, it should be noted that cellular protein profiles may vary depending on bacterial growth conditions.

Analysis of 5S and 16S rRNA. Over three decades ago, the conservative nature of ribosomal RNA genes was established. Genes encoding rRNA are highly conserved, meaning that rRNA changes very little during the course of evolution. These informational molecules are viewed as molecular chronometers that reflect the origin and development of microorganisms. Nucleotide sequence analysis of bacterial rRNA has revealed both unexpected differences and striking similarities. Notably, attempts were made to investigate the relationships among bacterial taxa based on 5S rRNA nucleotide sequences; however, this molecule proved too small for this task. Nowadays, identifying the phylogenetic position of prokaryotes relies heavily on 16S rRNA analysis. As noted by Academician G.O. Zavarzin in the Preface TO THE Russian-language ninth edition of Bergey's Manual of Determinative Bacteriology, this approach remains unrivaled for determining the generic affiliation of microorganisms.

Recently, Methods based on the Polymerase Chain Reaction (PCR) have been widely applied for 16S rRNA analysis. PCR is based on the Amplification (i.e., increasing the number of copies) of specific DNA fragments using primers—synthetic oligonucleotides 20–30 nucleotides in length that are complementary to the initiation site of Replication for a given DNA fragment. Consequently, The nucleotide sequence of the primers essentially mirrors the nucleotide sequence of the DNA molecule to be amplified.

The target DNA is denatured under conditions that prevent renaturation, in the presence of an excess of primers. Under these conditions, Two Types of primers hybridize with the complementary sequences of each of the two opposite DNA strands and serve as starting points for enzymatic synthesis. In the presence of DNA polymerase and a set of deoxynucleoside triphosphates, the synthesis of new DNA strands begins in the same direction as during normal replication. The final product of the reaction, consisting of two new double-stranded DNA molecules, is denatured again, and the amplification cycle is repeated. In this way, specific DNA regions are selectively multiplied (limited by the primer nucleotide sequences) to a concentration where they can be easily detected. Various DNA polymerases amplify the material through 20–50 cycles.

Amplification of 16S rRNA genes is carried out using universal eubacterial primers 27f and 1492r. The nucleotide sequence of the 27f primer (consisting of 20 nucleotides) is found in the DNA of most eubacteria, whereas The sequence of the 1492r primer (consisting of 22 nucleotides) is present in the DNA of most eubacteria and archaea. Therefore, these primers are termed universal eubacterial primers. Following the amplification of 16S rRNA genes, the nucleotide sequence of the resulting amplicon is determined using a Sequencer and compared against a computer database. To analyze 16S rRNA gene sequences of the studied bacteria, Databases such as EMBL, GenBank, and the Ribosomal Database Project are utilized. Sequenced fragments are compared with those of reference strains from related microorganisms using the BLAST program.