ABSTRACT
Microbial taxonomy is the scientific field for the classification of microorganisms based on their relationships and their evolutionary history. In the past, classification was based upon similarities or differences on the morphological and physiological features of the microorganisms. However, in several cases, close relative species have been shown to possess divergent characteristics, while unrelated species had identical phenotypes. The inaccuracy of the traditional methods of classification led to a molecular taxonomy approach based on hereditary material, which can provide a robust and more accurate relatedness among microorganisms and a better understanding of their evolution. Analysis of the genome using molecular techniques may provide an overview of microorganisms’ relationships on genus-, species- or even strain level. DNA fingerprinting is an important tool for taxonomy as it leads to the reduction of DNA complexity by the detection of several loci of the DNA molecule with specific patterns that can be used as molecular markers for the identification and further classification. In this regard, DNA-DNA hybridization based on complementarity of the double stranded DNA was proposed in order to provide an overview of the relevance of the genomes of two microorganisms, based on the degree of hybrid reassociation. Despite the fact that this method may effectively distinguish related and unrelated species, it requires large input of high-quality DNA and is, therefore, time consuming. Sequencing of specific targets of the genome, which encode the genes of ribosomal RNA, were proposed as an alternative approach to DNA-DNA hybridization. Restriction endonucleases, which recognize specific sequence motif, genetic loci, repetitive patterns of DNA sequence and gene sequences have been proposed as genetic markers which can be used for a microbial classification. Ribosomal rRNA gene contains highly conserved loci and is, thus, by far the most widely used approach for classification. 16S rRNA gene is a universal target for bacterial identification because of the fact that it has a large molecule which is highly conserved and its functions have not changed throughout its evolution. Despite its advantages, 16S rRNA gene sequence is not sufficient for classification at species level. Therefore, approaches such as multilocus sequence analysis have been developed, which together with alternative data processing pipelines aim to improve discrimination ability and reduce the effect of the bioinformatics approach employed to the quality of the result. Whole genome sequencing for classification can lead to higher resolution in a very short time by providing an overview of the whole genome. Next generation sequencing or “massively parallel sequencing” is the future of microbial taxonomy, as it can overcome the limitations of traditional Sanger method sequencing by providing a great number of reads in a single run and the creation of library fragments. In this chapter, the additional features offered by the application of the previous mentioned molecular approaches on the assessment of taxonomy and evolutionary relationships are discussed.
