The use of REP-PCR on members of the class Actinobacteria stackebrandt et al. 1997

Advances in molecular biology have provided highly specific methods for classifying, identifying and typing isolates based on nucleic acid sequence data. Taxonomic systems are becoming increasingly important for characterizing microorganisms since they can be applied in epidemiological investigations of disease outbreaks to identify a potential common source and/or to establish grouping strategies for isolates recovered from several environments. Typing systems based on phenotypic tests have limitations as key phenotypic traits of microorganisms may be incosistently expressed or may not provide enough discriminative power to separate closely related microorganisms. Therefore, shortcomings of phenotypically based methods have pushed towards the development and employment of methods based on microbial genotypes or DNA sequences minimising problems with respect to reproducibility and, in some cases, enabling the establishment of large databases (a biological-genotypic database) of characterized organisms. Several molecular typing systems have been used to study the relationships within high G+C bacteria (aka Actinobacteria) and the current chapter focuses on the genera Micromonospora and Streptomyces. Repetitive Extragenic Palindromic Polymerase Chain Reactions (Rep-PCR) fingerprints bacterial genomes based on strain-specific patterns derived from PCR amplification of repetitive DNA elements present within bacterial genomes. The palindromic nature of repetitive elements (ie. BOX, ERIC and REP primers) across the microbial genome and their ability to form stem-loop structures leads to the generation of unique fingerprint patterns. Members of the genus Micromonospora cannot be separated on basis of their phenotypic properties, therefore, the use of molecular fingerprinting methods when large numbers of isolates belonging to this genera are urgently needed. The chapter exemplifies its use on nearly 200 micromonosporae recovered from aquatic ecosystems. On the other hand, studies on members of the genus Streptomyces-which currently holds nearly 600 species-makes comparative studies difficult, hence also the need for a reliable fingerprinting method. The chapter then deals with studies on streptomycetes isolated from clinical material (11 strains) and their fingerprint relationships. The use of REP-PCR to generate fingerprint patterns and the construction of a biological-genotypic database are provided for these Actinobacteria genera. The accurate circumscription of subtypes within a species is becoming increasingly important in all branches of microbiology. Microbial fingerprinting is extensively used in diagnostic bacteriology (Oyarzabal et al., 1997), in ecological and evolutionary genetical studies (van Belkum et al., 2001) and in search and discovery programmes designed to detect new microbial products (Goodfellow & O'Donnell, 1989; Bull et al., 1992, 2000). The various molecular fingerprinting methods have advantages and disadvantages when applied to specific situations and objectives. Fingerprinting methods used to discriminate between strains within target species are easy to perform and data interpretation is relatively straighforward. Several molecular fingerprinting systems have been shown to be effective in the delineation of Actinobacteria, that is Gram positive bacteria of a high Guanine + Citosine content (> 55%), at and below the species level (Welsh & McClelland, 1990; Vaneechoutte et al., 1992; Gürtler & Stanisich, 1996; Yoon et al., 1997; Hall et al., 2001). The introduction and application of molecular taxonomic procedures such as 16S rRNA gene sequencing (Woese, 1987; Olsen & Woese, 1993; Ludwig & Klenk, 2001), DNA fingerprinting (Vaneechoutte et al., 1998; Rademaker et al., 2000; Gürtler & Mayall, 2001; van Belkum et al., 2001), DNA: DNA hybridisation (Grimont, 1981; Stackebrandt et al., 2002), multilocus sequence typing (Maiden et al., 1998; Sails et al., 2003; Tavanti et al., 2003) and sequence analyses of complete genomes (Õmura et al., 2001; Bentley et al., 2002; Ikeda et al., 2003) are providing new insights into prokaryotic systematics (Woese, 1987; Olsen et al., 1994; Ludwig & Schleifer, 1999; Palys et al., 2000; Gürtler & Mayall, 2001; Kim et al., 2001; Stackebrandt et al., 2002), including the classification and identification of the Actinobacteria (Stackebrandt et al., 1997; Kim et al., 1999; Salazar et al., 2000; Zhang et al., 2001; Stach et al., 2003; Stevens et al., 2007). The development of molecular taxonomic methods based on DNA analyses has made it possible to undertake extensive, rapid and precise characterisation of representatives of bacterial taxa isolated from diverse habitats (Moyer et al., 1994; Bull et al., 2000; Stach et al., 2003; Maldonado et al., 2005b, 2008, 2009; Stevens et al., 2007). Such techniques tend to give results that are more robust than those from chemosystematic studies which are often sensitive to small changes in cultivation conditions (Goodfellow & Minnikin, 1985; Goodfellow & O'Donnell, 1994; Hugenholtz et al., 1998). Besides, such techniques provide an insight of the whole genomic information from each strain. Many molecular taxonomic procedures are based on the use of the polymerase chain reaction, which is used to amplify target genes from either culturable isolates or microbial community DNA (Lane, 1991; Stackebrandt et al., 1997; Muyzer, 1999; Bull et al., 2000; Stach et al., 2003; Stevens et al., 2007). The application of different molecular fingerprinting techniques to determine the degree of sequence conservation between bacterial genomes is based on the detection of naturally occurring DNA polymorphisms which are the result of either point mutations or rearrangements in genomic DNA (i.e. insertions or deletions) or fragments of DNA. DNA polymorphisms can be detected by scoring band presence against band absence in banding patterns generated either by restriction enzyme digestion or DNA amplification procedures (Versalovic et al., 1991; Moyer et al., 1994; Vaneechoutte et al., 1998; van Belkum et al., 2001). The underlying premise is that variations in banding patterns are a direct reflection of genetic relationships between the tested strains hence banding patterns can be considered as genomic fingerprints for characterisation and identification purposes (Versalovic et al., 1994; Gürtler & Mayall, 2001). A robust fingerprinting technique can then become a dereplication molecular procedure to indicate whether a specific clinical and/or environmental isolate has been studied (cultured) previously (Maldonado et al., 2008).