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Chapter: Basic Concept of Biotechnology - Antibiotics: Microbial Sources, Production and Optimization

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Identification of actinomycetes

1. Molecular Approach 2. Chemotaxonomical Approach 3. Classical Approach 4. Numerical Taxonomic Approach

Identification of actinomycetes

The traditional methods used for the identification of the aerobic filamentous actinomycetes are laborious, time consuming and oftenrequire a series of specialized tests (Steingrubeet al., 1995; Wilson et al., 1998; Harvey et al., 2001). Chemical criteria, such as the isomer of diaminopimelic acid (DAP) present in the cell wall and the diagnostic sugar(s) present in the whole-cell hydrolysate, have been used to separate the actinomycete genera into broad chemotaxonomic groups. However, determination of these characteristics is time-consuming and, in most cases, cannot identify an isolate to a single genus (Lechevalier, 1989). PCR-based methods have provided a rapid and accurate way to identify these bacteria (Gurtleret al., 1991; Kohler et al., 1991; Beyazova and Lechevalier, 1993; Telentiet al., 1993; Soiniet al., 1994; Mehlingetal., 1995; Steingrubeet al., 1997; Wilson et al., 1998; Laurent et al.,1999).

By conventional isolation methods, members of the genus Streptomyces comprise more than 95% of the filamentous actinomycete population in soil (Lacey, 1973; Elander, 1987). The Streptomycetes produce more antibiotics than any other genus of bacteria and, therefore, have been heavily exploited as a source of novel antimicrobial agents (Watveet al., 2001). The probability of isolating known species of Streptomyces from the environment is thus great and the probability of isolating novel antibacterial molecules from such species is very low. The isolation of the rarer, non- Streptomyces actinomycetes greatly increases the probability of isolating novel antibacterial molecules (Lazzariniet al., 2000). Therefore, a rapid method to distinguish Streptomycetes from other actinomycetes and to identify the non Streptomycetes to the genus level would be extremely useful. This would be of particular value in discerning between Streptomycetes and non Streptomycetes, such as Actinomadura, Nocardia and Nocardiopsis, whose colonies may be morphologically similar on agar plates.


1.  Molecular Approach

The most powerful approaches to taxonomy are through the study of nucleic acids. Because these are either direct gene products or the genes themselves and comparisons of nucleic acids yield considerable information about true relatedness. Molecular systematics, which includes both classification and identification, has its origin in the early nucleic acid hybridization studies, but has achieved a new status following the introduction of nucleic acid sequencing techniques (O’Donnell et al., 1993). Significance of phylogenetic studies based on 16S rDNA sequences is increasing in the systematics of bacteria and actinomycetes (Yokota, 1997). Sequences of 16S ribosomal DNA have provided actinomycetologists with a phylogenetic tree that allows the investigation of evolution of actinomycetes and also provides the basis for identification. Analysis of the 16S rDNA begins by isolating DNA (Hapwood, 1985) and amplifying the gene coding for 16S rRNA using the polymerase chain reaction (e.g. SivaKumar, 2001). The purified DNA fragments are directly sequenced. The sequencing reactions are performed using DNA sequencer in order to determine the order in which the bases are arranged within the length of sample (Xu LiHua etal., 1999) and a computer is then used for studying the sequence foridentification using phylogenetic analysis procedures.


2. Chemotaxonomical Approach

Chemotaxonomy is the study of chemical variation in organisms and the use of chemical characters in the classification and identification. It is one of the valuable methods to identify the genera of actinomycetes. Studies of Cummins and Harris (1956) established that actinomycetes have a cell wall composition akin to that of grampositive bacteria, and also indicated that the chemical composition of the cell wall might furnish practical methods of differentiating various types of actinomycetes. This is because of the fact that chemical components ofthe organisms that satisfy the following conditions have significant meaning in systematics.


i.          They should be distributed universally among the microorganisms studied; and,


ii.        The components should be homologous among the strains Within a taxon, while significant differences exist between the taxa to be differentiated. Presence of Diaminopimelic acid (DAP) isomers is one of the most important cell-wall properties of gram-positive bacteria and actinomycetes. Most bacteria have a characteristic wall envelope, composed of peptidoglycan. The 2, 6- Diaminopimelic Acid (DAP) is widely distributed as a key aminoacid and it has optical isomers. The systematic significance lies mostly in the key aminoacid with two amino bases, and determination of the key aminoacid is usually sufficient for characterisation. If DAP is present, bacteria generally contain one of the isomers, the LL-form or the meso-form, mostly located in the peptidoglycan.

The sugar composition often provides valuable information on the classification and identification of actinomycetes. Actinomycete cells contain some kinds of sugars, in addition to the glucosamine and muramic acid of peptidoglycan. The sugar pattern plays a key role in the identification of sporulatingactinomycetes which have meso-DAP in their cell walls. However, the actinomycetes which have LL-DAP along with glycine have no characteristic pattern of sugars (Lechevalier and Lechevalier, 1970) and hence the whole cell sugar test has not received much attention here.


3. Classical Approach

Classical approaches for classification make use of morpho logical, physiological, and biochemical characters. The classical method described in the identification key by Nonomura (1974) and Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974) isvery much useful in the identification of Streptomycetes. These characteristics have been commonly employed in taxonomy of Streptomycetes for many years. They are quite useful in routine identification. They are as follows, Aerial Mass Color, Melanoid Pigments, Reverse Side Pigments, Soluble Pigments, Spore Chain Morphology, Spore Surface morphology, Assimilation of Carbon Source.


4. Numerical Taxonomic Approach

Numerical taxonomy involves examining many strains for a large number of characters prior to assigning the test organism to a cluster based on shared features. The numerically defined taxa are polythetic, so no single property is either indispensable or sufficient to entitle an organism for membership of a group. Once classification has been achieved, cluster specific or predictive characters can be selected for identification (Williams et al., 1983). Numerical taxonomy was first applied to Streptomyces by Silvestriet al. (1962). The numerical taxonomic study of the genus Streptomycesby Williams et al. (1983) involves determination of 139 unit characters for 394 type cultures of Streptomyces. Clusters were defined at 77.5% or 81% Ssm and 63% Sjsimilarity levels, and the former coefficient is being used to define the clusters. His study includes 23 major, 20 minor and 25 single member clusters.

The numerical classification of the genus Streptomyces by Kampferet al. (1991) involves determination of 329 physiological tests. His study includes 15 major clusters, 34 minor clusters and 40 single member clusters which are defined at 81.5% similarity level Ssm using the simple matching coefficient (Sokal and Michener, 1958) and 59.6 to 64.6% similarity level Sj using Jaccard coefficient (Sneath, 1957). Thus, numerical taxonomy provides us with an invaluable framework for Streptomyces taxonomy, including identification of species.

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