Actinomycetes are one of the most effi cient groups of secondary metabolite producers and are very important from an industrial point of view. Among its various genera, Streptomyces, Saccharopolyspora, Amycolatopsis, Micromonospora and Actinoplanes are the major producers of commercially important biomolecules. Several species have been isolated and screened from the soil in the past decades. Consequently the chance of isolating a novel actinomycete strain from a terrestrial habitat, which would produce new biologically active metabolites, has reduced. The most relevant reason for discovering novel secondary metabolites is to circumvent the problem of resistant pathogens, which are no longer susceptible to the currently used drugs. Existence of actinomycetes has been reported in the hitherto untapped marine ecosystem. Marine actinomycetes are effi cient producers of new secondary metabolites that show a range of biological activities including antibacterial, antifungal, anticancer, insecticidal and enzyme inhibition. Bioactive compounds from marine actinomycetes possess distinct chemical structures that may form the basis for synthesis of new drugs that could be used to combat resistant pathogens.

Classification of microorganisms on the basis of fi traditional microbiological methods (morphological, physiological and biochemical) creates a blurred image about their taxonomic status and thus needs further clarification. It fi should be based on a more pragmatic approach of deploying a number of methods for the complete characterization of microbes. Hence, the methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. Collectively these genotypic, chemotaxonomic and phenotypic methods for determining taxonomic position of microbes constitute what is known as the 'polyphasic approach' for bacterial systematics. This approach is currently the most popular choice for classifying bacteria and several microbes, which were previously placed under invalid taxa have now been resolved into new genera and species. This has been possible owing to rapid development in molecular biological techniques, automation of DNA sequencing coupled with advances in bioinformatic tools and access to sequence databases. Several DNA-based typing methods are known; these provide information for delineating bacteria into different genera and species and have the potential to resolve differences among the strains of a species. Therefore, newly isolated strains must be classifi ed fi on the basis of the polyphasic approach. Also previously classifi ed organisms, as and when required, can be reclasfi sified on this ground in order to obtain information about fi their accurate position in the microbial world. Thus, current techniques enable microbiologists to decipher the natural phylogenetic relationships between microbes.

A versatile plasmid cloning vector pRL60 carrying kanamycin/neomycin resistance replicates in various Amycolatopsis mediterranei strains and Escherichia coli has been constructed. This cloning vector has been derived from a hybrid plasmid pRL50, which was developed by cloning ermE from pIJ4026 into a pRL1 derivative pULAM2. While cloning ermE into the BamHI site of pULAM2, only a hybrid plasmid pRL50 with an additional copy of pULAM2 was selected. Thus pRL50 (18.7kb) contained two copies A. mediterranei DSM 40773 through electroporation and selected under erythromycin resistance, the plasmid underwent a spontaneous deletion of 8.5kb fragment resulting in the formation of plasmid pRL60. pRL60 (10.2kb) is a shuttle vector between A. mediterranei is also expressed in A. mediterranei DSM 40773 and its activity can be easily detected on starch containing medium after iodine staining. Most critical parameters evaluated for electrotransformation using pRL60 in A. mediterranei were growth phase, electrical field strength, pulse length, pretreatment of mycelia with lysozyme and use of salt free water.

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Rifamycins are primarily produced by Gram-positive bacterium Amycolatopsis mediterranei, which belongs to the order Actinomycetales. These antibiotics, apart from their application against pathogens of tuberculosis and leprosy, have also been found to be effective against several other pathogens including Mycobacterium avium and Pneumococcus. Because of the importance of rifamycin, the producer strain A. mediterranei has been genetically manipulated since 1957 in order to develop a strain that can either produce larger amounts of rifamycin or derivatives of rifamycin. In this article, the importance of the producer strain, traditional methods (mutations and recombination) of strain improvement, their limitations, and the development of a cloning vector and transformation methods that have made recombinant DNA techniques accessible for genetic manipulations of A mediterranei are discussed.