Intergeneric conjugation: A practical method for genetic manipulation in actinomycetes

Abstract

The system for intergeneric conjugation has been developed for many actinomycetespecies including Streptomyces and rare actinomycetes. E. coli donor strains generallyused are the methylation proficient strain, S17-1 harbouring tra genes on its chromosomeand the methylation-deficient strain, ET12567 containing tra genes in a self-transmissibleplasmid pUB307 or a non-transmissible plasmid pUZ8002. Integrative plasmids used arenormally non-replicative plasmids carrying the oriT for the propose of gene disruption(e.g. pSET151, pIJ8671) or with additional ΦC31 attP-int system in the case of geneexpression (e.g. pSET152, pIJ8600). Actinomycete recipients used for mating with E.coli could be in spore suspension with or without pre-germination process, or mycelia inliquid culture. Several agar media including MS and ISP 4 were generally used for streptomycete strains while other more specific media were proposed for rareactinomycetes. Concentration of MgCl2, ratio of donor and recipient and other parametersare significantly important to increase conjugation efficiency. The intergenericconjugation approach has become a powerful method to genetically manipulateactinomycete species with high efficiency. It turns out to be a common tool tocharacterise functions of genes/gene clusters in these pharmaceutically importantbacteria. © 2013 by Nova Science Publishers, Inc. All rights reserved.