Slovakia的链霉菌基因破坏实验.pdfVIP

Journal of GeneralMicrobiology (1993), 139,2525-2529. Printed in Great Britain 2525 Optimization of Streptomyces aureofaciens transformation and disruption of the krdA gene encoding ahomologue of the principal tr factor JANKORMANEC,* BRONISLAVA~ ~ U C H O VandA MARIANFARKA~OVSK+ Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 84251Bratislava, Slovakia (Received11 February 1993;revised 19April 1993; accepted 10May 1993) ~~~ ~ Conditions for the preparation of protoplastsfromStreptomyces aureofaciensandtransformation with several replicative and integrative plasmids were optimized. Of all the replicative plasmids used, carrying various Streptomyces replicationorigins,onlyplasmidpGM9producedtransformants,withatransformation efficiency of 2x 103(pgDNA)-. When plasmid DNA isolatedfrom S.aureofacienswas used, the transformation efficiency increasedto lo5transformants(pgDNA)-. Transformation of protoplastswith integrativeplasmids containing partofthehrdAgene gaverisetotransformantswithanefficiencyofapproximately30transformants(pgDNA)-. Integration frequency was stronglyreducedwhen plasmid DNA was modified by dam or dcm methylase.By integrative transformation, via a double crossover, a stablenull mutant of the hrdA gene was prepared. This mutation appeared to have no obvious effect on growth, morphology, differentiation and production of chlortetracycline. Introduction Matsushima, 1981;Ogawaet al., 1983; Hopwoodet al., 1985).However,we couldnot prepare protoplasts of S. In Streptomyces aureofaciens CCM3239 (ATCC 10762), aureofaciens CCM3239by any of the techniquesmen- four genes (hrdA, hrdB, hrdD and h