Hoechst Aktiengesellschaft

Gunter Muth

Alfred Puhler

Wolfgang Wohlleben

Gerhard Wohner

Rudiger Marquardt

Gunther J Rieb

The kil-kor system from the plasmid pIJ101 can be utilized for regulated gene expression in Streptomycetes. For this, the KorA protein is either inactivated or eliminated to 'switch on' a particular gene.

Regulated gene expression in streptomycetes

Starting from the Streptomycetes plasmids pSG2, pSG5 and pSVH1, 'shuttle vectors' which, in additon to a E. coli replicon, also contain suitable selection markers for E. coli and Streptomycetes are constructed. These shuttle vectors replicate both in E. coli and in Streptomycetes, and they can be used for cloning and expression of foreign DNA.

Hybrid plasmids having a streptomycetes replicon and an escherichia coli

Intensely crimson colonies are obtained from Streptomyces coelicolor DSM 3030 by total digestion of the total DNA, cloning into a suitable vector, and transformation of Streptomyces lividans. Re-isolation of the plasmid DNA and cutting with BamHI results in fragments about 3.4 to 9 kb in length, on which is located the gene which determines production of the coloring agent. This gene is suitable as marker, in particular as inactivation marker, in Streptomycetes.

Color marker for clonings in Streptomyces lividans

Intensely blue-black colonies are obtained from Streptomyces coelicolor DSM 3030 by total digestion of the total DNA, cloning into a suitable vector, and transformation of a streptomycetes recipient strain. Re-isolation of the plasmid DNA and cutting with BamHI results in a 5.5 kb fragment on which the gene for the coloring agent is located. This gene is suitable as marker, in particular as inactivation marker, in Streptomycetes.

Color marker in streptomycetes plasmids

S. ghanaensis DSM 2932 is resistant to gentamicin at up to 20 .mu.g/ml. Total digestion of the genomic DNA with BglII, incorporation of the restriction fragments into a suitable plasmid, and selection using gentamicin results in gentamicin-resistant clones which contain a 7 kb fragment with the gentamicin-resistance gene. The plasmid pPH1JI likewise contains a gentamicin-resistance gene located on a 2.3 kb HindIII-BamHI fragment. These genes are suitable as markers, in particular for Streptomycetes vectors.

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Bielefeld, Germany

Gunter Muth