Critical variations of conjugational DNA transfer into secondary metabolite multiproducing Sorangium cellulosum strains So ce12 and So ce56: development of a mariner-based transposon mutagenesis system.
Myxobacteria increasingly gain attention as a source of bioactive natural products. The genus Sorangium produces almost half of the secondary metabolites isolated from these microorganisms. Nevertheless, genetic systems for Sorangium strains are poorly developed, which makes the identification of the genes directing natural product biosynthesis difficult. Using biparental and triparental mating, we have developed methodologies for DNA transfer from Escherichia coli via conjugation for the genome sequencing model strain So ce56 and the secondary metabolite multiproducing strain So ce12. The conjugation protocol developed for strain So ce56 is not applicable to other Sorangium strains. Crucial points for the conjugation are the ratio of E. coli and Sorangium cellulosum cells, the choice of liquid or solid medium, the time used for the conjugation process and antibiotic selection in liquid medium prior to the plating of cells. A mariner-based transposon containing a hygromycin resistance gene was generated and used as the selectable marker for S. cellulosum. The transposon randomly integrates into the chromosome of both strains. As a proof of principle, S. cellulosum So ce12 transposon mutants were screened using an overlay assay to target the chivosazole biosynthetic gene cluster.
Kopp
Maren
M
Gesellschaft für Biotechnologische Forschung mbH (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany.
Irschik
Herbert
H
Gross
Frank
F
Perlova
Olena
O
Sandmann
Axel
A
Gerth
Klaus
K
Müller
Rolf
R
D003227:Conjugation, Genetic / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D018014:Gene Transfer Techniques; D018942:Macrolides / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009231:Myxococcales / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D018942:Macrolides; D011994:Recombinant Proteins; C096623:chivosazol A; C111652:mariner transposases; D019895:Transposases