Journal
GENOME RESEARCH
Volume 14, Issue 5, Pages 893-900Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gr.1710304
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- Biotechnology and Biological Sciences Research Council [EGH18395] Funding Source: Medline
- Biotechnology and Biological Sciences Research Council [EGH18395] Funding Source: researchfish
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The model organism Streptomyces coelicolor represents a genus that produces a vast range of bioactive secondary metabolites. We describe a versatile procedure for systematic and comprehensive mutagenesis of the S. coelicolor genome. The high-throughput process relies on in vitro transposon mutagenesis of an ordered cosmid library; mutagenized cosmids with fully characterized insertions are then transferred by intergeneric conjugation into Streptomyces, where gene replacement is selected. The procedure can yield insertions in upward of 90% of genes, and its application to the entire genome is underway. The methodology could be applied to many other organisms that can receive DNA via RK2/RP4-mediated intergeneric conjugation. The system permits introduction of mutations into different genetic backgrounds and qualitative measurement of the expression of disrupted genes as demonstrated in the analysis of a hybrid histidine kinase and response regulator gene pair, osaAB, involved in osmoadaptation in Streptomyces. The independently transcribed response regulator gene, osaB, is essential for osmoadaptation; when grown with supplementary osmolyte, an osaB mutant cannot erect aerial hyphae and produces Lip to fivefold greater antibiotic yields than the wild-type strain.
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