Systematic Insertional Mutagenesis of a Streptomycete Genome: A Link Between Osmoadaptation and Antibiotic Production
Abstract
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 up to fivefold greater antibiotic yields than the wild-type strain.
Footnotes
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[The sequence data from this study have been submitted to EMBL under accession nos. AJ565873 and AJ566337.]
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Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1710304. Article published online before print in April 2004.
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↵1 Corresponding author. E-MAIL p.j.dyson{at}swansea.ac.uk; FAX 44-1792-295447.
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- Accepted January 12, 2004.
- Received July 1, 2003.
- Cold Spring Harbor Laboratory Press