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Published online before print April 12, 2004
Genome Research, DOI: 10.1101/gr.1710304
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Methods

Systematic Insertional Mutagenesis of a Streptomycete Genome: A Link Between Osmoadaptation and Antibiotic Production

Amy Bishop, Sue Fielding, Paul Dyson1 and Paul Herron

School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK

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.

[The sequence data from this study have been submitted to EMBL under accession nos. AJ565873 and AJ566337.]

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1710304. Article published online before print in April 2004.

1 Corresponding author.
E-MAIL p.j.dyson{at}swansea.ac.uk; FAX 44-1792-295447.


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