Bishop, A. and Fielding, S. and Dyson, P.J. and Herron, P.R. (2004) Systematic insertional mutagenesis of a streptomycete genome: a link between osmoadaptation and antibiotic production. Genome Research, 14. pp. 893-900. ISSN 1088-9051Full text not available in this repository. (Request a copy from the Strathclyde author)
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.
|Keywords:||antibiotic production , insertional mutagenesis , streptomycete genome , osmoadaptation, Pharmacy and materia medica, Genetics, Genetics(clinical)|
|Subjects:||Medicine > Pharmacy and materia medica|
|Department:||Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences|
|Depositing user:||Pure Administrator|
|Date Deposited:||24 Feb 2012 14:07|
|Last modified:||20 Jan 2017 03:10|