Systematic insertional mutagenesis of a streptomycete genome: a link between osmoadaptation and antibiotic production.
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.
Bishop
Amy
A
School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK.
Fielding
Sue
S
Dyson
Paul
P
Herron
Paul
P
D000222:Adaptation, Physiological / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D018014:Gene Transfer Techniques; D008040:Genetic Linkage / Q000235:genetics* / Q000276:immunology*; D016680:Genome, Bacterial; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics / Q000502:physiology; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics* / Q000502:physiology; D013302:Streptomyces / Q000235:genetics* / Q000502:physiology; D014169:Transformation, Bacterial / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011494:Protein Kinases; D000071677:Histidine Kinase