RT Journal
A1 Bishop, Amy
A1 Fielding, Sue
A1 Dyson, Paul
A1 Herron, Paul
T1 Systematic Insertional Mutagenesis of a Streptomycete Genome: A Link Between Osmoadaptation and Antibiotic Production
JF Genome Research 
JO Genome Research 
YR 2004 
FD May 01 
VO 14 
IS 5 
SP 893 
OP 900 
DO 10.1101/gr.1710304 
UL http://genome.cshlp.org/content/14/5/893.abstract 
AB 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.