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.

journal_name

Genome Res

journal_title

Genome research

authors

Bishop A,Fielding S,Dyson P,Herron P

doi

10.1101/gr.1710304

subject

Has Abstract

pub_date

2004-05-01 00:00:00

pages

893-900

issue

5

eissn

1088-9051

issn

1549-5469

pii

1710304

journal_volume

14

pub_type

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