Systematic insertional mutagenesis of a streptomycete genome: a link between osmoadaptation and antibiotic production

A. Bishop, S. Fielding, P.J. Dyson, P.R. Herron

Research output: Contribution to journalArticle

66 Citations (Scopus)

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.
LanguageEnglish
Pages893-900
Number of pages8
JournalGenome Research
Volume14
DOIs
Publication statusPublished - 2004

Fingerprint

Streptomyces coelicolor
Cosmids
Insertional Mutagenesis
Streptomyces
Regulator Genes
Mutagenesis
Genome
Anti-Bacterial Agents
Hyphae
Genes
Gene Expression
Mutation
DNA
In Vitro Techniques
Histidine Kinase
Genetic Background

Keywords

  • antibiotic production
  • insertional mutagenesis
  • streptomycete genome
  • osmoadaptation

Cite this

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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.",
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Systematic insertional mutagenesis of a streptomycete genome: a link between osmoadaptation and antibiotic production. / Bishop, A.; Fielding, S.; Dyson, P.J.; Herron, P.R.

In: Genome Research, Vol. 14, 2004, p. 893-900.

Research output: Contribution to journalArticle

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AU - Fielding, S.

AU - Dyson, P.J.

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