Manipulating and understanding antibiotic production in Streptomyces coelicolor A3(2) with decoy oligonucleotides.
We have adapted and extended the decoy oligonucleotide technique for use in prokaryotes. To identify cis-acting regulatory elements within a promoter, we developed a DNase I/T7 exonuclease footprinting technique and applied it to actII-orf4 from Streptomyces coelicolor A3(2), which encodes the pathway-specific activator for production of the antibiotic actinorhodin. Our in vivo mapping data allowed us to create decoy oligonucleotides incorporating the identified regulatory elements and to test whether their introduction into S. coelicolor affected antibiotic production. We mapped the promoter region when in a transcriptionally inactive state before the onset of actinorhodin production with the aim of designing decoy oligonucleotides capable of interfering with potential repressor binding and so stimulate actinorhodin production. Mapping identified five candidates for decoy oligonucleotides, and these were tested in a plate-based assay to rapidly validate their activity. A transfection protocol was developed for liquid cultures that enabled efficient uptake of decoys, and quantitative real-time PCR demonstrated decoy persistence for >70 h. Measurement of the effects on growth, expression of actII-orf4, and antibiotic production demonstrated that one of the decoys, in concordance with the plate assay, was more efficacious than the others in increasing actinorhodin production. Two of the identified regulatory elements occurred upstream of gene SCO5812, deletion of which reduced actinorhodin production, confirming that experimental analysis of regulatory motifs can provide new insights into factors influencing antibiotic production in streptomycetes.
McArthur
Michael
M
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom. michael.mcarthur@bbsrc.ac.uk
Bibb
Mervyn J
MJ
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors
C431645:ACTII protein, Echinococcus granulosus; D000199:Actins; D000900:Anti-Bacterial Agents; D015801:Helminth Proteins; D017874:Immediate-Early Proteins; D009841:Oligonucleotides