Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2).
The enhanced green fluorescent protein (EGFP) gene is a modified version of the green fluorescent protein gene of the jellyfish Aequorea victoria with a codon usage that corresponds well to that found in many GC-rich streptomycete genes. Here the use of EGFP as a reporter for the analysis of spatially and temporally regulated gene expression in Streptomyces coelicolor A3(2) is demonstrated. The EGFP gene was inserted into plasmids that can replicate in Escherichia coli, greatly facilitating the construction of EGFP gene fusions. The plasmids can be transferred readily to S. coelicolor by conjugation, whereupon two of them (pIJ8630 and pIJ8660) integrate at the chromosomal attachment site for the temperate phage phiC31. These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a sigma factor required for spore maturation, and of redD, a pathway-specific regulatory gene for the production of undecylprodigiosin, one of the four antibiotics made by S. coelicolor. While transcription of sigF appeared to be confined to developing and mature spore chains, transcription of redD occurred only in ageing substrate mycelium. A further plasmid derivative (pIJ8668) was made that lacks the phiC31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations.
Sun
Jongho
J
Department of Genetics, John Innes Centre, Norwich NR4 7UH, UK1.
Kelemen
Gabriella H
GH
Department of Genetics, John Innes Centre, Norwich NR4 7UH, UK1.
Fernández-Abalos
José Manuel
JM
Departamento de Microbiologı́a y Genética, Instituto de Microbiologı́a Bioquı́mica, CSIC/Universidad de Salamanca, Edificio Departmental, Campus 'Miguel de Unamuno', 37007 Salamanca, Spain2.
Bibb
Mervyn J
MJ
Department of Genetics, John Innes Centre, Norwich NR4 7UH, UK1.
D001426:Bacterial Proteins; D001483:Base Sequence; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D013171:Spores, Bacterial / Q000502:physiology; D013302:Streptomyces / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C077706:redD protein, Streptomyces coelicolor; D049452:Green Fluorescent Proteins