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Molecular Genetics of Streptomycetes |
Department of Genetics, John Innes Centre, Norwich NR4 7UH, UK1
Departamento de Microbiología y Genética, Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Edificio Departmental, Campus ‘Miguel de Unamuno’, 37007 Salamanca, Spain2
Author for correspondence: Mervyn J. Bibb. Tel: +44 1603 452571. Fax: +44 1603 456844. e-mail: bibb{at}bbsrc.ac.uk
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 
C31. These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a 
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 C31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations.
Keywords: Streptomyces coelicolor A3(2), green fluorescent protein (GFP), sigF, redD, tipAp
Abbreviations: EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; MCS, multiple cloning site; Thio, thiostrepton; tipAp, the Thio-inducible tipA promoter of Streptomyces lividans 66
This paper is dedicated to the memory of Kathy Kendrick, whose devotion to understanding the biology of Streptomyces was unsurpassed.
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