Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2) This paper is dedicated to the memory of Kathy Kendrick, whose devotion to understanding the biology of Streptomyces was unsurpassed.

Abstract

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 φC31. These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a σ 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 φC31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations.

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels

“…7). It is noteworthy that integration of pIJ8600 does not appear to affect growth or antibiotic production in S. coelicolor (Sun et al, 1999;Hesketh et al, 2001). Thus, the effects of integration may be species specific and our data suggest that experiments in which the wC31 attachment site is used for integration should always involve an analysis of the effects of integration on the physiology of the host organism.…”

“…Plasmids for overexpression of S. antibioticus pnp were constructed using the streptomycete expression vector pIJ8600 (Sun et al, 1999;Kieser et al, 2000). For the construction of pJSE340 a 3?2 kb PCR fragment was prepared, extending from the beginning of the intergenic region between a putative integral membrane protein and the rpsO gene upstream of pnp, to a BamHI site in a putative protease gene downstream of pnp (see Fig.…”

“…pIJ8600 is a streptomycete expression vector based on the thiostrepton-inducible promoter tipA (Holmes et al, 1993;Sun et al, 1999). The vector also contains a ribosomebinding site downstream of tipA, the attachment site for the streptomycete phage wC31 and genes conferring resistance to apramycin and thiostrepton (Kieser et al, 2000).…”

Identification of DNA-binding proteins involved in regulation of expression of the Streptomyces aureofaciens sigF gene, which encodes sporulation sigma factor σF

“…Expression of the sigF gene, encoding the late-sporulation-specific σ F , is temporally committed to sporulation in both strains (Kelemen et al, 1996 ;Kormanec et al, 1996). Sun et al (1999) have recently shown that expression of sigF is spatially limited to the spore compartment, with no evidence of its expression before sporulation septa are formed in S. coelicolor. The dependence of sigF expression upon early-sporulation-specific σ factor gene whiG\rpoZ in both strains indicated a cascade of activation, similar to that occurring in B. subtilis (Kelemen et al, 1996 ;Kormanec et al, 1996).…”

Role of nsdA in negative regulation of antibiotic production and morphological differentiation in Streptomyces bingchengensis

“…33 E. coliStreptomyces shuttle plasmid, pHZ1358 (provided by Dr Yinghua Zheng), was used to construct the nsdA gene-replacement vector. pIJ8600, which integrated into the S. bingchengensis_226541 chromosome by site-specific recombination at the bacteriophage FC31 attachment site, attB, 34,35 was used to introduce single copies of genes into the S. bingchengensis_226541 chromosome.…”