The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters
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Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the −35 and −10 consensus promoter sequences were completely randomized and some variability was introduced in the −35 (position 6) and −10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different concentrations of neomycin (20, 50, and 100 μg ml−1). Promoter strengths varied up to 12-fold, in small increments of activity increase, as determined by reverse transcriptase-PCR. This collection of promoters of various strengths can be useful for the fine-tuning of gene expression in genetic engineering projects. Thirty-eight promoters were sequenced, and the sequences of the 14 weakest and 14 strongest promoters were compared using the WebLogo software with small sample correction. This comparison revealed that the −10 box, the −10 extended motif as well as the spacer of the strong Streptomyces promoters are more G rich than those of the weak promoters.
KeywordsStreptomyces Synthetic promoters Promoter strength aphII Neomycin
This work was supported by the European program ACTINOGEN (http://www.swan.ac.uk/ils/Research/BioMed/ActinoGen/), the Centre National de la Recherche Scientifique (http://www.cnrs.fr/), the University Paris Sud 11 (http://www.u-psud.fr), and the Pôle de Recherche et d'Enseignement supérieur UniverSud Paris (http://www.universud-paris.fr).
The authors wish to thank Guislaine Refregier and Barry Holland for stimulating discussions, and we are most grateful to Jeffrey Mellin for correction of the manuscript.
- Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, Bateman A, Brown S, Chandra G, Chen CW, Collins M, Cronin A, Fraser A, Goble A, Hidalgo J, Hornsby T, Howarth S, Huang CH, Kieser T, Larke L, Murphy L, Oliver K, O'Neil S, Rabbinowitsch E, Rajandream MA, Rutherford K, Rutter S, Seeger K, Saunders D, Sharp S, Squares R, Squares S, Taylor K, Warren T, Wietzorrek A, Woodward J, Barrell BG, Parkhill J, Hopwood DA (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417(6885):141–147CrossRefGoogle Scholar
- Buttner MJ, Chater KF, Bibb MJ (1990) Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). J Bacteriol 172(6):3367–3378Google Scholar
- Hook-Barnard IG, Hinton DM (2007) Transcription initiation by mix and match elements: flexibility for polymerase binding to bacterial promoters. Gene Regul Syst Bio 1:275–293Google Scholar
- Jensen PR, Hammer K (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 64(1):82–87Google Scholar
- Jeppsson M, Johansson B, Jensen PR, Hahn-Hagerdal B, Gorwa-Grauslund MF (2003) The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains. Yeast Chichester 20(15):1263–1272CrossRefGoogle Scholar
- Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Centre, Norwich Research Park, ColneyGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar