The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters.
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 μgml(-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.
Institut de Génétique et Microbiologie, UMR8621 CNRS Université Paris Sud, 91405, Orsay, France.
Nicolas
N
Seghezzi
Patrick
P
Amar
Brian
B
Koebmann
Peter R
PR
Jensen
Marie-Joëlle
MJ
Virolle
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009355:Neomycin / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012808:Sigma Factor; D013302:Streptomyces / Q000235:genetics*
D004269:DNA, Bacterial; D012808:Sigma Factor; D009355:Neomycin