The sequence upstream of the -10 consensus sequence modulates the strength and induction time of stationary-phase promoters in Escherichia coli.
We constructed a library of synthetic stationary-phase promoters for Escherichia coli. For designing the promoters, the known -10 consensus sequence, as well as the extended -10 region, and an A/T-rich region downstream of the -10 region were kept constant, whereas sequences from -37 to -14 were partially or completely randomised. For detection and selection of stationary-phase promoters, green fluorescent protein (GFP) with enhanced fluorescence was used. To establish the library, 33 promoters were selected, which differ in strength from 670 to more than 13,000 specific fluorescence units, indicating that the strength of promoters can be modulated by the sequence upstream of the -10 region. DNA sequencing revealed a preferential insertion of nucleotides depending on the position. By expressing the promoters in an rpoS-deficient strain, a special group of stationary-phase promoters was identified, which were expressed exclusively or preferentially by RNA polymerase holoenzyme Esigma(s). The DNA sequence of these promoters differed significantly in the region from -25 to -16. Furthermore, it was shown that the DNA curvature of the promoter region had no effect on promoter strength. The broad range of promoter activities make these promoters very suitable for fine-tuning of gene expression and for cost-effective large-scale applications in industrial bioprocesses.
Miksch
Gerhard
G
Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, 33594 Bielefeld, Germany. gmi@fermtech.techfak.uni-bielefeld.de
Bettenworth
Frank
F
Friehs
Karl
K
Flaschel
Erwin
E
D001483:Base Sequence; D016384:Consensus Sequence / Q000502:physiology*; D004269:DNA, Bacterial / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial