title: Cumate-inducible gene expression system for sphingomonads and other Alphaproteobacteria.
abstract: Tunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of the Pseudomonas putida F1 cym/cmt system. The resulting promoter, termed PQ5, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in other Alphaproteobacteria, such as the model organisms Caulobacter crescentus, Paracoccus denitrificans, and Methylobacterium extorquens. In the noninduced state, expression from PQ5 is low enough to allow gene depletion analysis, as demonstrated with the essential gene phyP of Sphingomonas sp. strain Fr1. A set of PQ5-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.
authors.affiliation: Institute of Microbiology, ETH Zurich, Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kaczmarczyk
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
authors.forename: Anne
authors.initials: A
authors.lastname: Francez-Charlot
mesh_terms: D001483:Base Sequence; D001565:Benzoates / Q000494:pharmacology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D042301:Sphingomonadaceae / Q000235:genetics* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001565:Benzoates; C055607:cumic acid; D001616:beta-Galactosidase