Cumate - inducible gene expression system for sphingomonads 1 and other Alphaproteobacteria 2 3

11 Tunable promoters represent a pivotal genetic tool for a wide range of applications. 12 Here we present such a system for sphingomonads, a phylogenetically diverse group of 13 bacteria that have gained much interest for their potential in bioremediation and their 14 use in industry and for which no dedicated inducible gene expression system has been 15 described so far. A strong, constitutive synthetic promoter was first identified through a 16 genetic screen and subsequently combined with the repressor and the operator sites of 17 the Pseudomonas putida F1 cym/cmt system. The resulting promoter, termed PQ5, re18 sponds rapidly to the inducer cumate and shows a maximal induction ratio of two to 19 three orders of magnitude in the different sphingomonads tested. Moreover, it was also 20 functional in other Alphaproteobacteria, such as the model organisms Caulobacter 21 crescentus, Paracoccus denitrificans and Methylobacterium extorquens. In the non22 induced state, PQ5 is tight enough to allow gene depletion analysis as demonstrated with 23 the essential gene, phyP, of Sphingomonas sp. strain Fr1. A set of PQ5-based plasmids has 24 been constructed allowing fusions to affinity tags or fluorescent proteins. 25 AEM Accepts, published online ahead of print on 30 August 2013 Appl. Environ. Microbiol. doi:10.1128/AEM.02296-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved. on N ovem er 4, 2017 by gest ht://aem .sm .rg/ D ow nladed fom