New broad-host-range promoter probe vectors based on the plasmid RK2 replicon

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucl eotide sequence were constructed. In the absence of an upstream promoter, t he expression of two tested reporter genes (luc and lacZ) in Escherichia co li was virtually zero, while insertion of the Ptre promoter resulted in str ong inducer-dependent expression. The lacZ-based Vectors were mobilized int o Pseudomonas fluorescens ST. Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400. and expression analyses indicated that the pro perties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology all ows easy manipulations of features such as plasmid copy number, further ext ending the application potential of the vectors. (C) 2001 Federation of Eur opean Microbiological Societies. Published by Elsevier Science B.V. All rig hts reserved.