Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria.

This report describes the construction and use of improved broad-host-range expression vectors based on the previously constructed pJB137 and pJB653 plasmids (Blatny et al., 1997). These vectors contain the minimal replicon of RK2 and the inducible Pu or Pm promoters together with their regulatory xylR or xylS genes, respectively, from the Pseudomonas putida TOL plasmid pWWO. A set of ATG vectors were derived from pJB653, and these vectors are characterized by the relatively small size, the presence of multiple cloning sites downstream of Pm, the establishment of their nucleotide sequence, the presence of RK2 oriT, and different antibiotic selection markers. The copy numbers of all the vectors can easily be modified by using copy-up mutations of the trfA gene, required for initiation of replication of RK2 replicons. The vectors were used to study the expression levels of the Acetobacter xylinum phosphoglucomutase gene celB and the two commonly used reporter genes luc and cat in Escherichia coli, Pseudomonas aeruginosa, and Xanthomonas campestris. Good induction properties and tight regulation of Pm were achieved in all three species tested, and higher gene expression levels were obtained by using the ATG vectors compared to pJB653. By introducing different trfA copy-up mutations into the vectors, a wide range of gene expression levels from Pu and Pm were obtained in E. coli. Induced expression levels of luc, cat, and celB from Pm were found to be comparable to or higher than those from the Ptrc and PT7 promoters located on high copy number plasmids. The induced levels of Luc activity were higher in P. aeruginosa than in E. coli, indicating that these vectors may be useful for maximization of gene expression in strains other than E. coli. We believe that the well-characterized vectors described here are useful for gene expression studies and routine cloning experiments in many Gram-negative bacteria.