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 an d pJB653 plasmids (Blatny et al., 1997). These vectors contain the min imal replicon of RK2 and the inducible Pu or Pm promoters together wit h their regulatory xylR or xylS genes, respectively, from the Pseudomo nas putida TOL plasmid pWWO. A set of ATG vectors were derived from pJ B653, and these vectors are characterized by the relatively small size , the presence of multiple cloning sites downstream of Pm, the establi shment of their nucleotide sequence, the presence of RK2 oriT, and dif ferent antibiotic selection markers. The copy numbers of all the vecto rs 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 ph osphoglucomutase gene celB and the two commonly used reporter genes lu c and car 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 leve ls were obtained by using the ATG vectors compared to pJB653. By intro ducing different trfA copy-up mutations into the vectors, a wide range of gene expression levels from Pu and Pm were obtained in E. coli. In duced expression levels of luc, cat, and celB from Pm were found to be comparable to or higher than those from the Ptrc and P-T7 promoters l ocated on high copy number plasmids. The induced levels of Luc activit y were higher in P. aeruginosa than in E. coli, indicating that these vectors may be useful for maximization of gene expression in strains o ther than E. coli. We believe that the well-characterized vectors desc ribed here are useful for gene expression studies and routine cloning experiments in many Gram-negative bacteria. (C) 1997 Academic Press.