Improved Broad-Host-Range RK2 Vectors Useful for High and Low Regulated Gene Expression Levels in Gram-Negative Bacteria

Abstract

This report describes the construction and use of improved broad-host-range expression vectors based on the previously constructed pJB137 and pJB653 plasmids (Blatnyet al.,1997). These vectors contain the minimal replicon of RK2 and the induciblePuorPmpromoters together with their regulatoryxylRorxylSgenes, respectively, from thePseudomonas putidaTOL 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 ofPm,the establishment of their nucleotide sequence, the presence of RK2oriT,and different antibiotic selection markers. The copy numbers of all the vectors can easily be modified by using copy-up mutations of thetrfAgene, required for initiation of replication of RK2 replicons. The vectors were used to study the expression levels of theAcetobacter xylinumphosphoglucomutase genecelBand the two commonly used reporter geneslucandcatinEscherichia coli, Pseudomonas aeruginosa,andXanthomonas campestris.Good induction properties and tight regulation ofPmwere achieved in all three species tested, and higher gene expression levels were obtained by using the ATG vectors compared to pJB653. By introducing differenttrfAcopy-up mutations into the vectors, a wide range of gene expression levels fromPuandPmwere obtained inE. coli.Induced expression levels ofluc, cat,andcelBfromPmwere found to be comparable to or higher than those from thePtrcandPT7promoters located on high copy number plasmids. The induced levels of Luc activity were higher inP. aeruginosathan inE. coli,indicating that these vectors may be useful for maximization of gene expression in strains other thanE. 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.