A novel vector allowing the expression of genes in a wide range of Gram-negative bacteria

Abstract

The construction and use of a novel vector allowing the expression of genes in a wide range of Gram-negative bacteria is described. The vector utilizes the regulatory region from IS 50. The 70-bp promoter region was isolated from one of the terminal inverted repeats of Tn 5 by creating EcoRI and SalI or PstI restriction sites by in vitro mutagenesis. This 70-bp region was shown to direct the expression of cat and lacZ genes in different bacterial genera including Alcaligenes, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens. Different strains containing the cat gene behind the regulatory elements of IS 50 were able to tolerate high concentrations (300 μg/ml) ofchloramphenicol in the medium. The 70-bp promoter region was cloned into a broad-host-range plasmid behind multiple cloning sites to create pAV10, which has unique restriction sites for BamHI, KpnI, SstI, and XbaI. Genes cloned into pAV10 can be expressed in a variety of Gram-negative bacteria.