Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria

Abstract

We describe several plasmids that are designed to create fusions between chromosomal or plasmid-encoded genes and the lacZ, phoA or gfp reporter genes. These plasmids all contain the vegetative origin of R6K, but lack the R6K pir gene, and therefore fail to replicate in strains lacking pir. Fragments of target genes are introduced into these plasmids, and fusions are created in a single step as a consequence of (Campbell-type) integration of the entire plasmid by homologous recombination. Cloned fragments containing either an intact 5′-end of the target gene including its promoter or an intact 3′-end of the gene preserve a functional copy of that gene, while fragments lacking both 5′- and 3′-ends of the target gene cause a gene disruption. In addition to facilitating measurements of gene expression, some plasmids create translational fusions to β-galactosidase or alkaline phosphatase and are therefore useful in studying the membrane topology of a target protein. We demonstrate the utility of these plasmids by constructing and testing two operon fusions and two protein fusions between the virG gene of Agrobacterium tumefaciens and lacZ.