SUICIDE PLASMIDS CONTAINING PROMOTERLESS REPORTER GENES CAN SIMULTANEOUSLY DISRUPT AND CREATE FUSIONS TO TARGET GENES OF DIVERSE BACTERIA

We describe several plasmids that are designed to create fusions betwe en chromosomal or plasmid-encoded genes and the lacZ, phoA or gfp repo rter genes. These plasmids all contain the vegetative origin of R6K, b ut lack the R6K pir gene, and therefore fail to replicate in strains l acking pir. Fragments of target genes are introduced into these plasmi ds, and fusions are created in a single step as a consequence of (Camp bell-type) integration of the entire plasmid by homologous recombinati on. 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 facil itating measurements of gene expression, some plasmids create translat ional fusions to beta-galactosidase or alkaline phosphatase and are th erefore useful in studying the membrane topology of a target protein. We demonstrate the utility of these plasmids by constructing and testi ng two operon fusions and two protein fusions between the virG gene of Agrobacterium tumefaciens and lacZ.