Construction of an efficient expression system for Agrobacterium tumefaciens based on the coliphage T5 promoter

Abstract

A versatile expression vector utilizing a promoter of coliphage T5, P N25 (Gentz and Bujard, 1985. J. Bacteriol. 164, 70–77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78–89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (Δ1–284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (Δ1–284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P N25 driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.