Construction and characterization of Tn5virB, a transposon that generates nonpolar mutations, and its use to define virB8 as an essential virulence gene in Agrobacterium tumefaciens

Abstract

Transfer of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant cells involves movement of a single-stranded DNA-protein intermediate across several membrane and cell wall barriers. The 11 VirB proteins encoded by the Ti plasmid are hypothesized to form at least part of a membrane-localized T-DNA transport apparatus. Although available genetic and biochemical analyses support this hypothesis, detailed study of this transport apparatus is hindered by the fact that most available mutations in the virB operon are in the form of transposon insertions that have polar effects. In this study we constructed a transposon, Tn5virB, that can be used to generate nonpolar insertions in operons of A. tumefaciens and used it to demonstrate that virB8 is an essential virulence gene.