Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli

Agrobacterium tumefaciens is a soil bacterium which infects plant wound sites and induces tumor formation. The bacterium harbors a large tumor inducing plasmid (Ti plasmid) encoding virulence genes and transfer DNA (T-DNA). The function of the virulence genes is the processing and transfer of the T-DNA from the Ti plasmid into susceptible plant cells, with subsequent integration into the host genome (for recent reviews, see references 27 and 56). Located on the T-DNA are genes that direct the biosynthesis of the plant growth regulators auxin and cytokinin in the infected cells (1, 49). The synthesis of these plant growth regulators results in a rapid, uncontrolled cell division leading to production of a characteristic tumor at the site of infection. In addition, the T-DNA contains genes for the synthesis of a unique class of compounds called opines, which A. tumefaciens can utilize as a carbon and energy source (36). In A. tumefaciens, the expression of virulence genes is under the control of a two-component regulatory system comprised of VirA and VirG (45, 52). VirA is an inner membrane histidine kinase (34, 54) which autophosphorylates in response to certain phenolic compounds released from wounded plants (44) with subsequent transfer of the phosphate moiety to the response regulator VirG (16, 22, 24). Once phosphorylated, VirG activates transcription from promoters containing a specific 12-bp sequence called the vir box, which is present in the promoters of all vir genes (25, 40). This expression is augmented by the presence of certain monosaccharides (5, 43) and an acidic pH (32), which is characteristic of plant wound sites. A periplasmic sugar-binding protein, ChvE, which is highly homologous to glucose-binding protein of Escherichia coli, interacts with the periplasmic portion of the VirA molecule in the presence of certain monosaccharides, including glucose and arabinose (2, 15). This interaction alone does not induce vir gene expression, but it sensitizes the VirA molecule to the phenolic inducers. While A. tumefaciens is a potentially serious plant pathogen, the main interest in this organism is due primarily to its ability to transform plant cells. Researchers have developed delivery systems based on T-DNA transfer to engineer new traits into selected plant species (14). However, the exact mechanism of T-DNA transfer is still not well understood. The ability to use E. coli as a heterologous host in which to study the regulation of A. tumefaciens virulence genes and the mechanism of T-DNA transfer would constitute an excellent model system given the degree of characterization at both the biochemical and the genetic levels and the relative ease of genetic manipulation. However, all previous attempts to reconstitute inducer-mediated vir gene expression in E. coli have not been successful. Previously, we reported the identification of the rpoA gene, encoding the alpha subunit of RNA polymerase from A. tumefaciens, and that it was required for transcription of a virB promoter fused to lacZ (virBp::lacZ) fusion in E. coli using a constitutively active VirG mutant [VirG(Con)] which can activate vir gene expression independent of VirA and inducers (31). In this study, we report the successful reconstitution of inducer-dependent expression of a virBp::lacZ fusion in E. coli utilizing lac promoter-driven virA and virG. Effects of various environmental conditions on virulence gene activation in E. coli are also described. Furthermore, by the gene fusion approach, the C-terminal domain of RpoA from A. tumefaciens is shown to be required for interaction with the transcriptional activator VirG.