Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A-tumefaciens rpoA gene, encoding the alpha subunit of RNA polymerase

The two-component regulatory system, composed of virA and virG, is indispen sable for transcription of virulence genes within Agrobacterium tumefaciens . However, virA and virG are insufficient to activate transcription from vi rulence gene promoters within Escherichia coli cells, indicating a requirem ent for additional A. tumefaciens genes. In a search for these additional g enes, we have identified the rpoA gene, encoding the alpha subunit of RNA p olymerase (RNAP), which confers significant expression of a virB promoter ( virBp)::lacZ fusion in E. coli in the presence of an active transcriptional regulator virG gene. We conducted in vitro transcription assays using eith er reconstituted E. call RNAP or hybrid RNAP in which the alpha subunit was derived from A. tumefaciens. The two forms of RNAP were equally efficient in transcription from a sigma(70)- dependent E. coli galP1 promoter; howeve r, only the hybrid RNAP was able to transcribe virBp in a virG-dependent ma nner. In addition, we provide evidence that the alpha subunit from A. tumef aciens, but not from E. coli, is able to interact with the VirG protein. Th ese data suggest that transcription of virulence genes requires specific in teraction between VirG and the alpha subunit of A. tumefaciens and that the alpha subunit from E. coli is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to exami ne vir gene expression as well as the T-DNA transfer process in E. coli.