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Journal of Bacteriology, August 1999, p. 4533-4539, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Transcriptional Activation of Agrobacterium tumefaciens Virulence Gene Promoters in Escherichia coli Requires the A. tumefaciens rpoA Gene, Encoding the Alpha Subunit of RNA Polymerase

S. M. Lohrke,1 S. Nechaev,2 H. Yang,1 K. Severinov,2 and S. J. Jin1,*

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,1 and Waksman Institute, Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey 088542

Received 12 March 1999/Accepted 21 May 1999

The two-component regulatory system, composed of virA and virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens. However, virA and virG are insufficient to activate transcription from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A. tumefaciens genes. In a search for these additional genes, we have identified the rpoA gene, encoding the alpha  subunit of RNA polymerase (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 either reconstituted E. coli 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; however, only the hybrid RNAP was able to transcribe virBp in a virG-dependent manner. In addition, we provide evidence that the alpha  subunit from A. tumefaciens, but not from E. coli, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction 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 examine vir gene expression as well as the T-DNA transfer process in E. coli.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205. Phone: (501) 296-1396. Fax: (501) 686-5359. E-mail: jinshouguang{at}exchange.uams.edu.

Journal of Bacteriology, August 1999, p. 4533-4539, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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