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Journal of Bacteriology, June 2001, p. 3704-3711, Vol. 183, No. 12
Department of Molecular Genetics and
Microbiology, University of Florida, Gainesville, Florida 32610
Received 26 January 2001/Accepted 30 March 2001
The ability to utilize Escherichia coli as a
heterologous system in which to study the regulation of
Agrobacterium tumefaciens virulence genes and the mechanism
of transfer DNA (T-DNA) transfer would provide an important tool to our
understanding and manipulation of these processes. We have previously
reported that the rpoA gene encoding the alpha subunit of
RNA polymerase is required for the expression of lacZ gene
under the control of virB promoter (virBp::lacZ) in E. coli
containing a constitutively active virG gene
[virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal
89 residues from A. tumefaciens was able to significantly
express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization of
lac promoter-driven virA and virG
in combination with the A. tumefaciens rpoA construct
resulted in significant inducer-mediated expression of the
virBp::lacZ fusion, and the level of
virBp::lacZ expression was positively
correlated to the copy number of the rpoA construct. This
expression was dependent on VirA, VirG, temperature, and, to a lesser
extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on vir
gene expression was observed only in the presence of the
chvE gene, suggesting that the glucose-binding protein of
E. coli, a homologue of ChvE, does not interact with the
VirA molecule. We also evaluated other phenolic compounds in induction
assays and observed significant expression with syringealdehyde, a low
level of expression with acetovanillone, and no expression with
hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was
derived. These data support the notion that VirA directly senses the
phenolic inducer. However, the overall level of expression of the
vir genes in E. coli is less than what is
observed in A. tumefaciens, suggesting that additional
gene(s) from A. tumefaciens may be required for the full
expression of virulence genes in E. coli.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3704-3711.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Reconstitution of Acetosyringone-Mediated Agrobacterium
tumefaciens Virulence Gene Expression in the Heterologous
Host Escherichia coli

and
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Corresponding author. Mailing address: Department of
Molecular Genetics and Microbiology, P.O. Box 100266, University of
Florida, Gainesville, FL 32610. Phone: (352) 392-8323. Fax: (352)
392-3133. E-mail: sjin{at}ufl.edu.
Present address: Biocontrol of Plant Disease Laboratory, USDA,
Beltsville, MD 20705-2350.
Present address: PDF Biotech, Inc., Tianjin, People's
Republic of China.
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