ABSTRACT
A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985. J. Bacteriol. 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78-89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.
Subject(s)
Agrobacterium tumefaciens/genetics , Genetic Vectors/genetics , Promoter Regions, Genetic , T-Phages/genetics , Virulence Factors , Acetophenones/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , DNA, Recombinant , DNA, Viral/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression , Gene Expression Regulation/drug effects , Histidine/genetics , Molecular Sequence Data , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Transcriptional activation of the Agrobacterium tumefaciens vir regulon is regulated by phenolics such as acetosyringone (AS), certain monosaccharides, and acidic conditions produced by wounded plant cells. The transmembrane protein VirA acts as an environmental sensor, mediating signal transduction upon perception of these stimuli. Although the periplasmic domain of VirA is not absolutely required for AS-dependent vir gene induction, it is needed for interactions with the periplasmic sugar-binding protein ChvE that result in sugar-induced enhancement of phenolic sensitivity. In this report, we demonstrate that mutations within the periplasmic domain but outside the predicted ChvE binding region can drastically alter the sensitivity of VirA to As. Using site-directed mutagenesis, we have characterized the roles of three individual amino acids in sugar-dependent AS sensitivity and have correlated the induction phenotype with the tumorigenic capacity of strains expressing mutant versions of VirA. Substitution of leucine for Glu-255 abolishes sugar enhancement while replacement with aspartic acid results in a wild-type phenotype. This residue lies outside the predicted ChvE binding site and thus identifies a new region of the VirA periplasmic domain crucial for the enhancement of vir gene induction by carbohydrates. In the absence of inducing sugar, wild-type VirA protein appears to be subject to some form of inhibition that suppresses the maximal level of transcriptional activation; deletions within the periplasmic region relieve this suppression.
