GLU-255 OUTSIDE THE PREDICTED CHVE BINDING-SITE IN VIRA IS CRUCIAL FOR SUGAR ENHANCEMENT OF ACETOSYRINGONE PERCEPTION BY AGROBACTERIUM-TUMEFACIENS

Transcriptional activation of the Agrobacterium tumefaciens vir regulo n is regulated by phenolics such as acetosyringone (AS), certain monos accharides, 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 pe riplasmic domain of VirA is not absolutely required for AS-dependent v ir 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 w ithin the periplasmic domain but outside the predicted ChvE binding re gion can drastically alter the sensitivity of VirA to AS. Using site-d irected mutagenesis, we have characterized the roles of three individu al amino acids in sugar-dependent AS sensitivity and have correlated t he induction phenotype with the tumorigenic capacity of strains expres sing mutant versions of VirA. Substitution of leucine for Glu-255 abol ishes sugar enhancement while replacement with aspartic acid results i n 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 carbohydra tes. In the absence of inducing sugar, wild-type VirA protein appears to be subject to some form of inhibition that suppresses the maximal l evel of transcriptional activation; deletions within the periplasmic r egion relieve this suppression.