MUTATIONAL ANALYSIS OF THE INPUT DOMAIN OF THE VIRA PROTEIN OF AGROBACTERIUM-TUMEFACIENS

The transmembrane sensor protein VirA activates VirG in response to hi gh levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and mono saccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 vir A gene was randomly mutagenize d, and 14 mutants defective in vir gene induction and containing mutat ions which mapped to the input domain of VirA were isolated, Six mutan ts had single missense mutations in three widely separated areas of th e periplasmic domain. Eight mutants had mutations in or near an amphip athic helix, TIMI, or TM2. Four of the mutations in the periplasmic do main, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose, This suggests t hat most of the periplasmic domain is required for the interaction wit h, or response to, ChvE. Three of the mutations from outside the perip lasmic domain, one from each transmembrane domain and one from the amp hipathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS, These mutations did not affect the stabil ity or topology of VirA or prevent dimerization; therefore, they may i nterfere with detection of AS or transmission of the signals to the ki nase domain, Characterization of C58 chvE mutants revealed that, unlik e A6 VirA, C58 VirA requires ChvE for activation of the vir genes.