Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of Agrobacterium tumefaciens

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes tha t are essential for the formation of the crown gall tumor. VirA also respon ds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homol ogous to the chemoreceptor Trg, and the phenolics are thought to be detecte d by a part of the cytoplasmic linker domain, while the second transmembran e domain (TM2) is reported to be nonessential. To define regions of VirA th at are essential for signal sensing, we introduced base-substitution and de letion mutations into coding regions that are conserved among the respectiv e domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The re sults show that the Trg-homologous region in the peri-plasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutat ions in the TM2 domain also failed to influence enhancement by sugars and r educed the level of vir induction, but a mutation in the TM2 region adjacen t to the cytoplasmic linker abolished induction of the vir genes. In the li nker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation o f a membrane-anchored VirA protein. Complementation analysis with two diffe rent VirA mutants suggested that intermolecular phosphorylation between Vir A molecules occurs in vivo, and that two intact periplasmic regions in a Vi rA dimer are required for the enhancement of vir induction by sugars.