Ratcheting up vir gene expression in Agrobacterium tumefaciens: coiled coils in histidine kinase signal transduction.

The transmembrane histidine kinase VirA is responsible for the recognition of information from several plant-derived xenognostic signals that control gene transfer between Agrobacterium tumefaciens and its eukaryotic host. As with other histidine autokinases, VirA appears to exist as a homodimer within the inner membrane of the bacterium. In this study, we identify the putative homodimeric coiled-coil-like motifs Helix TM2 (amino acids (aa) 259-288) and Helix C (aa 293-327) within the previously assigned signal input domain. The functional importance of these coiled-coil interactions in signal-mediated VirA activation is investigated by the construction of fusion proteins with the leucine zipper domain of the transcription factor GCN4. Replacement of the membrane-spanning and periplasmic domains of VirA with the GCN4 leucine zipper gave functional proteins with increased signal-induced vir gene expression. When the GCN4 fusion was used to conformationally bias the interface of the Helix C coiled coil, constitutively active chimeras were created. The activity of these constructs was dependent on the interface of the Helix C coiled coil, and a ratchet model is proposed in which VirA activation is achieved by signal-induced switching of the interfaces of the homodimer. Since VirA functions as a transducer and integrates various host cues indirectly, these data highlight its role as an "antenna" for the tumor-inducing (Ti) plasmid, able to monitor the host proteome so as to select for successful xenognostic signaling strategies.