Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein

Abstract

The VirA protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites. VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module. We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation. In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant protein lacking the receiver module. We also show that ablation of the receiver module restores activity to the inactive VirA (⌬324-413) mutant, which has a deletion within a region designated the linker module. This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module. These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously. The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression.

Environmental pH Sensing: Resolving the VirA/VirG Two-Component System Inputs for Agrobacterium Pathogenesis

“…2A. Consistent with previous reports implicating the linker domain's involvement in AS sensing (12,14), the kinase alone (K) displayed AS-independent activity while LKR and LK remained AS-inducible. When the pH was reduced from 7.0 to 5.5, all strains showed an increase of vir expression, but a far greater increase was observed for the full-length VirA strain.…”

“…pRG118, pRG119, and pRG120 were made by replacing the HindIII fragment of pRG100 with HindIII fragments from pAM23 (coding for stop codon after aa 711), pAM28 (coding for wild-type kinase domain and stop codon after aa 711), and pYW45 (coding for wild-type kinase and receiver domains of VirA) (46), respectively. The VirA with the truncated receiver domain (aa 1 to 711) was released from pCH355 (14) as a KpnI fragment and inserted into KpnI-digested pYW15 to make pRG135.…”

“…They concluded that the cytoplasmic linker domain of VirA was responsible for pH input. However, a later report (14) showed that an allele product of VirA, of which the linker domain was truncated, maintaining the periplasmic and kinase domains, still responded to a low-pH stimulus. Other investigators constructed fusion proteins with the periplasmic domain replaced by one of the E. coli membrane chemoreceptors, Tar (30).…”

Distribution and evolution of multiple-step phosphorelay in prokaryotes: lateral domain recruitment involved in the formation of hybrid-type histidine kinases

“…tumefaciens, where the attached receiver domain of VirA, a transmembrane hybrid HK, functions as an autoinhibitory domain. In its unphosphorylated state, this receiver domain interacts with the transmitter module and prevents the transmitter from autophosphorylating and serving as a phosphodonor to its cognate response regulator VirG (Chang et al, 1996;Appleby et al, 1996).…”

Genetic Analysis of the RcsC Sensor Kinase from Escherichia coli K-12

“…Only two other prokaryotic sensors with similar H1-D1 arrangements have been characterized, i.e., the VirA sensor protein of Agrobacterium tumefaciens and the LuxN sensor from Vibrio harveyi (1,3,8). In VirA the D1 domain is not required for signaling and this domain has a regulatory role whereby it appears to inhibit the kinase activity of VirA (4). On the other hand, the D1 domain of LuxN has been shown to play a direct role in the transfer of the phosphoryl group from LuxN to the response regulator LuxO via the HPt-containing protein LuxU (8).…”