The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation

Abstract

The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. We overproduced truncated VirA proteins in Escherichia coli by deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [y-32PJATP but not with [y-32PJGTP or [a-32P]ATP, which suggests an ATP -y-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens.Crown gall tumors of plants are induced by Agrobacterium tumefaciens by a process of natural genetic engineering. The bacterium contains a large tumor-inducing plasmid (Ti plasmid) bearing two sets of genes that are vital for tumorigenesis. One set is termed vir (virulence) genes and the other is termed the T-DNA or transferred DNA (for reviews, see references 18 and 37). The vir genes, consisting of seven genetically identified operons,

A Two-component System Mediates Developmental Regulation of Biosynthesis of a Heterocyst Polysaccharide

“…Protein-histidine kinases VirA, ArcB, and EnvZ autophosphorylate in vitro when truncated by removal of transmembrane regions but not when intact (54,55,58). Similarly, we observed phosphorylation in vitro by truncated but not by intact H 6 HepK (Fig.…”

Bacterial community assembly based on functional genes rather than species

“…The high proportion of environmental signal transducers and transcriptional regulators could be indicative of the need to respond to changes in the host environment (e.g., osmotic; see above), to control the complex steps of colonization or biofilm formation and to mediate the interactions with other community members. For example, there are many homologs of COG0642 (histidine kinase), members of which are known to be involved in osmoregulation (49), multidrug export (50), sporulation (51), nitrate reduction (52), cell differentiation (53), and plant virulence (54). Also overrepresented is COG0583 (transcriptional regulator), which contains proteins that regulate transcription in response to plant exudates (55), virulence, motility, and quorum sensing (56).…”

Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG

“…However, Melchers et al have shown that the periplasmic portion is not required for acetosyringonemediated vir gene induction; rather, the two transmembrane sequences may be important (19). The C-terminal half of the VirA protein was shown to possess an autophosphorylating activity in vitro (11,14), and this autophosphorylation is required for its biological activity (14). The phosphorylation site on the VirA protein was identified as a specific histidine residue (14).…”

“…The C-terminal half of the VirA protein was shown to possess an autophosphorylating activity in vitro (11,14), and this autophosphorylation is required for its biological activity (14). The phosphorylation site on the VirA protein was identified as a specific histidine residue (14). The phosphohistidine is a high-energy phosphate bond and usually exists as a transient state (23).…”