Demonstration of 3 functional domains responsible for a kinase activity in VirA, a transmembrane sensory protein encoded by the Ti plasmid of Agrobacterium tumefaciens

High-level expression of chimeric virA genes were obtained by replacing the first codons of virA with the first codons of trpE. The fusion proteins encoded by these constructs were partially purified and one of them exhibited autokinase activity. Therefore, protein phosphorylation may be an important feature of VirA function. This allowed us to define the existence of three functional domains inside VirA.     

Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.     

Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens.

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.     

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

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 [gamma-32P]ATP but not with [gamma-32P]GTP or [alpha-32P]ATP, which suggests an ATP gamma-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.     

Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein.

VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.     

Variable efficiency of a Ti plasmid-encoded VirA protein in different agrobacterial hosts.

The transconjugant CB100, harboring the Ti plasmid from the Agrobacterium tumefaciens biovar 2 strain D10B/87 in the chromosomal background of the biovar 1 strain C58, was defective in vir gene induction. This defect was corrected in the presence of virA from pTiA6. Based on this complementation result and an analysis of the induction requirements of the transconjugant CB100 and its parent strains, it was hypothesized that the defective vir gene induction in CB100 was related to a dysfunctional interaction between the pTi-encoded D10B/87 VirA and the chromosome-encoded C58 ChvE. To verify this hypothesis, D10B/87 and C58 virA were compared, and conclusions from this first set of analyses were then corroborated by comparing D10B/87 and C58 chvE. Whereas only a few nucleotide differences were identified in the promoters and 5' ends of the coding regions of D10B/87 and C58 virA, analysis of hybrid virA genes showed that these differences collectively accounted for the poor vir gene induction of strain CB100. In contrast with the sequence similarity of the VirA proteins, extensive divergence was seen between the chromosome-encoded D10B/87 and C58 ChvE. Although D10B/87 chvE introduced in trans had little effect on vir gene induction of CB100, it enhanced the induction response of a strain CB100 derivative in which the chromosomal C58 chvE had been inactivated by marker exchange. These results suggest that chromosomal backgrounds provided by different strains of A. tumefaciens are not equivalent for VirA function. Following conjugative transfer of certain Ti plasmids to a new agrobacterial host, evolution of the newly introduced virA, or coevolution of chvE and virA, may lead to optimization of ChvE-VirA interaction and vir gene induction levels.     

The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression.     

Shigella deliver an effector protein to trigger host microtubule destabilization,which promotes Rac1 activity and efficient bacterial internalization

Shigella deliver a subset of effectors into the host cell via the type III secretion system, that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells. Here we show that one of the Shigella effectors, called VirA, can interact with tubulin to promote microtubule (MT) destabilization, and elicit protrusions of membrane ruffling. Under in vitro conditions, VirA inhibited polymerization of tubulin and stimulated MT destabilization. Upon microinjection of VirA into HeLa cells, a localized membrane ruffling was induced rapidly. Overexpression of VirA in host cells caused MT destruction and protruding membrane ruffles which were absent when VirA was co-expressed with a dominant-negative Rac1 mutant. Indeed, Shigella but not the virA mutant stimulated Rac1, including the formation of membrane ruffles in infected cells. Importantly, the MT structure beneath the protruding ruffling was destroyed. Furthermore, drug-induced MT growth in HeLa cells greatly enhanced the Shigella entry. These results indicate that VirA is a novel type of bacterial effector capable of inducing membrane ruffling through the stimulation of MT destabilization.     

Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon.

Hydroxylamine-induced mutations in the virA gene of Agrobacterium tumefaciens that do not require the plant phenolic-inducing compound acetosyringone for vir regulon induction were isolated. The isolation was based on the activation of both virB::lacZ and virE::cat fusions by mutant virA loci in the absence of acetosyringone. Three of these virA(Ais) (acetosyringone-independent signaling) mutants were characterized. All three mutants expressed a virB::lacZ fusion at high levels in the absence of acetosyringone. One virA (Ais) mutant, virA112, exhibited vir gene expression in the absence of inducing monosaccharides and acidic growth conditions, both of which are normally required for vir gene induction. The phenotype of the virA112 mutant resulted from a glycine to glutamic acid change near His-474, the site of VirA autophosphorylation.     

Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia coli

The ability to utilize Escherichia coli as a heterologous system in which to study the regulation of Agrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. coli containing a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization of lac promoter-driven virA and virG in combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of the virBp::lacZ fusion, and the level of virBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on vir gene expression was observed only in the presence of the chvE gene, suggesting that the glucose-binding protein of E. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the vir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for the full expression of virulence genes in E. coli.     

Localization of the VirA domain involved in acetosyringone-mediatedvir gene induction inAgrobacterium tumefaciens

The VirA protein ofAgrobacterium tumefaciens is thought to be a receptor for plant phenolic compounds such as acetosyringone. Although it is not known whether the interaction between VirA and the phenolics is direct or requires other phenolic-binding proteins, it is shown in this study that the first 280 amino acids of the VirA protein are not essential for the acetosyringone mediatedvir gene induction response. Considering the fact that the cytoplasmic region between the amino acids 283 and 304 is highly conserved between the different VirA proteins, and that deletion of this region abolishes VirA activity, we suggest that the acetosyringone receptor domain is located in this cytoplasmic domain of the VirA protein.     

Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave alpha-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 A resolution. The shape of the molecule resembles the letter "V," with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.