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.     

Molecular Basis of ChvE Function in Sugar Binding,Sugar Utilization,and Virulence in Agrobacterium tumefaciens

ChvE is a chromosomally encoded protein in Agrobacterium tumefaciens that mediates a sugar-induced increase in virulence (vir) gene expression through the activities of the VirA/VirG two-component system and has also been suggested to be involved in sugar utilization. The ChvE protein has homology to several bacterial periplasmic sugar-binding proteins, such as the ribose-binding protein and the galactose/glucose-binding protein of Escherichia coli. In this study, we provide direct evidence that ChvE specifically binds the vir gene-inducing sugar d-glucose with high affinity. Furthermore, ChvE mutations resulting in altered vir gene expression phenotypes have been isolated and characterized. Three distinct categories of mutants have been identified. Strains expressing the first class are defective in both virulence and d-glucose utilization as a result of mutations to residues lining the sugar-binding cleft. Strains expressing a second class of mutants are not adversely affected in sugar binding but are defective in virulence, presumably due to impaired interactions with the sensor kinase VirA. A subset of this second class of mutants includes variants of ChvE that also result in defective sugar utilization. We propose that these mutations affect not only interactions with VirA but also interactions with a sugar transport system. Examination of a homology model of ChvE shows that the mutated residues associated with the latter two phenotypes lie in two overlapping solvent-exposed sites adjacent to the sugar-binding cleft where conformational changes associated with the binding of sugar might have a maximal effect on ChvE''s interactions with its distinct protein partners.Virulent strains of Agrobacterium tumefaciens contain the tumor-inducing (Ti) plasmid that carries virulence (vir) operons. Products of vir operons are involved in infecting wound sites of dicotyledonous plants and initiating tumor formation. The expression of vir genes in A. tumefaciens is activated by plant-released signals, namely, phenolic derivatives, acidic pH, and monosaccharides (for a review, see reference 6), via the combined activities of the periplasmic protein ChvE and the VirA/VirG two-component regulatory system. Upon perception of these plant signals, autophosphorylated VirA, a transmembrane histidine kinase, transfers a phosphoryl group to VirG, a response regulator, and then the phosphorylated VirG activates the expression of vir genes by binding vir boxes in their promoters (8, 19, 24, 31, 52).Perception and transduction of the sugar signals is crucial to the virulence of A. tumefaciens: strains lacking ChvE, a chromosomally encoded putative sugar-binding protein, are significantly less virulent than wild-type strains (17, 18). Previous studies have shown that, in fact, sugar signaling is neither sufficient for nor absolutely required for vir gene expression. Rather, sugars vastly increase both the sensitivity of VirA to phenol derivatives, such as acetosyringone (AS), and the maximal levels of vir gene expression observed at saturating levels of such compounds (for a review, see reference 26). The periplasmic domain of VirA is required for transduction of the sugar and pH signals (7, 8, 16, 41), whereas the so-called “linker” domain, located in the cytoplasm between the second transmembrane domain and the kinase domain, is required for perception and transduction of the phenolic signals (8, 46, 47).A working model for the ChvE/sugar/VirA signaling pathway suggests that monosaccharide-bound ChvE interacts with the periplasmic domain of VirA to relieve periplasmic repression, resulting in maximal sensitivity of VirA to phenolic signals (7, 11, 32, 41). However, limited evidence has been presented to reveal how ChvE recognizes monosaccharides and how it interacts with the periplasmic domain of VirA. Shimoda et al. (41) identified a mutant chvE allele [chvE(T211M)] that is able to suppress a sugar-insensitive virA allele [virA(E210V)], thereby restoring the sugar-sensing ability. The suppressing effect of chvE(T211M) was then proposed to be the result of the specific restoration of the capacity of VirA^E210V to bind ChvE^T211M. However, ChvE^T211M also activated wild-type VirA in the absence of sugars (32), suggesting that this mutant may not be a site-specific suppressor of VirA^E210V. Based on a homology model of ChvE, a recent study (16) does predict, though, that the residue T211 is located on the surface of the ChvE protein, consistent with the model that T211 is in a position to interact with the periplasmic domain of VirA.Based on sequence similarity, ChvE is a member of the periplasmic sugar-binding protein (PSBP) family. The structures of some PSBPs, including two ChvE homologues in Escherichia coli, ribose-binding protein (RBP) and glucose/galactose-binding protein (GBP), have been solved. The family of PSBPs shares very similar structural features, and each of them contains two similar but distinct globular domains connected by a flexible hinge (38). A sugar-binding site is located at the cleft between the two domains. PSBPs play an important role in active sugar transport, and some of them also serve as an initial receptor for sugar chemotaxis (45). A wealth of evidence has demonstrated that some specialized regions located on the surfaces of PSBPs are important for transport and chemotactic functions. In the case of RBP, four distinct regions spanning the N-terminal and C-terminal domains are involved in interaction with its permease (a transport partner), its chemotransducer (a chemotactic partner), or both (5, 15). In GBP, one residue was identified as being specifically involved in chemotaxis but not transport (36, 49). For maltose-binding protein (MBP), which is also a member of the PSBP family, two well-defined regions located on each domain of the protein are involved in interaction with its chemotransducer (54). These regions partially overlap with the regions involved in interaction with its permease (25, 54). Structural analysis indicates that both domains of MBP have direct interactions with its transport partners (35).ChvE also appears to be a highly versatile protein: not only does it play an important role in virulence, but as in the case of the PSPBs described above, it has been indicated to be a primary receptor for transport of and chemotaxis toward some sugars (7). This raises important biological/biochemical questions. How can ChvE interact with three presumably different periplasmic components of systems that are respectively involved in virulence, sugar utilization, and chemotaxis? How are the interactions of ChvE with these periplasmic components structurally segregated: do the interactions occur on the same or different regions of ChvE? To address these issues, we employed genetic and biophysical approaches to identify the residues of ChvE involved in sugar utilization versus the residues involved in virulence. The residues of both groups were mapped onto a homology model of ChvE based on a high-resolution crystal structure of E. coli GBP (PDB ID, 2ipn). Our results identify an extended surface spanning both the N-terminal and C-terminal domains of ChvE that is essential for interacting with VirA and that partially overlaps the surface responsible for the interaction of ChvE with a putative ABC sugar transport protein.     

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.     

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.     

Integration of rotation and piston motions in coiled-coil signal transduction

A coordinated response to a complex and dynamic environment requires an organism to simultaneously monitor and interpret multiple signaling cues. In bacteria and some eukaryotes, environmental responses depend on the histidine autokinases (HKs). For example, VirA, a large integral membrane HK from Agrobacterium tumefaciens, regulates the expression of virulence genes in response to signals from multiple molecular classes (phenol, pH, and sugar). The ability of this pathogen to perceive inputs from different known host signals within a single protein receptor provides an opportunity to understand the mechanisms of signal integration. Here we exploited the conserved domain organization of the HKs and engineered chimeric kinases to explore the signaling mechanisms of phenol sensing and pH/sugar integration. Our data implicate a piston-assisted rotation of coiled coils for integration of multiple inputs and regulation of critical responses during pathogenesis.     

Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site.     

The chimeric VirA-tar receptor protein is locked into a highly responsive state.

The wild-type VirA protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE protein (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA 87:6708-6712, 1990, and N. Shimoda, A. Toyoda-Yamamoto, J. Nagamine, S. Usami, M. Katayama, Y. Sakagami, and Y. Machida, Proc. Natl. Acad. Sci. USA 87:6684-6688, 1990). It is shown here that the mutant VirA(Ser-44, Arg-45) protein and the chimeric VirA-Tar protein are no longer responsive to sugars and the ChvE protein. However, whereas the chimeric VirA-Tar protein was found to be locked in a highly responsive state, the VirA(Ser-44, Arg-45) mutant protein appeared to be locked in a low responsive state. This difference turned out to be important for tumorigenicity of the host strains in virulence assays on Kalanchoë daigremontiana.     

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.     

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

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 (delta324-413) 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.     

The genetics of phytochrome signalling in Arabidopsis

The application of Arabidopsis genetics to research into the responses of plants to light has enabled rapid recent advances in this field. The plant photoreceptor phytochrome mediates well-defined responses that can be exploited to provide elegant and specific genetic screens. By this means, not only have mutants affecting the phytochromes themselves been isolated, but also mutants affecting the transduction of phytochrome signals. The genes involved in these processes have now begun to be characterized by using this genetic approach to isolate signal transduction components. Most of the components characterized so far are capable of being translocated to the cell nucleus, and they may help to define a new system of regulation of gene expression. This review summarises the ongoing contribution made by genetics to our understanding of light perception and signal transduction by the phytochrome system.     

VirA of Agrobacterium tumefaciens is an intradimer transphosphorylase and can actively block vir gene expression in the absence of phenolic signals

The VirA-VirG two-component system regulates the 30-gene vir regulon in response to host-released chemical signals. VirA is a homodimeric membrane-spanning histidine protein kinase. Here, we show that mutations in two essential VirA residues, His-474 and Gly-657, can be complemented by the formation of mixed heterodimers, indicating that each subunit of a VirA dimer transphosphorylates the opposite subunit. VirA contains a receiver domain that inhibits kinase activity. We use the forced heterodimer system to show that the two receiver domains of a VirA dimer act independently and that each inhibits the phosphoacceptor subdomain of the opposite subunit. We also demonstrate that merodiploid strains co-expressing constitutive VirA mutants and wild-type VirA show levels of vir gene expression far lower than haploid strains expressing just the constitutive alleles. The fact that wild-type VirA can actively block vir gene expression in the absence of phenolic signals suggests that it might have a phospho-VirG phosphatase activity. The receiver domain of VirA is essential for this activity, whereas residues H474 and G657 of the kinase domain are not required. Merodiploid strains co-expressing a constitutive VirA allele and an allele that is kinase inactive but proficient in the inhibitory activity show strongly inducible vir gene expression, indicating that the inhibitory activity is modulated by environmental signals.