Self-assembly of the Agrobacterium tumefaciens VirB11 traffic ATPase

The Agrobacterium tumefaciens VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells. In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo. A chimeric protein composed of VirB11 fused to the DNA binding domain of lambda cI repressor protein formed dimers, as shown by immunity of Escherichia coli to lambda superinfection. An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type A. tumefaciens cells. Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP. In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized A. tumefaciens cell extracts. VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5' and 3' halves of virB11. Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (Delta GKT174-176). By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein. Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein. Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers.     

Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA-DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.     

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.     

Identification of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systems

Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili.     

Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter

The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to lambda phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His-VirB4 in A. tumefaciens, indicating that ATP binding is not required for self-association. A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4'312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.     

Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells.

The 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells. The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecular-weight complexes after treatment with a chemical cross-linker. Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex. VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins. Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence. Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced. We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking.     

An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.     

An essential virulence protein of Agrobacterium tumefaciens,VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.     

The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer

Agrobacterium tumefaciens is a plant pathogen that utilizes a type IV secretion system (T4SS) to transfer DNA and effector proteins into host cells. In this study we discovered that an α-crystallin type small heat-shock protein (α-Hsp), HspL, is a molecular chaperone for VirB8, a T4SS assembly factor. HspL is a typical α-Hsp capable of protecting the heat-labile model substrate citrate synthase from thermal aggregation. It forms oligomers in a concentration-dependent manner in vitro. Biochemical fractionation revealed that HspL is mainly localized in the inner membrane and formed large complexes with certain VirB protein subassemblies. Protein-protein interaction studies indicated that HspL interacts with VirB8, a bitopic integral inner membrane protein that is essential for T4SS assembly. Most importantly, HspL is able to prevent the aggregation of VirB8 fused with glutathione S-transferase in vitro, suggesting that it plays a role as VirB8 chaperone. The chaperone activity of two HspL variants with amino acid substitutions (F98A and G118A) for both citrate synthase and glutathione S-transferase-VirB8 was reduced and correlated with HspL functions in T4SS-mediated DNA transfer and virulence. This study directly links in vitro and in vivo functions of an α-Hsp and reveals a novel α-Hsp function in T4SS stability and bacterial virulence.     

Interactions of the Integrase Protein of the Conjugative Transposon Tn916 with Its Specific DNA Binding Sites

The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn916 Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.     

Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T-DNA transfer proteins, we constructed a transposon, Tn 3phoA . The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest. For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis. A plasmid containing virB and virD4 was used as a target for mutagenesis. Twenty-eight unique phoA -positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm. Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic domain. No insertions mapped to VirD4 either. To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N-terminus, and most of the protein lies in the cytoplasm. A hypothetical model for the T-DNA transport pore is presented.     

Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore

Agrobacterium tumefaciens transforms plants by transferring DNA to the plant cell nucleus. The VirB membrane proteins are postulated to form a pore for the transport of the DNA across the bacterial membranes. Immunofluorescence and immunoelectron microscopy were used to study the transport pore complex. Three likely components of the transport pore, VirB8, VirB9 and VirB10, localized primarily to the inner membrane, outer membrane and periplasm respectively. A significant amount of VirB10 was also found associated with the outer membrane. When expressed alone VirB9 and VirB10 were randomly distributed along the cell membrane. Subcellular location of both proteins changed dramatically in the presence of the other VirB proteins. Both proteins localized to fewer sites and most of the gold particles representing protein molecules were found in clusters suggesting that the two proteins are in a protein complex. VirB8, on the other hand, localized to clusters even in the absence of the other VirB proteins. To investigate the role of VirB8 in the formation of VirB9 and VirB10 protein complexes, we studied the effect of deletion of virB8 on the subcellular location of VirB9 and VirB10. In a virB8 deletion mutant both proteins were distributed randomly on the cell membrane indicating that VirB8 is essential for complex assembly.     

The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.     

Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion

Bacteria use type IV secretion systems (T4SS) to translocate DNA (T-DNA) and protein substrates across the cell envelope. By transfer DNA immunoprecipitation (TrIP), we recently showed that T-DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9. Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis. In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP-dependent mechanism. By co-immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10. We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co-synthesis of previously identified 'core' components of the VirB/D4 T4SS. Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway.     

The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus for the transfer of DNA and proteins to plant cells. To study the role of the VirB6 protein in the assembly and function of the type IV apparatus, we determined its subcellular location by immunofluorescence microscopy. In wild-type bacteria VirB6 localized to the cell poles but in the absence of the tumour-inducing plasmid it localized to random sites on the cell membranes. Five of the 11 VirB proteins, VirB7-VirB11, are required for the polar localization of VirB6. We identified two regions of VirB6, a conserved tryptophan residue at position 197 and the extreme C-terminus, that are essential for its polar localization. Topology determination by PhoA fusion analysis placed both regions in the cell cytoplasm. Alteration of tryptophan 197 or the deletion of the extreme C-terminus led to the mislocalization of the mutant protein. The mutations abolished the DNA transfer function of the protein as well. The C-terminus of VirB6, in silico, can form an amphipathic helix that may encode a protein-protein interaction domain essential for targeting the protein to a cell pole. We previously reported that another DNA transfer protein, VirD4, localizes to a cell pole. To determine whether VirB6 and VirD4 localize to the same pole, we performed colocalization experiments. Both proteins localized to the same pole indicating that VirB6 and VirD4 are in close proximity and VirB6 is probably a component of the transport apparatus.