Autoinhibitory regulation of TrwK, an essential VirB4 ATPase in type IV secretion systems

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS.     

ATPase activity and oligomeric state of TrwK, the VirB4 homologue of the plasmid R388 type IV secretion system

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.     

An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes

The 9.5kb virB operon is the largest of the six major operons in the Ti plasmid vir region. This operon contains eleven genes, the largest of which is virB4. This gene encodes an 84kDa protein whose function has not been identified. Its roles in conferring virulence on Agrobacterium tumefaciens and in the T-DNA transfer process were determined by generating non-polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion. Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis. These mutants were tested for T-DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura. All mutants were agroinfection- and tumorigenicity-negative. These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T-DNA and virulence. Furthermore, by using anti-VirB4 serum, the protein product of virB4 was localized to the inner-membrane fraction of A. tumefaciens. Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti-VirB4 serum. The energy generated by VirB4 ATPase therefore may be used to transfer T-DNA or to assemble the T-DNA transfer apparatus on the bacterial membrane. Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, repectively. These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T-DNA from A. tumefaciens to plant cells.     

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.     

Identification of new protein-protein interactions involving the products of the chromosome- and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. citri

The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.     

Enzymology of type IV macromolecule secretion systems: the conjugative transfer regions of plasmids RP4 and R388 and the cag pathogenicity island of Helicobacter pylori encode structurally and functionally related nucleoside triphosphate hydrolases

Type IV secretion systems direct transport of protein or nucleoprotein complexes across the cell envelopes of prokaryotic donor and eukaryotic or prokaryotic recipient cells. The process is mediated by a membrane-spanning multiprotein assembly. Potential NTPases belonging to the VirB11 family are an essential part of the membrane-spanning complex. Three representatives of these NTPases originating from the conjugative transfer regions of plasmids RP4 (TrbB) and R388 (TrwD) and from the cag pathogenicity island of Helicobacter pylori (HP0525) were overproduced and purified in native form. The proteins display NTPase activity with distinct substrate specificities in vitro. TrbB shows its highest specific hydrolase activity with dATP, and the preferred substrate for HP0525 is ATP. Analysis of defined TrbB mutations altered in motifs conserved within the VirB11 protein family shows that there is a correlation between the loss or reduction of NTPase activity and transfer frequency. Tryptophan fluorescence spectroscopy of TrbB and HP0525 suggests that both interact with phospholipid membranes, changing their conformation. NTPase activity of both proteins was stimulated by the addition of certain phospholipids. According to our results, Virb11-like proteins seem to most likely be involved in the assembly of the membrane-spanning multiprotein complex.     

Topology of the VirB4 C terminus in the Agrobacterium tumefaciens VirB/D4 type IV secretion system

Gram-negative type IV secretion systems (T4SSs) transfer proteins and DNA to eukaryotic and/or prokaryotic recipients resulting in pathogenesis or conjugative DNA transfer. VirB4, one of the most conserved proteins in these systems, has both energetic and structural roles in substrate translocation. We previously predicted a structural model for the large C-terminal domain (residues 425-789) of VirB4 of Agrobacterium tumefaciens. Here we have defined a homology-based structural model for Agrobacterium VirB11. Both VirB4 and VirB11 models predict hexameric oligomers. Yeast two-hybrid interactions define peptides in the C terminus of VirB4 and the N terminus of VirB11 that interact with each other. These interactions were mapped onto the homology models to predict direct interactions between the hexameric interfaces of VirB4 and VirB11 such that the VirB4 C terminus stacks above VirB11 in the periplasm. In support of this, fractionation and Western blotting show that the VirB4 C terminus is localized to the membrane and periplasm rather than the cytoplasm of cells. Additional high resolution yeast two-hybrid results demonstrate interactions between the C terminus of VirB4 and the periplasmic portions of VirB1, VirB8, and VirB10. Genetic studies reveal dominant negative interactions and thus function of the VirB4 C terminus in vivo. The above data are integrated with the existing body of literature to propose a structural, periplasmic role for the C-terminal half of the Agrobacterium VirB4 protein.     

The Hexameric Structure of a Conjugative VirB4 Protein ATPase Provides New Insights for a Functional and Phylogenetic Relationship with DNA Translocases

VirB4 proteins are ATPases essential for pilus biogenesis and protein transport in type IV secretion systems. These proteins contain a motor domain that shares structural similarities with the motor domains of DNA translocases, such as the VirD4/TrwB conjugative coupling proteins and the chromosome segregation pump FtsK. Here, we report the three-dimensional structure of full-length TrwK, the VirB4 homologue in the conjugative plasmid R388, determined by single-particle electron microscopy. The structure consists of a hexameric double ring with a barrel-shaped structure. The C-terminal half of VirB4 proteins shares a striking structural similarity with the DNA translocase TrwB. Docking the atomic coordinates of the crystal structures of TrwB and FtsK into the EM map revealed a better fit for FtsK. Interestingly, we have found that like TrwB, TrwK is able to bind DNA with a higher affinity for G4 quadruplex structures than for single-stranded DNA. Furthermore, TrwK exerts a dominant negative effect on the ATPase activity of TrwB, which reflects an interaction between the two proteins. Our studies provide new insights into the structure-function relationship and the evolution of these DNA and protein translocases.     

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.     

The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein.

The process of T-DNA transfer from Agrobacterium tumefaciens to plant cells is thought to involve passage of a DNA-protein complex through a specialized structure in the bacterial membrane. The virB operon of A. tumefaciens encodes 11 proteins, of which 9 are known to be located in the membranes and 10 have been shown to be essential for virulence. Sequence comparisons between proteins encoded by the virB operon and those encoded by operons from conjugative plasmids indicated that VirB proteins may form a structure similar to a conjugative pilus. Here, we examine the effects of mutations in virB4 on the accumulation and localization of other VirB proteins. VirB4 shares amino acid sequence similarity with the TraC protein of plasmid F, which is essential for pilus formation in Escherichia coli, and with the PtlC protein of Bordetella pertussis, which is required for toxin secretion. Polar and nonpolar virB4 mutants were examined, and all were shown to be unable to accumulate VirB3 protein to wild-type levels. A low level of VirB3 protein which was present in induced NT1RE cells harboring virB4 nonpolar mutant pBM1130 was found to associate with the inner membrane fraction only, whereas in wild-type cells VirB3 associated with both inner and outer membranes. The results indicate that for VirB3 to accumulate in the outer membrane, VirB4 must also be present, and it is possible that one role of VirB4 is in the correct assembly of a VirB protein membrane structure.     

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.     

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.     

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.     

TraG-like proteins of DNA transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane gate for exported substrates?

TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.     

Bacterial secrets of secretion: EuroConference on the biology of type IV secretion processes

Type IV secretion systems (TFSS) mediate secretion or direct cell-to-cell transfer of virulence factors (proteins or protein-DNA complexes) from many Gram-negative animal, human and plant pathogens, such as Agrobacterium tumefaciens, Bartonella tribocorum, Bordetella pertussis, Brucella suis, Helicobacter pylori, Legionella pneumophila and Rickettsia prowazekii, into eukaryotic cells. Bacterial conjugation is also classified as a TFSS-like process mediating the spread of broad-host-range plasmids between Gram-negative bacteria such as RP4 and R388, which carry antibiotic resistance genes. Genetic, biochemical, cell biological and structural biology experiments led to significant progress in the understanding of several aspects of TFSS processes. X-ray crystallography revealed that homologues of the A. tumefaciens inner membrane-associated proteins VirB11 and VirD4 from H. pylori and R388, respectively, may form channels for substrate translocation or assembly of the transmembrane TFSS machinery. Biochemical and cell biological experiments revealed interactions between components of the periplasmic core components VirB8, VirB9 and VirB10, which may form the translocation channel. Analysis of A. tumefaciens virulence proteins VirE2 and VirF suggested that the periplasmic translocation route of the pertussis toxin from B. pertussis may be more generally valid than previously anticipated. Secretion and modification of toxins from H. pylori and L. pneumophila profoundly affect host cell metabolism, thus entering the discipline of cellular microbiology. Finally, results from genome sequencing projects revealed the presence of up to three TFSS in a single organism, and the analysis of their interplay and adaptation to different functions will be a future challenge. TFSS-carrying plasmids were discovered in different ecosystems, suggesting that genetic exchange may speed up their evolution and adaptation to different cell-cell interactions.     

Agrobacterium tumefaciens oncogenic suppressors inhibit T-DNA and VirE2 protein substrate binding to the VirD4 coupling protein

Agrobacterium tumefaciens uses a type IV secretion (T4S) system composed of VirB proteins and VirD4 to deliver oncogenic DNA (T-DNA) and protein substrates to susceptible plant cells during the course of infection. Here, by use of the Transfer DNA ImmunoPrecipitation (TrIP) assay, we present evidence that the mobilizable plasmid RSF1010 (IncQ) follows the same translocation pathway through the VirB/D4 secretion channel as described previously for the T-DNA. The RSF1010 transfer intermediate and the Osa protein of plasmid pSa (IncW), related in sequence to the FiwA fertility inhibition factor of plasmid RP1 (IncPalpha), render A. tumefaciens host cells nearly avirulent. By use of a semi-quantitative TrIP assay, we show that both of these 'oncogenic suppressor factors' inhibit binding of T-DNA to the VirD4 substrate receptor. Both factors also inhibit binding of the VirE2 protein substrate to VirD4, as shown by coimmunoprecipitation and bimolecular fluorescence complementation assays. Osa fused to the green fluorescent protein (GFP) also blocks T-DNA and VirE2 binding to VirD4, and Osa-GFP colocalizes with VirD4 at A. tumefaciens cell poles. RSF1010 and Osa interfere specifically with VirD4 receptor function and not with VirB channel activity, as shown by (i) TrIP and (ii) a genetic screen for effects of the oncogenic suppressors on pCloDF13 translocation through a chimeric secretion channel composed of the pCloDF13-encoded MobB receptor and VirB channel subunits. Our findings establish that a competing plasmid substrate and a plasmid fertility inhibition factor act on a common target, the T4S receptor, to inhibit docking of DNA and protein substrates to the translocation apparatus.     

Functional subsets of the virB type IV transport complex proteins involved in the capacity of Agrobacterium tumefaciens to serve as a recipient in virB-mediated conjugal transfer of plasmid RSF1010

The virB-encoded type IV transport complex of Agrobacterium tumefaciens mediates the transfer of DNA and proteins into plant cells, as well as the conjugal transfer of IncQ plasmids, such as RSF1010, between Agrobacterium strains. While several studies have indicated that there are physical interactions among the 11 VirB proteins, the functional significance of the interactions has been difficult to establish since all of the proteins are required for substrate transfer. Our previous studies, however, indicated that although all of the VirB proteins are required for the capacity of a strain to serve as an RSF1010 donor, only a subset of these proteins in the recipient is necessary to increase the conjugal frequency by 3 to 4 logs. The roles of particular groups of VirB proteins in this increased recipient activity were examined in the study reported here. Examination of the expression of subgroups of virB genes revealed that translation of virB6 is necessary for expression of downstream open reading frames. Expression of limited subsets of the VirB proteins in a recipient strain lacking the Ti plasmid revealed that the VirB7 to VirB10 proteins yield a subcomplex that is functional in the recipient assay but that the VirB1 to VirB4 proteins, as a group, dramatically increase this activity in strains expressing VirB7 to VirB10. Finally, the membrane distribution and cross-linking patterns of VirB10, but not of VirB8 or VirB9, in a strain expressing only VirB7 to VirB10 are significantly altered compared to the patterns of the wild type. These characteristics are, however, restored to the wild-type status by coexpression of VirB1 to VirB3. Taken together, these results define subsets of type IV transport complex proteins that are critical in allowing a strain to participate as a recipient in virB-mediated conjugal RSF1010 transfer.     

The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface.

The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid. VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and 'phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2). Conversely, VirB4::beta-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low beta-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high beta-galactosidase activities. Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K treatment of A. tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of approximately 35 and approximately 45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains.