INTRODUCTION

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Agrobacterium tumefaciens, the causative agent of crown gall disease, induces tumors on most dicotyledonous plants. During infection, A. tumefaciens transfers DNA in a DNA-protein complex (T complex) into plant cells (reviewed in references 12, 18, 58, 60, and 62). The complex comprises a single-stranded copy (T strand) of a segment (T-DNA) of the tumor-inducing plasmid (Ti), as well as the Agrobacterium proteins VirD2 and VirE2. A single molecule of VirD2 is covalently attached to the 5' end of the T strand (32, 41). The single-strand binding protein VirE2 coats the length of the T strand (14, 29, 46), although whether binding of VirE2 occurs in the bacterium (13) or in the plant cell (8, 49) is currently unresolved. After import into the plant cell nucleus (15, 33, 52, 61), the T strand becomes integrated into a plant chromosome (43, 53). The gene products that mediate T-strand production and transfer, as well as provide structural components of the T complex, are encoded in the vir region, a nontransferred segment of the Ti plasmid. Five complementation groups, virA, virB, virD, virE, and virG, are essential for DNA transfer (reviewed in reference 58). In the plant cell, gene products of the transferred DNA promote the unregulated production of plant growth regulators that induce increased rates of cell division in the transformed cells, resulting in neoplastic growth. The T-DNA also encodes enzymes for the biosynthesis of sugar derivatives called opines. As the infecting strain also carries genes for opine metabolism on the Ti plasmid, these compounds can be specifically utilized as a carbon source (reviewed in references 20 and 31). Thus, A. tumefaciens genetically manipulates plant cells to produce a unique habitat that it is specifically equipped to exploit.

Export of the T complex is thought to be mediated by a multimeric, transmembrane apparatus assembled from 11 VirB proteins and VirD4. This apparatus belongs to a growing family of transporters called type IV secretion systems (9). Type IV systems constitute the transfer machinery of many broad-host-range (BHR) and narrow-host-range conjugal plasmids (e.g., IncF, IncW, IncP, and IncN) (12, 37, 57, 62). In addition, the ptl operon of Bordetella pertussis encodes nine proteins required for the secretion of pertussis toxin that are homologous to VirB proteins and the transfer proteins of plasmid conjugation systems (56). Homologs of VirB4, VirB7, VirB9, VirB10, VirB11, and VirD4 are implicated in transfer of a factor from Helicobacter pylori that induces secretion of interleukin-8 by epithelial cells (16), while pathogenicity of Legionella pneumophila requires homologs of VirB10 and VirB11 (54). Recently, homologs of virB4, virB8, virB9, virB10, virB11, and virD4 were identified in the genome of Rickettsia prowazekii, but the functions of their products are unknown (2). Finally, an operon in Brucella suis that is required for virulence contains homologs of all 11 virB genes (16, 40). These homologies suggest that type IV secretion systems share a common ancestor and that the secretion or transfer of substrates as diverse as single-stranded-DNA-protein complexes and proteinaceous pathogenesis factors involves common mechanisms.

Little is known about the assembly of the VirB DNA transfer apparatus or the molecular details of its operation. VirB2 to VirB11 and VirD4 are all required for virulence (7). The apparatus has a pilus (27) and may form a transmembrane channel for cell-to-cell trafficking of the T complex (12). It is unknown whether these two structures are coupled physically or functionally.

The major structural component of the pilus is VirB2 (36). VirB5 also cofractionates with VirB2 through pilus purification and may be a minor pilus component (45). VirB3 and VirB4 are homologous to TraC and TraE, respectively, which are accessory pilus proteins in the IncF system required for pilus assembly but are not structural components (34). Evidence to date for the Agrobacterium protein VirB4, however, suggests that it is a structural component of the transmembrane channel (17).

The remainder of the VirB proteins form the putative transmembrane channel (12). The core of the apparatus likely is composed of VirB7-VirB9 heterodimers that are linked by a disulfide bridge and anchored in the outer membrane by lipid modification of VirB7 (1, 4, 23, 24, 47). The VirB7-B9 heterodimer interacts, either directly or indirectly, with VirB10 (6) and is genetically required for the stability of VirB4, VirB8, VirB10, and VirB11 (24). Coordinate overexpression of VirB9, VirB10, and VirB11 relieves the dominant negative phenotype of specific VirB11 mutations, suggesting that these proteins may be required in stoichiometric amounts for the assembly of VirB transporters (59). VirB6 is firmly embedded in the inner membrane with five transmembrane regions, and its presence is required for the stability of several VirB proteins (S. Hapfelmeier, N. Domke, P. C. Zambryski, and C. Baron, unpublished data). Thus, VirB6 was suggested to form a pore in the inner membrane (12) and may anchor the VirB transfer apparatus to the inner membrane. VirB8 localizes to the inner membrane (50, 51), but a specific role in transporter assembly has not been assigned to it. Finally, VirD4, by analogy to its homologs in conjugal type IV systems, which are termed "coupling" proteins (10), may mediate delivery of the T complex to the VirB transfer apparatus.

The first product of the VirB operon, VirB1, is not absolutely required for virulence, although its deletion reduces DNA transfer 100- to 1,000-fold depending on the assay (7, 25). Sequence similarity between the N terminus of VirB1 and chicken egg white lysozyme as well as lytic transglycosylases (21, 39) suggests that it may provide local lysis of the peptidoglycan cell wall to create channels large enough for assembly of the transporter. Regions of lysozyme homology are present in proteins from many type II, III, and IV secretion systems (5, 39, 48), which suggests a broad requirement for this activity in the assembly of membrane-spanning complexes. Mutations of putative active-site residues within the region of lysozyme homology of VirB1 reduce virulence (39). Furthermore, low cellular levels of VirB4 and VirB11 (7) and the lack of T pili (26, 45) observed in virB1 deletion strains suggest that transporter assembly across an intact bacterial cell wall is inefficient or unstable. A second role for VirB1 was suggested by the observation that the C-terminal third of the protein, VirB1*, is secreted and loosely associated with the exterior of Agrobacterium cells (3). Chemical cross-linking and coimmunoprecipitation demonstrated an association between VirB1* and VirB9 (3). It has not been determined whether VirB1* has a postsecretion function.

In the present study, we further characterized the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1*. Complementation of virB1 deletion strains with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a virB1 deletion strain. Thus, each domain has a unique role in tumorigenesis. While processing and secretion of VirB1* occur in the absence of other vir functions, they do require signal peptide-mediated export into the periplasm. Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized between the inner and outer membranes and are not subject to vir regulation.

	MATERIALS AND METHODS

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Strains and growth conditions. Two common laboratory A. tumefaciens strains, C58 and A348, and their derivatives were used. C58 carries the nopaline Ti plasmid pTiC58. A348 (28) was produced by introducing the octopine Ti plasmid pTiA6NC into A136 (C58 cured of its Ti plasmid [C58NT1] and then screened for resistance to rifampin and nalidixic acid [A136]). C58 and A348 strains with in-frame deletions of virB1 are CB1001 (45) and A348B1 (7), respectively.

Construction of vectors for expression of wild-type VirB1 and VirB1 mutants. All procedures for plasmid DNA isolation and manipulations, such as digestion with restriction endonucleases or ligation, were as described by Sambrook et al. (44). Diagrams of VirB1 and derivative proteins are shown in Fig. 1.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   virB1 and virB1 deletion constructs in pBP2N, a BHR vector with the nopaline virB promoter. pSW213:virB1 is also a BHR vector that contains virB1 under the control of the IPTG-inducible lactose promoter. N, coding sequences from nopaline virB1; O, coding sequences from octopine virB1; I, II, and III, the three conserved motifs in the superfamily of glycosidases that includes VirB1 and chicken egg white lysozyme (39); AA, amino acid; SP, signal peptide.

Tumor assays. Virulence assays were performed on Kalanchoe diagremontiana. One-centimeter-long wound sites, created by carefully scratching the surface of a leaf with a toothpick, were inoculated with 10⁹ CFU of the strains described above. Virulence was assayed by tumor size and time course of tumor development. The virulence of each strain carrying different constructs was assayed at least 10 times in independent experiments. Photographs were taken 6 to 7 weeks after inoculation.

Protein analysis. Preparation of the cell lysates and supernatant fraction by precipitation with trichloroacetic acid as well as analysis of VirB1 products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting was performed as previously described (3).

	RESULTS

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Complementation with N- and C-terminal domains. Deletion of virB1 severely attenuates but does not abolish virulence (7). In the present experiments with K. diagremontiana as a host, a strain of A. tumefaciens (A348B1) carrying octopine pTiA6 with an in-frame deletion of virB1 only rarely formed a small tumor (Table 2 and Fig. 2A). In contrast, virB1 deletion from nopaline pTiC58 in CB1001 had only a slight effect on virulence regardless of whether the host was K. diagremontiana or Nicotiana tabacum (data not shown). pTiC58 may be inherently more tumorigenic than pTiA6NC, and so the deletion of virB1 has a smaller effect on virulence. Alternatively, other pTi factors, most likely non-vir, play a role in determining the requirement for VirB1 during tumorigenesis.

View larger version (80K): [in this window] [in a new window]   FIG. 2.   Complementation of virB1 deletion strain A348B1 assessed by testing virulence on K. diagremontiana. (A) Complementation with full-length VirB1, either octopine (pMTX124) or nopaline (pMTX106), restored tumorigenicity completely. (B) Complementation of virB1 deletion strain A348B1 by constructs expressing either the VirB1 region of lysozyme homology (pMTX110) or VirB1* (pMTX107) partially restored tumorigenicity. Wound sites are labeled with the strain used for inoculation. Plasmids and encoded VirB1 fragments (Fig. 1) are indicated below the strain names.

Processing and secretion of VirB1*. As both domains of VirB1 have some function in tumorigenesis, the processing and secretion events that produce VirB1* were further studied to characterize the requirements for these reactions.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Processing of VirB1 and secretion of VirB1*. Samples from vir-induced cultures of the indicated strains were taken 18 to 20 h after induction. Cell lysates (A) and supernatants (B) were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore Corp.), followed by detection with VirB1-specific antiserum. pMTX, the plasmid used for complementation; VirB1, the VirB1 fragment expressed (Fig. 1); WT, wild type. The arrow indicates the position of VirB1; the arrowheads indicate the positions of VirB1*. Numbers to the left are molecular mass markers, in kilodaltons.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Synthesis and secretion of VirB1 are vir independent. Expression of VirB1 was regulated by the plac promoter in CB1001 (pSW213:virB1). Numbers above lanes indicate that samples from IPTG-induced cultures were taken 0, 3, 5, and 18 h after induction. CB1001 carrying vector alone (pSW213) was the negative control. Endogenous vir genes were not acetosyringone induced. Supernatants were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride membranes, followed by detection with VirB1-specific antiserum.

View larger version (42K): [in this window] [in a new window]   FIG. 5.   The signal peptide is required for processing and secretion of VirB1*. CB1001, a virB1 deletion strain of C58, was transformed with pMTX128, in which the coding sequence for the signal peptide was deleted. Samples from vir-induced cultures were taken 18 to 20 h after induction. Cell lysates (A) and supernatants (B) were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride membranes, followed by detection with VirB1-specific antiserum. pMTX, the plasmid used for trans-complementation; VirB, the VirB1 fragment expressed by that plasmid (Fig. 1); WT, wild type. The arrow indicates the position of VirB1; the arrowhead indicates VirB1*; the circle indicates VirB1SP. Numbers are molecular mass markers, in kilodaltons. In panel B, the supernatant fraction from CB1001(pMTX128) was applied at two concentrations: equivalent to that of other lanes (1×) and 10 times that of other lanes (10×).

Complementation requires the signal peptide. The clone for expression of VirB1SP (pMTX128) was introduced into A348B1 and did not restore this strain to virulence (Fig. 6). Thus, complementation of the virB1 deletion requires the sec-dependent export of VirB1 into the periplasm. The partial restoration of virulence to A348B1 by expression of VirB1* (pMTX107) (Fig. 3B) was not observed when the VirB1* was expressed without a signal peptide (pMTX129) (Fig. 6). Thus, formation of smaller tumors, induced by expression of VirB1* in A348B1, also requires sec-dependent export of VirB1* to the periplasm. This suggests that VirB1 and VirB1* function in the periplasm or at the exterior of the cell during DNA transfer.

View larger version (77K): [in this window] [in a new window]   FIG. 6.   The signal peptide is required for full complementation of the virB1 deletion by VirB1 or partial complementation by VirB1*. Complementation of A348B1 with pMTX128 (VirB1SP) and pMTX129 (VirB1*SP) were assessed by testing virulence on K. diagremontiana. Wound sites are labeled with the strain used for inoculation. Plasmids and encoded VirB1 fragments (Fig. 1) are indicated below the strain names.

	DISCUSSION

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Here evidence is provided that nopaline VirB1 has two distinct functions that enhance tumorigenesis. The ability of either octopine or nopaline virB1 coding sequences to complement the deletion of virB1 from an octopine Ti plasmid and to restore virulence (Fig. 2A) suggests that these proteins provide identical functions. The lack of effect on tumorigenesis of deleting virB1 from the nopaline pTiC58 may reflect either the greater tumorigenicity of pTiC58 or the presence of a cryptic pTiC58 protein(s) that is functionally redundant. For example, B. suis has a virB-like operon that encodes homologs of all VirB proteins, including VirB1 (40), but also has a homolog corresponding to lytic transglycosylases, located 5' upstream of the virB-like operon (D. O'Callaghan, personal communication).

Each VirB1 domain's performance of a unique function derives from the ability of the N- and C-terminal domains to restore partial virulence to A348B1 (Fig. 2B). The formation of tumors intermediate in size between those incited by A348B1 and A348B1(pMTX106) expressing full-length VirB1 suggests that the functions are distinct and necessary for complete complementation. The dependence of virulence on the signal peptide of VirB1 (Fig. 6) suggests that both VirB1 and VirB1* act in the periplasm or the outer membrane or outside the cell.

The role of the VirB1 N-terminal domain, suggested by its homology to lysozyme, has been discussed extensively (3, 12, 21, 39). Assembly of a membrane-spanning, multimeric complex requires penetration of the bacterial cell wall. The size of a complex composed of even a few VirB proteins would prohibit its insertion through naturally occurring channels in the peptidoglycan layer, which permit diffusion of molecules up to ca. 50 kDa (21). The N terminus of VirB1 is predicted to hydrolyze the murein layer to provide channels large enough to accommodate assembly of the VirB transporter (3, 39). The hypothesis that VirB1 provides hydrolytic activity is supported by specific mutagenesis of putative active-site residues (39). These mutants partially restore the ability to incite tumors when used to complement a virB1 deletion strain (39). The partial restoration may result from functions provided by VirB1*, which is not affected by mutations at the putative active site for polysaccharide hydrolysis.

The function provided by nopaline VirB1* is unknown. VirB1* is most likely not a component of the pilus (45). By virtue of its secretion, however, it would be in the proper location to play an early, transient role mediating pilus formation, e.g., providing chaperone activity for VirB2. Alternatively, it may modify the surface of the Agrobacterium cell during transporter assembly as a prerequisite for attachment. Association of VirB1* with VirB9 is consistent with such a role (3). Finally, the loose association of VirB1* with the exterior of Agrobacterium suggests that it may be available to interact with the site of attachment on the plant cell surface.

Processing of nopaline VirB1 to VirB1* and subsequent secretion of VirB1* to the exterior of the cell are independent events (Fig. 3) critical to the promotion of tumorigenesis by VirB1, as shown by the partial restoration of virulence (Fig. 2B). The synthesis of VirB1* from derivatives of VirB1 with deletions in the lysozyme-homologous region (Fig. 3B) suggests that full-length VirB1 is not required for VirB1* processing. The wild-type context at the signal peptidase I site and the VirB1* processing site, however, does enhance the efficiency of processing and secretion (Fig. 3B, lanes 4 and 5). Neither processing nor secretion requires any factors encoded in the vir region (Fig. 4). The specific factors involved, however, are not known. The processing and secretion that follow IPTG-induced expression of VirB1 (Fig. 4) suggest two possibilities: either VirB1 is autocatalytic for both functions or these activities are constitutively expressed.

Many extracellular virulence factors are translated as preproteins with domains that act as intramolecular chaperones (38, 42, 55). These domains assist in folding of the mature protein in the periplasm and are proteolytically removed prior to translocation across the outer membrane. Proteolysis of some intramolecular chaperones is autocatalytic. By analogy, the C terminus of VirB1 may potentiate enzymatic activity of the N terminus by ensuring proper folding after sec-dependent secretion into the periplasm. The conditions in the periplasm might then induce the autocatalytic liberation of VirB1*. If processing and secretion are not functions of VirB1 itself, they may be provided by constitutively expressed bacterial proteins. This would require a protease localized to the periplasm that has not yet been identified. In addition, a secretion system is needed. As VirB1* is delivered into the periplasm by the general secretory pathway, a type II secretion system may transport VirB1* to the exterior of the cell. This would be analogous to secretion of elastase by Pseudomonas aeruginosa (38). Elastase is exported into the periplasm by the general secretory pathway, where an intramolecular domain, which serves as a chaperone, is cleaved autoproteolytically but remains associated with the elastase. Secretion of the elastase across the outer membrane is mediated by the Xcp apparatus, a type II secretion apparatus required for pathogenicity (38).

Until recently, non-vir functions for pathogenesis were primarily associated with attachment of Agrobacterium to plant cells. This list, however, is expanding. In the final step of maturation, the T-pilin VirB2 is cyclized by the formation of an intramolecular peptide bond between the N and C termini (22). Cyclization does not require any products encoded by the Ti plasmid other than VirB2 (22). The formation of VirB7-VirB7 or VirB7-VirB9 dimers may require specific chaperones or Dsb (disulfide bond formation)-like enzymes that are not encoded within the vir region (4, 47). During infection of a plant, VirB1 is transported into the periplasm by the general secretory pathway and is processed to generate VirB1*, which is secreted to the exterior of the cell. The VirB1-VirB1* processing and secretion events, which also do not require any functions encoded by the vir region of the Ti plasmid, rely on factors encoded outside the vir region or on the bacterial chromosome.