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.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens.

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.

The Ctp type IVb pilus locus of Agrobacterium tumefaciens directs formation of the common pili and contributes to reversible surface attachment.

Agrobacterium tumefaciens can adhere to plant tissues and abiotic surfaces and forms biofilms. Cell surface appendages called pili play an important role in adhesion and biofilm formation in diverse bacterial systems. The A. tumefaciens C58 genome sequence revealed the presence of the ctpABCDEFGHI genes (cluster of type IV pili; Atu0216 to Atu0224), homologous to tad-type pilus systems from several bacteria, including Aggregatibacter actinomycetemcomitans and Caulobacter crescentus. These systems fall into the type IVb pilus group, which can function in bacterial adhesion. Transmission electron microscopy of A. tumefaciens revealed the presence of filaments, significantly thinner than flagella and often bundled, associated with cell surfaces and shed into the external milieu. In-frame deletion mutations of all of the ctp genes, with the exception of ctpF, resulted in nonpiliated derivatives. Mutations in ctpA (a pilin homologue), ctpB, and ctpG decreased early attachment and biofilm formation. The adherence of the ctpA mutant could be restored by ectopic expression of the paralogous pilA gene. The ΔctpA ΔpilA double pilin mutant displayed a diminished biovolume and lower biofilm height than the wild type under flowing conditions. Surprisingly, however, the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants formed normal biofilms and showed enhanced reversible attachment. In-frame deletion of the ctpA pilin gene in the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants caused the same attachment-deficient phenotype as the ctpA single mutant. Collectively, these findings indicate that the ctp locus is involved in pilus assembly and that nonpiliated mutants, which retain the CtpA pilin, are proficient in attachment and adherence.

Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells.

The genetic transformation of plant cells by Agrobacterium tumefaciens results from the transfer of DNA and proteins via a specific virulence (vir) -induced type IV secretion system (T4SS). To better understand T4SS function, we analyzed the localization of its structural components and substrates by deconvolution fluorescence microscopy. GFP fusions to T4SS proteins with cytoplasmic tails, VirB8 and VirD4, or cytoplasmic T4SS substrate proteins, VirD2, VirE2, and VirF, localize in a helical pattern of fluorescent foci around the perimeter of the bacterial cell. All fusion proteins were expressed at native levels of vir induction. Importantly, most fusion proteins are functional and do not exhibit dominant-negative effects on DNA transfer to plant cells. Further, GFP-VirB8 complements a virB8 deletion strain. We also detect native VirB8 localization as a helical array of foci by immunofluorescence microscopy. T4SS foci likely use an existing helical scaffold during their assembly. Indeed, the bacterial cytoskeletal component MinD colocalizes with GFP-VirB8. Helical arrays of foci are found at all times investigated between 12 and 48 h post vir induction at 19 degrees C. These data lead to a model with multiple T4SSs around the bacterial cell that likely facilitate host cell attachment and DNA transfer. In support, we find multiple T pili around vir-induced bacterial cells.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Pilus assembly by Agrobacterium T-DNA transfer genes.

Agrobacterium tumefaciens can genetically transform eukaryotic cells. In many bacteria, pili are required for interbacterial DNA transfer. The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature. A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly. The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.

The type IV secretion system component VirB5 binds to the trans-zeatin biosynthetic enzyme Tzs and enables its translocation to the cell surface of Agrobacterium tumefaciens.

VirB5 is a minor component of the extracellular T pilus determined by the Agrobacterium tumefaciens type IV secretion system. To identify proteins that interact with VirB5 during the pilus assembly process, we purified VirB5 as a recombinant fusion protein and, by using a gel overlay assay, we detected a 26-kDa interacting protein in Agrobacterium cell lysates. The VirB5-binding protein was purified from A. tumefaciens and identified as the cytokinin biosynthetic enzyme Tzs. The VirB5-Tzs interaction was confirmed using pulldown assays with purified proteins and the yeast two-hybrid system. An analysis of the subcellular localization in A. tumefaciens showed that Tzs was present in the soluble as well as the membrane fraction. Tzs was extracted from the membranes with the mild detergent dodecyl-beta-D-maltoside in complexes of different molecular masses, and this association was strongly reduced in the absence of VirB5. Using immunoelectron microscopy, we also detected Tzs on the Agrobacterium cell surface. A functional type IV secretion system was required for efficient translocation to the surface, but Tzs was not secreted into the cell supernatant. The fact that Tzs localizes on the cell surface suggests that it may contribute to the interaction of Agrobacterium with plants.

Atomic force microscopy measurements of bacterial adhesion and biofilm formation onto clay-sized particles.

Bacterial adhesion onto mineral surfaces and subsequent biofilm formation play key roles in aggregate stability, mineral weathering, and the fate of contaminants in soils. However, the mechanisms of bacteria-mineral interactions are not fully understood. Atomic force microscopy (AFM) was used to determine the adhesion forces between bacteria and goethite in water and to gain insight into the nanoscale surface morphology of the bacteria-mineral aggregates and biofilms formed on clay-sized minerals. This study yields direct evidence of a range of different association mechanisms between bacteria and minerals. All strains studied adhered predominantly to the edge surfaces of kaolinite rather than to the basal surfaces. Bacteria rarely formed aggregates with montmorillonite, but were more tightly adsorbed onto goethite surfaces. This study reports the first measured interaction force between bacteria and a clay surface, and the approach curves exhibited jump-in events with attractive forces of 97 ± 34 pN between E. coli and goethite. Bond strengthening between them occurred within 4 s to the maximum adhesion forces and energies of -3.0 ± 0.4 nN and -330 ± 43 aJ (10(-18) J), respectively. Under the conditions studied, bacteria tended to form more extensive biofilms on minerals under low rather than high nutrient conditions.

[Formation of extracellular structures in conjugated cultures of agrobacteria]. / Formirovanie vnekletochnykh struktur v kon''iugiruiushchikh kul'turakh agrobakterii.

Electron microscopy of noncentrifugated agrobacterial cells on a nitrocellulose membrane labeled with colloid gold-conjugated antibodies to VirB1 showed that the labeled complex bound to acetosyringone (AS)-induced cells but failed to form red-colored stains during incubation with Ti aplasmid cells. Supramembrane structures of AS-treated A. tumefaciens cells were for the first time visualized by transmission electron microscopy. Colloid gold labeling of VirB2-specific antibodies showed that VirB2 proteins produce long thin pilus structures emerging at the poles of AS-induced agrobacterial cells but never on the surface untreated with AS and Ti-plasmid-free agrobacterial cells. As a rule, one (or rarely two) thread-like connections and bridges were observed between the cells at the primary contact stage. The bridges were not destroyed by SDS, did not react with VirB2-specific antibodies, and remained visible at 30 degrees C. Visible close contacts between mating bacteria did not cease after SDS treatment. SDS pretreatment of donor cells or a mating cell suspension significantly modified the efficiency of pTd33 plasmid transfer from donor to recipient agrobacterial cells. In the presence of AS the optimal temperature for transfer was 25 degrees C. The frequency of plasmid pTd33 transfer from A. tumefaciens via vir-dependent pathway decreased 2-4-fold due to increase of temperature from 19.25 to 31 degrees C.

[Formation of TRA-dependent surface structures in Agrobacterium tumefaciens and their absence in R1 (deltatraR) mutant]. / Obrazovanie TRA-zavisimykh poverkhnostnykh struktur u Agrobacterium tumefaciens i ikh otsutstvie u mutanta R1 (deltatraR).

Agrobacteria have Ti plasmid DNA delivering systems for the transfer to recipient cells by the conjugation mechanism. This transfer is absolutely dependent on induction tra genes. It is not clear which tra-dependent surface (extracellular) proteins (structures) are involved in the transport mechanism and whether these proteins also play a role in the contact formation. SDS-PAGE electrophoresis of proteins released from the cell showed disappearance of 63 and 67 kD proteins in R1(delta traR) strain, which were found in the growth medium and triton extract from the outer membrane of Ti plasmid-harboring A. tumefaciens R10 strains. The traR defective mutant did not express these proteins and had a higher hemagglutination and flocculation capacity than the wild strain. On the other hand, the wild strain showed D-galactose and N-acetyl-galactosamine specific hemagglutination which was not shown by traR mutant. Motility and chemotactic behavior of traR mutant in semisolid medium were defective. As a rule, one (or rarely two) thread-like connections in vir(-) and tra(+) conditions were observed on the agrobacterial cell surface. SDS pretreatment of agrobacterial cells had a significant effect on the expression of tra-dependent surface structures.

[Colonization of wheat root hairs and roots by agrobacteria]. / Kolonizatsiia agrobakteriiami kornevikh voloskov i kornei pshenitsy.

Formation of extracellular structures in pure culture and in interaction with wheat root surface was studied by scanning and transmission electron microscopy. The effect of various factors (growth temperature as well as pretreatment of agrobacteria with kalanchoe extract, acetosyringone, and centrifugation) on formation of extracellular structures was tested. The data on Agrobacterium tumefaciens (wild strain C58 and mutants LBA2525 (virB2::lacZ) and LBA288 (without Ti plasmid)) adhesion to wheat root surface and root hairs after pretreatment of agrobacteria with inducer of virulence genes (vir) acetosyringone were obtained. Formation of agrobacterial cell aggregates on wheat root hair tips was demonstrated. The proportion of root hairs with agrobacterial aggregates on the root hair tip insignificantly changed after pretreatment with acetosyringone but considerably increased after treatment of A. tumefaciens C58 and LBA2525 with kalanchoe leaf extract. The most active colonization of root hairs and formation of agrobacterial aggregates on hair root tips was observed at 22 degrees C. The capacity of agrobacteria for adhesion on monocotyledon surface could be changed by pretreatment of bacteria with various surface-active substances. Bacterial cells subjected to centrifugation had a decreased capacity for attachment to both wheat root surface and root hairs. The relationship between the capacity for adhesion and pile production in agrobacteria was considered.

Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.

Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli.

Variable internal flexibility characterizes the helical capsid formed by agrobacterium VirE2 protein on single-stranded DNA.

Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity.

Identification of Beijerinckia fluminensis strains CIP 106281T and UQM 1685T as Rhizobium radiobacter strains, and proposal of Beijerinckia doebereinerae sp. nov. to accommodate Beijerinckia fluminensis LMG 2819.

During the course of a research project with free-living, nitrogen-fixing bacteria, we determined the 16S rRNA gene sequence of Beijerinckia fluminensis strains UQM 1685T and CIP 106281T and discovered that they were only 90.6-91.2% similar to the sequences of strains of other Beijerinckia species and subspecies. Moreover, the highest similarity to these sequences (99.7%) corresponded to strains of Rhizobium radiobacter (including Agrobacterium tumefaciens). Other diagnostic features confirmed that the two strains have the same origin but do not descend from the nomenclatural type. At the same time, B. fluminensis LMG 2819 was characterized and it was found that its properties also do not agree with the original description of the species, although it can be considered a member of the genus. Further characterization, including chemotaxonomic and other phenotypic traits, allows us to propose (i) the identification of B. fluminensis strains CIP 106281T and UQM 1685T as strains of Rhizobium radiobacter and (ii) the designation of strain LMG 2819T (=CECT 7311T) as the type strain of a novel species, Beijerinckia doebereinerae sp. nov.

Role of vitronectin-like protein in Agrobacterium attachment and transformation of Arabidopsis cells.

The role of plant vitronectin-like protein (Vn) in Agrobacterium-host plant interactions and receptor-specific bacterial attachment is unclear and still open to debate. Using a well-established Agrobacterium-mediated Arabidopsis transformation system, the marker gene beta-glucuronidase (GUS) of Escherichia coli, and biochemical and cytological methods, such as ELISA tests, immunoblots, immunolocalization, and functional in vitro binding assays, we have reassessed the role of Vn in receptor-specific bacterial attachment and transformation. We provide evidence that Vn is present in the host plant cells and anti-human vitronectin antibody cross-reacts with a 65-kDa protein from Arabidopsis cells. The specificity of the immunological cross-reactivity of anti-vitronectin antibodies was further demonstrated by ELISA competition experiments. Immunogold labeling showed that Vn is localized in the plant cell wall, and its level increased considerably after phytohormone treatment of the petiole explants. However, Agrobacterium attachment was unaffected, and no inhibition of petiole cell transformation was detected in the presence of human vitronectin and anti-vitronectin antibodies in the media. Additionally, no correlation between the occurrence of Vn, attachment of bacteria to the cells, and susceptibility to Agrobacterium-mediated transformation was observed. Taken together, our data do not support a functional role of plant Vn as the receptor for site-specific Agrobacterium attachment leading to the transformation of Arabidopsis cells.

[Conjugative transfer of pTd33-plasmid between strains of Agrobacterium]. / Izuchenie kon''iugativnogo perenosa pTd33-plazmidy mezhdu shtammami agrobacterii.

Supramembrane structures that connect conjugating agrobacterial cells were visualized for the first time by transmission electron microscopy. The primary contact of cells during conjugation was shown to occur through the formation of long pili containing no VirB1 protein. Pretreatment of agrobacterial cells with acetosyringone resulted in a six- to tenfold increase in the transfer frequency of the plasmid pTd33 at 19-25 degrees C and had almost no effect at 30 degrees C. The transfer of the plasmid pTd33 from A. tumefaciens strain GV3101 to plasmid-free A. tumefaciens strain UBAPF-2 was 16 times decreased after the centrifugation of cells. The transfer efficiency of the plasmid pTd33 from A. tumefaciens strain LBA2525 (virB2::lacZ) to plasmid-free A. tumefaciens strain UBAPF-2 was one order of magnitude lower than the transfer from the wild-type A. tumefaciens strain GV3101. Treatment of donor cells with 0.01% SDS before mating decreased the transfer efficiency by a factor of 26. The role of pili in the establishment of contact between conjugating cells of agrobacteria is discussed.

Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens.

Previous studies have implicated the obligatory requirement for the vir regulon (or "virulon") of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19 degreesC as compared with 28 degreesC, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107-1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the "T pilus" of A. tumefaciens.

Expression of virB2 protein-containing structures on Agrobacterium in mating cultures.

Exocellular structures containing VirB2 proteins were, for the first time, localized on the surface of Agrobacterium by transmission electron microscopy. Using colloidal gold (CG)-labeled VirB2-specific antibodies, it was shown that VirB2 proteins enter into the composition of short surface pili, which emerge at the poles of acetosyringone (AS)-inducedAgrobacterium cells. However, cells of the Ti plasmidless A. tumefaciens strain UBAPF-2 and cells not induced with AS were incapable of pilus synthesis. In suspension, mating Agrobacterium cells were connected together by short thick bridges. It was found that these bridges did not include as part of their structure CG-labeled VirB1 and VirB2 proteins. We did not find the tetracycline-resistant transconjugants after mating of A. tumefaciens donor cells harboring binary systems with plasmid-free A. tumefaciens GM-I9023 in vir-induced and vir-uninduced conditions. However, the same strains can transfer pSUP106 plasmid via a vir-dependent way. We found that activated vir genes slightly stimulate pTd33 plasmid transfer via a tra-dependent pathway to plasmid-free strain UBAPF-2. It seems, that vir-induced T-DNA/plasmid DNA transfer machinery is not essential for the conjugation process between agrobacterial cells but may participate in this process.

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.

Formation of extracellular structures containing VirB2 proteins in mating cultures of agrobacteria.

Using transmission electron immunomicroscopy, VirB2 protein has been revealed at the surface of acetosyringon-treated A. tumefaciens cells. VirB2 was seen within long flexible and short structures localized at the opposite poles of the cells. These structures were not observed in cells not treated with acetosyringon and in agrobacterial cells treated with this reagent but carrying no Ti-plasmid. Labeled complexes [antibodies to virB2 protein + (A protein + colloidal gold)] bound to pili at a certain periodicity.