[Study of extracellular structures of Agrobacterium involved in bacterial and plant interactions]. / Izuchenie vnekletochnykh struktur agrobakterii, uchastvuiushchikh v kontaktakh s bakterial'noi i rastitel'noi kletkami.

Agrobacterial cells produced straight microfibrils not only when in contact with wheat seedling roots, but also when in contact with each other. After 2 h of incubation, agrobacterial cells were found to form aggregates, in which the cells were in contact either directly or through thick straight microfibrils (bridges) of an unknown composition. The majority of the microfibrils were susceptible to attack by cellulase, although some of them showed resistance to this enzyme. Like the wild-type flagellated agrobacteria, their bald mutants produced long straight microfibrils. The cells surface structures of agrobacteria were examined by labeling them immunocytochemically with colloidal gold conjugated antibodies against O-specific lipopolysaccharides, Vir proteins, and cellulase. Agrobacterial cells treated with acetosyringone and brought into contact were found to contain subpolar and polar cell surface structures. Antibodies against the VirB2 protein were able to interact with a tuft of thin microfibrils located on one pole of the agrobacterial cell, whose vir genes were induced by acetosyringone, but were unable to interact with the surface structures of the agrobacterial cells aggregated in liquid medium in the absence of wheat seedlings.

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.

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.

[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.

[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.

Localization of VirB1 protein on the agrobacterial cell surface.

Using colloidal gold-labelled VirB1-specific antibodies, it was found that VirB1 proteins are included into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells.

Localization of the protein VirB1 involved in contact formation during conjugation among Agrobacterium cells.

Supramembrane structures of Agrobacterium, which link cells during mating, were for the first time visualized using transmission electron microscopy. The initial cell contact was found to be mediated by long pili. Using colloidal gold-labeled, VirB1-specific antibodies, it was established that VirB1 proteins enter into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells. Labeling of non-centrifuged agrobacterial cells on a nitrocellulose membrane using colloidal gold-conjugated antibodies to VirB1 showed that the labeled complex could bind to AS-induced cells, but failed to form red stains during incubation with cells of the Ti plasmidless A. tumefaciens strains LBA288 and UBAPF-2.