Localization of the protein VirB1 involved in contact formation during conjugation among<i>Agrobacterium</i>cells

Chumakov, M. I.; Kurbanova, I. V.

doi:10.1111/j.1574-6968.1998.tb13287.x

FEMS Microbiology Letters

1998

DOI: 10.1111/j.1574-6968.1998.tb13287.x

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Localization of the protein VirB1 involved in contact formation during conjugation amongAgrobacteriumcells

M. I. Chumakov

I. V. Kurbanova

Abstract: 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 … Show more

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Cited by 8 publications

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Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Volokhina¹,

Sazonova²,

Velikov³

et al. 2005

Microbiological Research

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Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.

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Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Volokhina¹,

Sazonova²,

Velikov³

et al. 2005

Microbiological Research

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Kurbanova

Velikov

Chumakov

2001

Antonie Van Leeuwenhoek

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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.

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Colonization of Wheat Root Hairs and Roots by Agrobacteria

et al. 2004

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Localization of the protein VirB1 involved in contact formation during conjugation amongAgrobacteriumcells

Cited by 8 publications

References 16 publications

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens

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Colonization of Wheat Root Hairs and Roots by Agrobacteria

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