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Journal of Bacteriology, June 2001, p. 3642-3651, Vol. 183, No. 12
Department of Microbiology and Molecular
Genetics, The University of Texas-Houston Medical School, Houston,
Texas 77030
Received 18 December 2000/Accepted 25 March 2001
Agrobacterium tumefaciens transfers oncogenic T-DNA and
effector proteins to plant cells via a type IV secretion pathway. This
transfer system, assembled from the products of the virB operon, is thought to consist of a transenvelope mating channel and the
T pilus. When screened for the presence of VirB and VirE proteins,
material sheared from the cell surface of octopine strain A348 was seen
to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer
membrane lipoprotein. Material sheared from the cell surface of most
virB gene deletion mutants also possessed VirB7, but not
VirB2 or VirB5. During purification of the T pilus from wild-type
cells, VirB2, VirB5, and VirB7 cofractionated through successive steps
of gel filtration chromatography and sucrose density gradient
centrifugation. A complex containing VirB2 and VirB7 was precipitated
from a gel filtration fraction enriched for T pilus with both
anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular
forms of VirB7 migrated as disulfide-cross-linked dimers and monomers
when samples were electrophoresed under nonreducing conditions. A
mutant synthesizing VirB7 with a Ser substitution of the lipid-modified
Cys15 residue failed to elaborate the T pilus, whereas a mutant
synthesizing VirB7 with a Ser substitution for the disulfide-reactive
Cys24 residue produced very low levels of T pilus. Together, these
findings establish that the VirB7 lipoprotein localizes exocellularly,
it associates with the T pilus, and both VirB7 lipid modification and
disulfide cross-linking are important for T-pilus assembly.
T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and
a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as
a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution
for the reactive Cys64 residue elaborated T pilus but exhibited an
attenuated virulence phenotype. Dithiothreitol-treated T pilus composed
of native VirB2 pilin and untreated T pilus composed of the VirB2C64S
mutant pilin distributed in sucrose gradients more predominantly in
regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3642-3651.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
VirB7 Lipoprotein Is Exocellular and Associates
with the Agrobacterium tumefaciens T Pilus
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics, The University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030. Phone: (713) 500-5440. Fax: (713) 500-5499. E-mail:
Peter.J.Christie{at}uth.tmc.edu.
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