Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB

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.Bacterial type IV secretion systems are of significant clinical concern. The type IV systems are composed of the well-known conjugation machines that are responsible for the rapid transmission of antibiotic resistance genes throughout bacterial populations under selective pressure (10, 27). Type IV systems also are composed of a more recently described group of secretion machines that mediate the delivery of effector molecules to the cytosols of eukaryotic cells during infection (8, 23). The list of medically important pathogens that utilize type IV systems for interkingdom macromolecular translocation now includes Helicobacter pylori, Bordetella pertussis, Legionella pneumophila, and Brucella and Bartonella species. All t...

Section: Cofractionation Of Virb2 and Virb7 During T-pilus Purificationcontrasting

confidence: 55%

VirB7 Lipoprotein Is Exocellular and Associates with the Agrobacterium tumefaciens T Pilus

Sagulenko

Jakubowski

et al. 2001

“…As shown in Fig. 7A and B, a small amount of the VirE2 secretion substrate was detectable in shockates independently of the VirB/VirD4 T4SS, as previously reported (12,17); even so, levels of released VirE2 were unaffected by VirB4 synthesis. Periplasmic ChvE accumulated at abundant levels in the shockates, and levels were also unaffected by VirB4 synthesis.…”

supporting

confidence: 53%

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Kerr

Christie

2010

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.

“…Although the essential virB operon encodes a pilus (20), termed the T-pilus (29), the notion that the T-pilus is the conduit for the transfer of T-DNA and proteins from the bacterial cytoplasm into host cells in a single step has not been demonstrated. In contrast, recent work in our laboratory suggests a two-step transfer process since the virulence proteins VirE2 and VirF and VirD2 are present in the periplasm of Agrobacterium strains lacking the VirB proteins (12).…”

mentioning

confidence: 45%

“…The virulence assays on Kalanchöe diagremontiana were performed as previously described (12). The plant cell culture infection assays were conducted as previously described (17), except that the plant culture was Ageratum conyzoides (27) and the detection plasmid used to monitor T-DNA transfer was pBISN1 (36).…”

Section: Methodsmentioning

confidence: 99%

A New Type IV Secretion System Promotes Conjugal Transfer inAgrobacterium tumefaciens

Chen

Wood

et al. 2002

Self Cite

Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulenceassociated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10-or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.