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1
den Hartigh, Andreas B., Hortensia G. Rolán, Maarten F. de Jong, and Renée M. Tsolis. "VirB3 to VirB6 and VirB8 to VirB11, but Not VirB7, Are Essential for Mediating Persistence of Brucella in the Reticuloendothelial System." Journal of Bacteriology 190, no. 13 (2008): 4427–36. http://dx.doi.org/10.1128/jb.00406-08.
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ABSTRACT The Brucella abortus virB locus contains 12 open reading frames, termed virB1 through virB12, which encode a type IV secretion system. Polar mutations in the virB locus markedly reduce the ability of B. abortus to survive in cultured macrophages or to persist in organs of mice. While a nonpolar deletion of the virB2 gene reduces survival in cultured macrophages and in organs of mice, a nonpolar deletion of virB1 only reduces survival in macrophages, whereas virB12 is dispensable for either virulence trait. Here we investigated the role of the remaining genes in the virB locus during survival in macrophages and virulence in mice. Mutants carrying nonpolar deletions of the virB3, virB4, virB5, virB6, virB7, virB8, virB9, virB10, or virB11 gene were constructed and characterized. All mutations reduced the ability of B. abortus to survive in J774A.1 mouse macrophage-like cells to a degree similar to that caused by a deletion of the entire virB locus. Deletion of virB3, virB4, virB5, virB6, virB8, virB9, virB10, or virB11 markedly reduced the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection. Interestingly, deletion of virB7 did not reduce the ability of B. abortus to persist in spleens of mice. We conclude that virB2, virB3, virB4, virB5, virB6, virB8, virB9, virB10, and virB11 are essential for virulence of B. abortus in mice, while functions encoded by the virB1, virB7, and virB12 genes are not required for persistence in organs with this animal model.
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Baron, Christian, Natalie Domke, Michael Beinhofer, and Siegfried Hapfelmeier. "Elevated Temperature Differentially Affects Virulence, VirB Protein Accumulation, and T-Pilus Formation in Different Agrobacterium tumefaciens andAgrobacterium vitis Strains." Journal of Bacteriology 183, no. 23 (2001): 6852–61. http://dx.doi.org/10.1128/jb.183.23.6852-6861.2001.
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ABSTRACT That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20°C to growth-inhibitory 37°C. Incubation at 28°C but not at 26°C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20°C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28°C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26°C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20°C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37°C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28°C. Levels of many virulence proteins were strongly reduced at 28°C compared to 20°C, and T-pilus assembly did not occur in all strains except “temperature-resistant” Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.
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Hapfelmeier, Siegfried, Natalie Domke, Patricia C. Zambryski, and Christian Baron. "VirB6 Is Required for Stabilization of VirB5 and VirB3 and Formation of VirB7 Homodimers in Agrobacterium tumefaciens." Journal of Bacteriology 182, no. 16 (2000): 4505–11. http://dx.doi.org/10.1128/jb.182.16.4505-4511.2000.
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ABSTRACT VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression ofvirB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.
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Judd, Paul K., David Mahli, and Anath Das. "Molecular characterization of the Agrobacterium tumefaciens DNA transfer protein VirB6." Microbiology 151, no. 11 (2005): 3483–92. http://dx.doi.org/10.1099/mic.0.28337-0.
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The VirB proteins of Agrobacterium tumefaciens assemble a T-pilus and a type IV secretion (T4S) apparatus for the transfer of DNA and proteins to plant cells. VirB6 is essential for DNA transfer and is a polytopic integral membrane protein with at least four membrane-spanning domains. VirB6 is postulated to function in T-pilus biogenesis and to be a component of the T4S apparatus. To identify amino acids required for VirB6 function, random mutations were introduced into virB6, and mutants that failed to complement a deletion in virB6 in tumour formation assays were isolated. Twenty-one non-functional mutants were identified, eleven of which had a point mutation that led to a substitution in a single amino acid. Characterization of the mutants indicated that the N-terminal large periplasmic domain and the transmembrane domain TM3 are required for VirB6 function. TM3 has an unusual sequence feature in that it is rich in bulky hydrophobic amino acids. This feature is found conserved in the VirB6 family of proteins. Studies on the effect of VirB6 on other VirB proteins showed that the octopine Ti-plasmid VirB6, unlike its nopaline Ti-plasmid counterpart, does not affect accumulation of VirB3 and VirB5, but has a strong negative effect on the accumulation of the VirB7-VirB7 dimer. Using indirect immunofluorescence microscopy the authors recently demonstrated that VirB6 localizes to a cell pole in a VirB-dependent manner. Mutations identified in the present study did not affect polar localization of the protein or the formation of the VirB7-VirB7 dimer. A VirB6-GFP fusion that contained the entire VirB6 ORF did not localize to a cell pole in either the presence or the absence of the other VirB proteins. IMF studies using dual labelling demonstrated that VirB6 colocalizes with VirB3 and VirB9, and not with VirB4, VirB5 and VirB11. These results support the conclusion that VirB6 is a structural component of the T4S apparatus.
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Mossey, Pamela, Andrew Hudacek, and Anath Das. "Agrobacterium tumefaciens Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization." Journal of Bacteriology 192, no. 11 (2010): 2830–38. http://dx.doi.org/10.1128/jb.01331-09.
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ABSTRACT Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.
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Cheng, Zhihui, Xueqi Wang, and Yasuko Rikihisa. "Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein." Journal of Bacteriology 190, no. 6 (2008): 2096–105. http://dx.doi.org/10.1128/jb.01813-07.
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ABSTRACT The type IV secretion (T4S) system is critical for the virulence of several pathogens. In the rickettsial pathogen Ehrlichia chaffeensis, the virBD genes are split into two operons, the virB3-virB6 (preceded by sodB) and virB8-virD4 operons. Between these two operons, there are duplications of virB4, virB8, and virB9. In this study we found that transcription of all five loci was downregulated prior to the release of E. chaffeensis from host THP-1 cells and was upregulated at the initiation of exponential growth. Electrophoretic mobility shift assays revealed an E. chaffeensis-encoded protein that specifically bound to the promoter regions upstream of the virBD loci. The protein was purified from the bacterial lysate by affinity chromatography using a biotinylated promoter region upstream of sodB. Mass spectrometry identified the protein as an E. chaffeensis 12.3-kDa hypothetical protein, which was designated EcxR. Recombinant EcxR bound to the promoter regions upstream of five individual virBD loci. EcxR also activated transcription of all five virBD loci in lacZ reporter constructs. The expression of ecxR was positively autoregulated by EcxR. These results suggest that the five virBD loci are coordinately regulated by EcxR to allow developmental stage-specific expression of the T4S system in E. chaffeensis.
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Liu, Zhenying, and Andrew N. Binns. "Functional Subsets of the VirB Type IV Transport Complex Proteins Involved in the Capacity of Agrobacterium tumefaciens To Serve as a Recipient in virB-Mediated Conjugal Transfer of Plasmid RSF1010." Journal of Bacteriology 185, no. 11 (2003): 3259–69. http://dx.doi.org/10.1128/jb.185.11.3259-3269.2003.
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ABSTRACT The virB-encoded type IV transport complex of Agrobacterium tumefaciens mediates the transfer of DNA and proteins into plant cells, as well as the conjugal transfer of IncQ plasmids, such as RSF1010, between Agrobacterium strains. While several studies have indicated that there are physical interactions among the 11 VirB proteins, the functional significance of the interactions has been difficult to establish since all of the proteins are required for substrate transfer. Our previous studies, however, indicated that although all of the VirB proteins are required for the capacity of a strain to serve as an RSF1010 donor, only a subset of these proteins in the recipient is necessary to increase the conjugal frequency by 3 to 4 logs. The roles of particular groups of VirB proteins in this increased recipient activity were examined in the study reported here. Examination of the expression of subgroups of virB genes revealed that translation of virB6 is necessary for expression of downstream open reading frames. Expression of limited subsets of the VirB proteins in a recipient strain lacking the Ti plasmid revealed that the VirB7 to VirB10 proteins yield a subcomplex that is functional in the recipient assay but that the VirB1 to VirB4 proteins, as a group, dramatically increase this activity in strains expressing VirB7 to VirB10. Finally, the membrane distribution and cross-linking patterns of VirB10, but not of VirB8 or VirB9, in a strain expressing only VirB7 to VirB10 are significantly altered compared to the patterns of the wild type. These characteristics are, however, restored to the wild-type status by coexpression of VirB1 to VirB3. Taken together, these results define subsets of type IV transport complex proteins that are critical in allowing a strain to participate as a recipient in virB-mediated conjugal RSF1010 transfer.
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Morse, Kaitlyn, Junzo Norimine, Guy H. Palmer, Eric L. Sutten, Timothy V. Baszler, and Wendy C. Brown. "Association and Evidence for Linked Recognition of Type IV Secretion System Proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale." Infection and Immunity 80, no. 1 (2011): 215–27. http://dx.doi.org/10.1128/iai.05798-11.
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ABSTRACTLike several other bacterial pathogens,Anaplasma marginalehas an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen.A. marginaleT4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine againstA. marginale.
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Rancès, Edwige, Denis Voronin, Van Tran-Van, and Patrick Mavingui. "Genetic and Functional Characterization of the Type IV Secretion System in Wolbachia." Journal of Bacteriology 190, no. 14 (2008): 5020–30. http://dx.doi.org/10.1128/jb.00377-08.
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ABSTRACT A type IV secretion system (T4SS) is used by many symbiotic and pathogenic intracellular bacteria for the successful infection of and survival, proliferation, and persistence within hosts. In this study, the presence and function of the T4SS in Wolbachia strains were investigated by a combination of genetic screening and immunofluorescence microscopy. Two operons of virB-virD4 loci were found in the genome of Wolbachia pipientis strain wAtab3, from the Hymenoptera Asobara tabida, and strain wRi, infecting Drosophila simulans. One operon consisted of five vir genes (virB8, virB9, virB10, virB11, and virD4) and the downstream wspB locus. The other operon was composed of three genes (virB3, virB4, and virB6) and included four additional open reading frames (orf1 to orf4) orientated in the same direction. In cell culture and insect hosts infected with different Wolbachia strains, the bona fide vir genes were polycistronically transcribed, together with the downstream adjacent loci, notably, as virB8 to virD4 and wspB and as virB3, virB4, virB6, and orf1 to orf4. Two peptides encompassing conserved C and N termini of the Wolbachia VirB6 protein were used for the production of polyclonal antibodies. Anti-VirB6 antibodies could detect the corresponding recombinant protein by chemifluorescence on Western blots of total proteins from Escherichia coli transformants and Wolbachia strains cultured in cell lines. Using immunofluorescence microscopy, we further demonstrated that the VirB6 protein was produced by Wolbachia strains in ovaries of insects harboring wAtab3 or wRi and cell lines infected with wAlbB or wMelPop. As VirB6 is known to associate with other VirB proteins to form a membrane-spanning structure, this finding suggests that a T4SS may function in Wolbachia.
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Jakubowski, Simon J., Eric Cascales, Vidhya Krishnamoorthy, and Peter J. Christie. "Agrobacterium tumefaciens VirB9, an Outer-Membrane-Associated Component of a Type IV Secretion System, Regulates Substrate Selection and T-Pilus Biogenesis." Journal of Bacteriology 187, no. 10 (2005): 3486–95. http://dx.doi.org/10.1128/jb.187.10.3486-3495.2005.
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ABSTRACT Agrobacterium tumefaciens translocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra+), completely transfer defective (Tra−), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the VirB2 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outer-membrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra+ strains failed to elaborate T pilus at detectable levels (Pil−), and three such Tra+ Pil− mutant strains were rendered Tra− upon deletion of virB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis.
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Jakubowski, Simon J., Vidhya Krishnamoorthy, and Peter J. Christie. "Agrobacterium tumefaciens VirB6 Protein Participates in Formation of VirB7 and VirB9 Complexes Required for Type IV Secretion." Journal of Bacteriology 185, no. 9 (2003): 2867–78. http://dx.doi.org/10.1128/jb.185.9.2867-2878.2003.
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ABSTRACT This study characterized the contribution of Agrobacterium tumefaciens VirB6, a polytopic inner membrane protein, to the formation of outer membrane VirB7 lipoprotein and VirB9 protein multimers required for type IV secretion. VirB7 assembles as a disulfide cross-linked homodimer that associates with the T pilus and a VirB7-VirB9 heterodimer that stabilizes other VirB proteins during biogenesis of the secretion machine. Two presumptive VirB protein complexes, composed of VirB6, VirB7, and VirB9 and of VirB7, VirB9, and VirB10, were isolated by immunoprecipitation or glutathione S-transferase pulldown assays from detergent-solubilized membrane extracts of wild-type A348 and a strain producing only VirB6 through VirB10 among the VirB proteins. To examine the biological importance of VirB6 complex formation for type IV secretion, we monitored the effects of nonstoichiometric VirB6 production and the synthesis of VirB6 derivatives with 4-residue insertions (VirB6.i4) on VirB7 and VirB9 multimerization, T-pilus assembly, and substrate transfer. A virB6 gene deletion mutant accumulated VirB7 dimers at diminished steady-state levels, whereas complementation with a plasmid bearing wild-type virB6 partially restored accumulation of the dimers. VirB6 overproduction was correlated with formation of higher-order VirB9 complexes or aggregates and also blocked substrate transfer without a detectable disruption of T-pilus production; these phenotypes were displayed by cells grown at 28°C, a temperature that favors VirB protein turnover, but not by cells grown at 20°C. Strains producing several VirB6.i4 mutant proteins assembled novel VirB7 and VirB9 complexes detectable by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two strains producing the D60.i4 and L191.i4 mutant proteins translocated IncQ plasmid and VirE2 effector protein substrates in the absence of a detectable T pilus. Our findings support a model that VirB6 mediates formation of VirB7 and VirB9 complexes required for biogenesis of the T pilus and the secretion channel.
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Schmidt-Eisenlohr, Heike, Natalie Domke, Christina Angerer, Gerhard Wanner, Patricia C. Zambryski, and Christian Baron. "Vir Proteins Stabilize VirB5 and Mediate Its Association with the T Pilus of Agrobacterium tumefaciens." Journal of Bacteriology 181, no. 24 (1999): 7485–92. http://dx.doi.org/10.1128/jb.181.24.7485-7492.1999.
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ABSTRACT Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711–2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genesvirB1, virB2, virB5, andvirB6 blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.
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Höppner, Christoph, Anna Carle, Durga Sivanesan, Sabine Hoeppner, and Christian Baron. "The putative lytic transglycosylase VirB1 from Brucella suis interacts with the type IV secretion system core components VirB8, VirB9 and VirB11." Microbiology 151, no. 11 (2005): 3469–82. http://dx.doi.org/10.1099/mic.0.28326-0.
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VirB1-like proteins are believed to act as lytic transglycosylases, which facilitate the assembly of type IV secretion systems via localized lysis of the peptidoglycan. This paper presents the biochemical analysis of interactions of purified Brucella suis VirB1 with core components of the type IV secretion system. Genes encoding VirB1, VirB8, VirB9, VirB10 and VirB11 were cloned into expression vectors; the affinity-tagged proteins were purified from Escherichia coli, and analyses by gel filtration chromatography showed that they form monomers or homo-multimers. Analysis of protein–protein interactions by affinity precipitation revealed that VirB1 bound to VirB9 and VirB11. The results of bicistron expression experiments followed by gel filtration further supported the VirB1–VirB9 interaction. Peptide array mapping identified regions of VirB1 that interact with VirB8, VirB9 and VirB11 and underscored the importance of the C-terminus, especially for the VirB1–VirB9 interaction. The binding sites were localized on a structure model of VirB1, suggesting that different portions of VirB1 may interact with other VirB proteins during assembly of the type IV secretion machinery.
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Das, Anath, and Yong-Hong Xie. "The Agrobacterium T-DNA Transport Pore Proteins VirB8, VirB9, and VirB10 Interact with One Another." Journal of Bacteriology 182, no. 3 (2000): 758–63. http://dx.doi.org/10.1128/jb.182.3.758-763.2000.
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ABSTRACT The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9. All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer. We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function. Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9. The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.
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Ohashi, Norio, Ning Zhi, Quan Lin, and Yasuko Rikihisa. "Characterization and Transcriptional Analysis of Gene Clusters for a Type IV Secretion Machinery in Human Granulocytic and Monocytic Ehrlichiosis Agents." Infection and Immunity 70, no. 4 (2002): 2128–38. http://dx.doi.org/10.1128/iai.70.4.2128-2138.2002.
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ABSTRACT Anaplasma (Ehrlichia) phagocytophila and Ehrlichia chaffeensis, the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight virB and virD genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight virB and virD genes were clustered at two separate loci in each genome. Five of the genes (virB8, virB9, virB10, virB11, and virD4) were located downstream from a ribA gene. These five genes in both A. phagocytophila and E. chaffeensis were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the virB8 gene in human leukemia cell lines. The virB9 gene of A. phagocytophila was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes (virB3, virB4, and virB6) of both A. phagocytophila and E. chaffeensis were arranged downstream from a sodB gene and cotranscribed with the sodB gene through one or more sodB promoters in human leukocytes. This suggests that transcription of the three virB genes in these two Anaplasma and Ehrlichia spp. is regulated by factors that influence the sodB gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of Anaplasma and Ehrlichia spp. in granulocytes or monocytes.
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Sagulenko, Vitaliya, Evgeniy Sagulenko, Simon Jakubowski, Elena Spudich, and Peter J. Christie. "VirB7 Lipoprotein Is Exocellular and Associates with the Agrobacterium tumefaciens T Pilus." Journal of Bacteriology 183, no. 12 (2001): 3642–51. http://dx.doi.org/10.1128/jb.183.12.3642-3651.2001.
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ABSTRACT 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 virBoperon, 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 mostvirB 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.
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Zupan, John, Cheryl A. Hackworth, Julieta Aguilar, Doyle Ward, and Patricia Zambryski. "VirB1* Promotes T-Pilus Formation in the vir-Type IV Secretion System of Agrobacterium tumefaciens." Journal of Bacteriology 189, no. 18 (2007): 6551–63. http://dx.doi.org/10.1128/jb.00480-07.
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ABSTRACT The vir-type IV secretion system of Agrobacterium is assembled from 12 proteins encoded by the virB operon and virD4. VirB1 is one of the least-studied proteins encoded by the virB operon. Its N terminus is a lytic transglycosylase. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell, suggesting an additional function. We show that both nopaline and octopine strains produce abundant amounts of VirB1* and perform detailed studies on nopaline VirB1*. Both domains are required for wild-type virulence. We show here that the nopaline type VirB1* is essential for the formation of the T pilus, a subassembly of the vir-T4SS composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit). A nopaline virB1 deletion strain does not produce T pili. Complementation with full-length VirB1 or C-terminal VirB1*, but not the N-terminal lytic transglycosylase domain, restores T pili containing VirB2 and VirB5. T-pilus preparations also contain extracellular VirB1*. Protein-protein interactions between VirB1* and VirB2 and VirB5 were detected in the yeast two-hybrid assay. We propose that VirB1 is a bifunctional protein required for virT4SS assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the T4SS. The C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.
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Karnholz, Arno, Claudia Hoefler, Stefan Odenbreit, Wolfgang Fischer, Dirk Hofreuter, and Rainer Haas. "Functional and Topological Characterization of Novel Components of the comB DNA Transformation Competence System in Helicobacter pylori." Journal of Bacteriology 188, no. 3 (2006): 882–93. http://dx.doi.org/10.1128/jb.188.3.882-893.2006.
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ABSTRACT Helicobacter pylori is one of the most diverse bacterial species known. A rational basis for this genetic variation may be provided by its natural competence for genetic transformation and high-frequency recombination. Many bacterial competence systems have homology with proteins that are involved in the assembly of type IV pili and type II secretion systems. In H. pylori, DNA uptake relies on a transport system related to type IV secretion systems (T4SS) designated the comB system. The prototype of a T4SS in Agrobacterium tumefaciens consists of 11 VirB proteins and VirD4, which form the core unit necessary for the delivery of single proteins or large nucleoprotein complexes into target cells. In the past we identified proteins ComB4 and ComB7 through ComB10 as being involved in the process of DNA uptake in H. pylori. In this study we identified and functionally characterized further (T4SS-homologous) components of the comB transformation competence system. By combining computer prediction modeling, experimental topology determination, generation of knockout strains, and genetic complementation studies we identified ComB2, ComB3, and ComB6 as essential components of the transformation apparatus, structurally and functionally homologous to VirB2, VirB3, and VirB6, respectively. comB2, comB3, and comB4 are organized as a separate operon. Thus, for the H. pylori comB system, all T4SS core components have been identified except for homologues to VirB1, VirD4, VirB5, and VirB11.
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den Hartigh, Andreas B., Yao-Hui Sun, David Sondervan, et al. "Differential Requirements for VirB1 and VirB2 during Brucella abortus Infection." Infection and Immunity 72, no. 9 (2004): 5143–49. http://dx.doi.org/10.1128/iai.72.9.5143-5149.2004.
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ABSTRACT The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.
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Yuan, Qing, Anna Carle, Chan Gao, et al. "Identification of the VirB4-VirB8-VirB5-VirB2 Pilus Assembly Sequence of Type IV Secretion Systems." Journal of Biological Chemistry 280, no. 28 (2005): 26349–59. http://dx.doi.org/10.1074/jbc.m502347200.
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Martínez-Núñez, Carola, Pamela Altamirano-Silva, Francisco Alvarado-Guillén, Edgardo Moreno, Caterina Guzmán-Verri, and Esteban Chaves-Olarte. "The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB in Brucella abortus." Journal of Bacteriology 192, no. 21 (2010): 5603–8. http://dx.doi.org/10.1128/jb.00567-10.
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ABSTRACT The pathogenesis of Brucella is related to the ability to multiply intracellularly, an event controlled by the two-component system BvrR/BvrS (TCS BvrRS) and the type IV secretion machinery VirB (T4SS VirB). We have hypothesized that the TCS BvrRS transcriptionally regulates the T4SS VirB. To test this hypothesis, we have compared the levels of VirB proteins in the wild-type strain Brucella abortus 2308 and mutant strains devoid of the sensor and regulator genes (bvrS and bvrR mutants, respectively). While the bvrR and bvrS mutants showed low levels of the VirB1, VirB5, VirB8, and VirB9 proteins, the same proteins were overexpressed in the bvrR mutant complemented with a plasmid carrying a functional bvrR gene. Quantitation of virB5 mRNA confirmed these data and indicated that the influence of the TCS BvrRS on the T4SS VirB occurs at the transcriptional level. The expression of the transcriptional activator VjbR also depended on the TCS BvrRS. In addition, we demonstrate a direct interaction between the promoter region of the VirB operon and the response regulator BvrR. Altogether these data demonstrate that the TCS BvrRS controls the expression of the T4SS VirB through direct and indirect mechanisms.
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Selbach, Matthias, Stefan Moese, Thomas F. Meyer, and Steffen Backert. "Functional Analysis of the Helicobacter pylori cag Pathogenicity Island Reveals Both VirD4-CagA-Dependent and VirD4-CagA-Independent Mechanisms." Infection and Immunity 70, no. 2 (2002): 665–71. http://dx.doi.org/10.1128/iai.70.2.665-671.2002.
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ABSTRACT The type IV secretion machinery encoded by the cag pathogenicity island (PAI) of Helicobacter pylori has been implicated in a series of host responses during infection. Here, we analyzed the function of 12 cag PAI genes from both cag I and cag II loci, including the complete virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, and virD4). We monitored interleukin-8 (IL-8) secretion, CagA translocation and tyrosine phosphorylation, and induction of a scattering (“hummingbird”) phenotype upon H. pylori infection of AGS gastric epithelial cells. For the first time, we have complemented individual cag PAI gene knockout mutants with their intact genes expressed from a shuttle vector and showed that complemented CagA and VirD4 restored wild-type function. Our results demonstrate that phenotypic changes and phosphorylation of CagA depended on all virB/D genes and several other genes of the cag PAI. Induction of IL-8 secretion depended largely on the same set of genes but was independent of CagA and VirD4. Thus, CagA translocation and induction of IL-8 secretion are regulated by VirD4-CagA-dependent and VirD4-CagA-independent mechanisms, respectively. The function of VirD4 as a possible adapter protein which guides CagA into the type IV secretion channel is presented in a model.
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Bao, Weichao, Yumi Kumagai, Hua Niu, Mamoru Yamaguchi, Koshiro Miura, and Yasuko Rikihisa. "Four VirB6 Paralogs and VirB9 Are Expressed and Interact in Ehrlichia chaffeensis-Containing Vacuoles." Journal of Bacteriology 191, no. 1 (2008): 278–86. http://dx.doi.org/10.1128/jb.01031-08.
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ABSTRACT The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.
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Kumar, Renu B., and Anath Das. "Functional Analysis of the Agrobacterium tumefaciens T-DNA Transport Pore Protein VirB8." Journal of Bacteriology 183, no. 12 (2001): 3636–41. http://dx.doi.org/10.1128/jb.183.12.3636-3641.2001.
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ABSTRACT The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8 S87Lpartially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8R107P is defective in interactions with both VirB9 and VirB10. A second mutant VirB8S87L is defective in interaction with VirB9.
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Sutten, Eric L., Junzo Norimine, Paul A. Beare, et al. "Anaplasma marginale Type IV Secretion System Proteins VirB2, VirB7, VirB11, and VirD4 Are Immunogenic Components of a Protective Bacterial Membrane Vaccine." Infection and Immunity 78, no. 3 (2010): 1314–25. http://dx.doi.org/10.1128/iai.01207-09.
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ABSTRACT Anaplasma and related Ehrlichia spp. are important tick-borne, Gram-negative bacterial pathogens of livestock and humans that cause acute infection and disease and can persist. Immunization of cattle with an Anaplasma marginale fraction enriched in outer membranes (OM) can provide complete protection against disease and persistent infection. Serological responses of OM vaccinees to the OM proteome previously identified over 20 antigenic proteins, including three type IV secretion system (T4SS) proteins, VirB9-1, VirB9-2, and VirB10. Subsequent studies showed that these three proteins also stimulated CD4+ T-cell responses in OM vaccinees. The T4SS, composed of a complex of proteins spanning the inner and outer membranes of certain bacteria, is an important virulence factor but is relatively unexplored as a vaccine target. The goal of this study was to determine if additional T4SS proteins are immunogenic for animals immunized with the protective OM fraction of A. marginale. T4SS proteins expressed by in vitro transcription and translation were screened for stimulating proliferation of T cells from OM vaccinees, and immunogenic proteins were expressed as recombinant proteins in Escherichia coli and their immunogenicity was verified. VirB2, a putative VirB7, VirB11, and VirD4 were immunogenic for OM vaccinees expressing several common major histocompatibility complex (MHC) class II haplotypes. VirB2 is encoded by multiple genes that share a conserved central region, and epitope mapping revealed T-cell epitopes in this region. The discovery of novel immunogenic T4SS proteins recognized by outbred individuals with common MHC haplotypes further justifies evaluating the T4SS as a potential vaccine candidate for pathogenic bacteria.
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Kyrova, Elena, Maria Egorova, and Alexander Ignatov. "Species of the genus Xanthomonas infecting cereals and oilseeds in the Russian Federation and its diagnostics." BIO Web of Conferences 18 (2020): 00017. http://dx.doi.org/10.1051/bioconf/20201800017.
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Plant pathogenic bacteria of the genus Xanthomonas display high levels of genetic diversity and cause remarkable damage to about 400 plant species. In 2001–2008, a new group of strains of Xanthomonas arboricola has been found as pathogens on novel host plants such as wheat, rye, barley, tomato, sunflower, and brassicas in Russia. Physiological tests and multilocus sequence typing (MLST) analysis confirmed their position within the Xanthomonas arboricola species. The obtained draft genome sequence of Xanthomonas arboricola strain 3004 from barley plants, also virulent to sunflower, brassicas, and chestnut, has demonstrated an evidence for the lateral gene transfer (LGT) of the virulence genes. It can be suggested that the virE and other genes of T4SS, obtained due to LGT, may contribute to the host range extension. Thus, T4SS genes can be used as the target for group-specific PCR analysis of this emerging pathogen of cereals and oilseeds. We propose to use virB3, virB4, and virB9 genes to design a detection system.
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Kerr, Jennifer E., and Peter J. Christie. "Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System." Journal of Bacteriology 192, no. 19 (2010): 4923–34. http://dx.doi.org/10.1128/jb.00557-10.
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ABSTRACT 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.
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Bauer, Theresa, Thomas Rösch, Mitsuhiro Itaya, and Peter L. Graumann. "Localization Pattern of Conjugation Machinery in a Gram-Positive Bacterium." Journal of Bacteriology 193, no. 22 (2011): 6244–56. http://dx.doi.org/10.1128/jb.00175-11.
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Conjugation is an efficient way for transfer of genetic information between bacteria, even between highly diverged species, and a major cause for the spreading of resistance genes. We have investigated the subcellular localization of several conserved conjugation proteins carried on plasmid pLS20 found inBacillus subtilis. We show that VirB1, VirB4, VirB11, VirD2, and VirD4 homologs assemble at a single cell pole, but also at other sites along the cell membrane, in cells during the lag phase of growth. Bimolecular fluorescence complementation analyses showed that VirB4 and VirD4 interact at the cell pole and, less frequently, at other sites along the membrane. VirB1 and VirB11 also colocalized at the cell pole. Total internal reflection fluorescence microscopy showed that pLS20 is largely membrane associated and is frequently found at the cell pole, indicating that transfer takes place at the pole, which is a preferred site for the assembly of the active conjugation apparatus, but not the sole site. VirD2, VirB4, and VirD4 started to localize to the pole or the membrane in stationary-phase cells, and VirB1 and VirB11 were observed as foci in cells resuspended in fresh medium but no longer in cells that had entered exponential growth, although at least VirB4 was still expressed. These data reveal an unusual assembly/disassembly timing for the pLS20 conjugation machinery and suggest that specific localization of conjugation proteins in lag-phase cells and delocalization during growth are the reasons why pLS20 conjugation occurs only during early exponential phase.
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Alegria, Marcos C., Diorge P. Souza, Maxuel O. Andrade, et al. "Identification of New Protein-Protein Interactions Involving the Products of the Chromosome- and Plasmid-Encoded Type IV Secretion Loci of the Phytopathogen Xanthomonas axonopodis pv. citri." Journal of Bacteriology 187, no. 7 (2005): 2315–25. http://dx.doi.org/10.1128/jb.187.7.2315-2325.2005.
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ABSTRACT The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.
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Zaragoza, Gabrijela Mecky. "Virgo und Virago: Zwei Frühneuzeitliche Judith-Figuren im Vergleich." Daphnis 31, no. 1-2 (2002): 107–26. http://dx.doi.org/10.1163/18796583-0310102005.
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Die These des Artikels ist, daß der konfessionell geprägte Paradigmenwechsel im frühneuzeitlichen weiblichen Gendermodell Auswirkungen darauf hat, wie die ersten beiden Judith-Texte des 16. Jahrhunderts – Marko Marulićs Judita (1501) und Sixt Bircks lvdith (1534) – mit ihren mörderischen Heldinnen umgehen. Während Marulićs Text selbstbewußt das Bild einer Renaissancemadonna feiert, macht sich in Bircks Text Verwirrung breit. Der Widerspruch zwischen dem verhäuslichten weiblichen Frauenbild der Zwingli-Ära und dem eigentlichen - von Martin Luther als “fein/ gut/ nuetzlich” klassifizierten – Judith-Plot, der eine keusche Witwe im Namen Gottes politisches Terrain betreten läßt, erweist sich in diesem Mikrokosmos Text als ein schwerwiegendes Problem, das nur auf absurde Weise gelöst werden kann: Judith muß zum Mannweib mutieren.
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Pollak, Cora N., María Magdalena Wanke, Silvia M. Estein, et al. "Immunization with Brucella VirB Proteins Reduces Organ Colonization in Mice through a Th1-Type Immune Response and Elicits a Similar Immune Response in Dogs." Clinical and Vaccine Immunology 22, no. 3 (2014): 274–81. http://dx.doi.org/10.1128/cvi.00653-14.
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ABSTRACTVirB proteins fromBrucellaspp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice fromBrucellainfection and whether this response can be induced in the dog, a natural host forBrucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with liveBrucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals uponin vitrostimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane ofBrucellaorganisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis ofB. caniswas assessedin vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization byBrucellain mice can be also elicited in dogs.
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Kyrova*, E. I., and A. N. Ignatov. "Selection of target genes for pcr diagnostics of Xanthomonas arboricola virulent for cereals and brassicas." PLANT PROTECTION NEWS 104, no. 2 (2021): 87–96. http://dx.doi.org/10.31993/2308-6459-2021-104-2-14962.
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Plant pathogenic xanthomonads virulent to wheat, rye, barley, tomato, sunflower, and brassicas were isolated in Russia in 2001–2008. Physiological tests and multilocus sequence typing analysis confirmed their position within the Xanthomonas arboricola species. The obtained draft genome sequence of representative strain 3004 from barley plants, which is also virulent to sunflower, brassicas, and chestnut, demonstrated an absence of the Type 3 Secretion System T3SS and an evidence for the lateral gene transfer of some other virulence genes from distantly related bacteria. It was concluded that T4SS genes can be used as the target for group-specific PCR analysis of the emerging pathogen. It was proposed to use virD4, virB3, virB4, and virB9 genes to design a detection system. After preliminary experiments with classic PCR for the chosen genes, primers and TaqMan(R) probe were designed to specifically amplify a 121 bp fragment of the VirD4 gene. Amplification products were obtained for all target Xanthomonas arboricola strains and were not detected in other Xanthomonas species, or in other pathogenic or epiphytic bacteria occurring on these host plants. The assay readily detected Xanthomonas arboricola infection in diseased plants and from bacterial colonies isolated on semi-selective media, and was more sensitive and specific than traditional plating methods.
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Fullner, Karla Jean. "Role of Agrobacterium virB Genes in Transfer of T Complexes and RSF1010." Journal of Bacteriology 180, no. 2 (1998): 430–34. http://dx.doi.org/10.1128/jb.180.2.430-434.1998.
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ABSTRACT Nonpolar virB mutants of Agrobacterium tumefaciens were tested for RSF1010 mobilization and extracellular complementation. virB2 to virB11were essential for transfer in both assays. virB1 was essential only for high frequency transfer of RSF1010 and VirE2. Coordinated transfer of a preassembled T complex is supported by these data and competition studies.
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Shamaei-Tousi, Alireza, Rachel Cahill, and Gad Frankel. "Interaction between Protein Subunits of the Type IV Secretion System of Bartonella henselae." Journal of Bacteriology 186, no. 14 (2004): 4796–801. http://dx.doi.org/10.1128/jb.186.14.4796-4801.2004.
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ABSTRACT In this study we used the yeast two-hybrid system to identify interactions between protein subunits of the virB type IV secretion system of Bartonella henselae. We report interactions between inner membrane and periplasmic proteins, the pilus polypeptide, and the core complex and a novel interaction between VirB3 and VirB5.
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Villamil Giraldo, Ana Maria, Durga Sivanesan, Anna Carle, et al. "Type IV Secretion System Core Component VirB8 fromBrucellaBinds to the Globular Domain of VirB5 and to a Periplasmic Domain of VirB6." Biochemistry 51, no. 18 (2012): 3881–90. http://dx.doi.org/10.1021/bi300298v.
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Rouot, Bruno, Maria-Teresa Alvarez-Martinez, Carine Marius, et al. "Production of the Type IV Secretion System Differs among Brucella Species as Revealed with VirB5- and VirB8-Specific Antisera." Infection and Immunity 71, no. 3 (2003): 1075–82. http://dx.doi.org/10.1128/iai.71.3.1075-1082.2003.
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ABSTRACT Expression of the virB operon, encoding the type IV secretion system required for Brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suis VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suis from which chvE was deleted was fully virulent in a mouse model. Comparative analyses of various Brucella species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canis and B. suis, as well as the Brucella abortus- and Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortus and B. melitensis strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virB expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virB operon are not uniformly regulated in different Brucella species. Since VirB proteins were shown to modulate Brucella phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.
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Rolán, Hortensia G., Andreas B. den Hartigh, Melissa Kahl-McDonagh, Thomas Ficht, L. Garry Adams, and Renée M. Tsolis. "VirB12 Is a Serological Marker of Brucella Infection in Experimental and Natural Hosts." Clinical and Vaccine Immunology 15, no. 2 (2007): 208–14. http://dx.doi.org/10.1128/cvi.00374-07.
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ABSTRACT The Brucella species type IV secretion system, encoded by the virB1-12 locus, is required for intracellular replication and persistent infection in vivo. The requirement of VirB proteins for infection suggests that they are expressed in vivo and may therefore represent serological markers of infection. To test this idea, we purified recombinant VirB1, VirB5, VirB11, and VirB12 and tested for their recognition by antibodies in sera from experimentally infected mice and goats by using an indirect enzyme-linked immunosorbent assay. Antibody responses to VirB12 but not to VirB1, VirB5, or VirB11 were detected in 20/20 mice experimentally inoculated with Brucella abortus and 12/12 goats experimentally infected with Brucella melitensis. The potential use of VirB12 as a serological tool for the diagnosis of brucellosis was evaluated in the natural bovine host. Serum samples from 145 cattle of known serology (29% negative and 71% positive) were analyzed for the production of antibody responses to VirB12. One hundred two cattle samples (70.3%) were positive for antibodies to VirB12, while 43 samples were negative (29.7%). A positive serological response to VirB12 correlated with positive serology to whole B. abortus antigen in 99% of samples tested. These results show that VirB12 is expressed during infection of both experimental and natural hosts of Brucella species, and they suggest that VirB12 may be a useful serodiagnostic marker for brucellosis.
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Praja, Ratih Novita, Didik Handijatno, Setiawan Koesdarto, and Aditya Yudhana. "Karakterisasi Protein VirB4 Brucella abortus Isolat Lokal dengan Teknik Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis." Jurnal Veteriner 18, no. 3 (2017): 416. http://dx.doi.org/10.19087/jveteriner.2017.18.3.416.
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Brucellosis is a zoonotic disease cause by pathogenic bacteria of the genus Brucella. The disease infects livestock mammals such as cows, goats, pigs, and including humans. Brucella abortus has several potential virulence factors, i.e. Proteins VirB. Type IV secretion system (T4SS) which is a combination of 12 proteins from VirB1-VirB11 and VirD4. Brucella can survive for long periods in the environment despite the limitations of nutrients and oxygen. This study aims to characterize the protein VirB4 of local isolate of B. abortus using SDS PAGE (Sodium Dodecly sulfate-polyacrylamide gel electrophoresis). The results showed that the protein contained 10 protein bands with a molecular weight of 158.93; 110.89; 99.931; 70.60; 64.61; 59.25; 45.32; 42.35; 23.63; and 16.70 kDa, respectively. Protein VirB4 of the local isolate of B. abortus have a molecular weight of 59.25 kDa.
 ABSTRAK
 Brucellosis merupakan salah satu penyakit zoonosis yang disebabkan oleh bakteri patogen genus Brucella. Penyakit ini menyerang hewan ternak mamalia seperti sapi, kambing, babi, dan dapat menular ke manusia. Bakteri Brucella abortus memiliki faktor virulensi potensial yaitu protein VirB. Type IV Secretion System (T4SS) merupakan gabungan dari 12 protein yaitu VirB1–VirB11 dan VirD4. Brucella dapat bertahan hidup lama di dalam lingkungan meskipun memiliki keterbatasan nutrisi dan oksigen. Penelitian ini dilakukan untuk karakterisasi protein VirB4 B. abortus isolat lokal dengan metode Sodium Dodecly Sulfate-Polyacrylamide Gel Electrophoresis (SDS PAGE). Hasil karakterisasi protein B. abortus isolat lokal dengan teknik SDS-PAGE terdapat 10 pita protein dengan bobot molekul 158,93; 110,89; 99,931; 70,60; 64,61; 59,25; 45,32; 42,35; 23,63; dan 16,70 kDa. Simpulan penelitian ini adalah terdapat protein VirB4 B. abortus isolat lokal yang mempunyai bobot molekul 59,25 kDa.
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Lee, Min, Ji Yang, Yoonjung Cho та ін. "Inhibitory Effects of Menadione on Helicobacter pylori Growth and Helicobacter pylori-Induced Inflammation via NF-κB Inhibition". International Journal of Molecular Sciences 20, № 5 (2019): 1169. http://dx.doi.org/10.3390/ijms20051169.
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H. pylori is classified as a group I carcinogen by WHO because of its involvement in gastric cancer development. Several reports have suggested anti-bacterial effects of menadione, although the effect of menadione on major virulence factors of H. pylori and H. pylori-induced inflammation is yet to be elucidated. In this study, therefore, we demonstrated that menadione has anti-H. pylori and anti-inflammatory effects. Menadione inhibited growth of H. pylori reference strains and clinical isolates. Menadione reduced expression of vacA in H. pylori, and translocation of VacA protein into AGS (gastric adenocarcinoma cell) was also decreased by menadione treatment. This result was concordant with decreased apoptosis in AGS cells infected with H. pylori. Moreover, cytotoxin-associated protein A (CagA) translocation into H. pylori-infected AGS cells was also decreased by menadione. Menadione inhibited expression of several type IV secretion system (T4SS) components, including virB2, virB7, virB8, and virB10, that are responsible for translocation of CagA into host cells. In particular, menadione inhibited nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and thereby reduced expression of the proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α in AGS as well as in THP-1 (monocytic leukemia cell) cell lines. Collectively, these results suggest the anti-bacterial and anti-inflammatory effects of menadione against H. pylori.
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Bourg, Gisèle, Romain Sube, David O'Callaghan, and Gilles Patey. "Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems." Journal of Bacteriology 191, no. 9 (2009): 2985–92. http://dx.doi.org/10.1128/jb.01426-08.
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ABSTRACT The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.
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Baron, Christian. "VirB8: a conserved type IV secretion system assembly factor and drug targetThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease." Biochemistry and Cell Biology 84, no. 6 (2006): 890–99. http://dx.doi.org/10.1139/o06-148.
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Type IV secretion systems are used by many Gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA–protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8–12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure–function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein–protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.
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Casu, Bastien, Charline Mary, Aleksandr Sverzhinsky, Aurélien Fouillen, Antonio Nanci, and Christian Baron. "VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD." Proceedings of the National Academy of Sciences 115, no. 23 (2018): 5950–55. http://dx.doi.org/10.1073/pnas.1802501115.
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Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.
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Anderson, L. B., A. V. Hertzel, and A. Das. "Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex." Proceedings of the National Academy of Sciences 93, no. 17 (1996): 8889–94. http://dx.doi.org/10.1073/pnas.93.17.8889.
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Sieira, Rodrigo, Diego J. Comerci, Daniel O. Sánchez, and Rodolfo A. Ugalde. "A Homologue of an Operon Required for DNA Transfer in Agrobacterium Is Required in Brucella abortusfor Virulence and Intracellular Multiplication." Journal of Bacteriology 182, no. 17 (2000): 4849–55. http://dx.doi.org/10.1128/jb.182.17.4849-4855.2000.
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ABSTRACT As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virBoperon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment.
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Lux-Sterritt, Laurence. "‘Virgo Becomes Virago’: Women in the Accounts of Seventeenth-Century English Catholic Missionaries." Recusant History 30, no. 4 (2011): 537–53. http://dx.doi.org/10.1017/s0034193200013170.
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In the account of his missionary work, the Jesuit John Gerard (1564–1637) famously explained how, after a few years of penury in ‘Mass equipment’, Catholic houses had become so well equipped that priests were able to set about their work immediately upon their arrival. He recalled that in the last two years of his work (1604–06), he no longer needed to lodge in taverns but always found friendly dwellings to shelter him on his way. Most of these were run by women, whose prominence in the activity of harbouring was pointed out in many documents, including the minutes of the confession given to the Privy Council by the appellant priest Anthony Sherlock, who turned informer after his capture in 1606:[Sherlock] grew into acquaintance with Lady Stonor near Henley-on-Thames and stayed with her three or four years, often saying mass in her house. Next he moved to Warwicks. and at Brailes and Welsford was with a widow named Margaret Bishop for two or three years. Then to Worcs., where he said Mass once or twice in the house of Lady Windsor and also at Mrs. Heath’s at Alchurch, at Hawkesley with Mr. Middlemore and at Tamworth in Warwicks. with Richard Dolphin two or three years. Then he was with widow Knowles at Ridware, with Mrs. Comberford at Wednesbury, with Mrs. Stanford at Parkington …
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Hwang, Hau-Hsuan, Yin-Tzu Liu, Si-Chi Huang, et al. "Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions." Phytopathology® 105, no. 2 (2015): 160–68. http://dx.doi.org/10.1094/phyto-05-14-0133-r.
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Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth.
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Backert, Steffen, Remi Fronzes, and Gabriel Waksman. "VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems?" Trends in Microbiology 16, no. 9 (2008): 409–13. http://dx.doi.org/10.1016/j.tim.2008.07.001.
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Baron, C., Y. R. Thorstenson, and P. C. Zambryski. "The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens." Journal of bacteriology 179, no. 4 (1997): 1211–18. http://dx.doi.org/10.1128/jb.179.4.1211-1218.1997.
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Huang, Fan-Chen, and Hau-Hsuan Hwang. "Arabidopsis RETICULON-LIKE4 (RTNLB4) Protein Participates in Agrobacterium Infection and VirB2 Peptide-Induced Plant Defense Response." International Journal of Molecular Sciences 21, no. 5 (2020): 1722. http://dx.doi.org/10.3390/ijms21051722.
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Agrobacterium tumefaciens uses the type IV secretion system, which consists of VirB1-B11 and VirD4 proteins, to deliver effectors into plant cells. The effectors manipulate plant proteins to assist in T-DNA transfer, integration, and expression in plant cells. The Arabidopsis reticulon-like (RTNLB) proteins are located in the endoplasmic reticulum and are involved in endomembrane trafficking in plant cells. The rtnlb4 mutants were recalcitrant to A. tumefaciens infection, but overexpression of RTNLB4 in transgenic plants resulted in hypersusceptibility to A. tumefaciens transformation, which suggests the involvement of RTNLB4 in A. tumefaciens infection. The expression of defense-related genes, including FRK1, PR1, WRKY22, and WRKY29, were less induced in RTNLB4 overexpression (O/E) transgenic plants after A. tumefaciens elf18 peptide treatment. Pretreatment with elf18 peptide decreased Agrobacterium-mediated transient expression efficiency more in wild-type seedlings than RTNLB4 O/E transgenic plants, which suggests that the induced defense responses in RTNLB4 O/E transgenic plants might be affected after bacterial elicitor treatments. Similarly, A. tumefaciens VirB2 peptide pretreatment reduced transient T-DNA expression in wild-type seedlings to a greater extent than in RTNLB4 O/E transgenic seedlings. Furthermore, the VirB2 peptides induced FRK1, WRKY22, and WRKY29 gene expression in wild-type seedlings but not efr-1 and bak1 mutants. The induced defense-related gene expression was lower in RTNLB4 O/E transgenic plants than wild-type seedlings after VirB2 peptide treatment. These data suggest that RTNLB4 may participate in elf18 and VirB2 peptide-induced defense responses and may therefore affect the A. tumefaciens infection process.
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Spudich, G. M., D. Fernandez, X. R. Zhou, and P. J. Christie. "Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus." Proceedings of the National Academy of Sciences 93, no. 15 (1996): 7512–17. http://dx.doi.org/10.1073/pnas.93.15.7512.
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