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  • 1
    Online Resource
    Online Resource
    Proceedings of the National Academy of Sciences ; 2005
    In:  Proceedings of the National Academy of Sciences Vol. 102, No. 3 ( 2005-01-18), p. 832-837
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 102, No. 3 ( 2005-01-18), p. 832-837
    Abstract: Several human pathogens and the plant pathogen Agrobacterium tumefaciens use a type IV secretion system for translocation of effector proteins into host cells. How effector proteins are selected for transport is unknown, but a C-terminal transport signal is present in the proteins translocated by the A. tumefaciens VirB/D4 type IV secretion system. We characterized this signal in the virulence protein VirF by alanine scanning and further site-directed mutagenesis. The Cre recombinase was used as a reporter to measure the translocation efficiency of Cre-Vir fusions from A. tumefaciens to Arabidopsis . The data unambiguously showed that positive charge is an essential characteristic of the C-terminal transport signal. We increased the sensitivity of this translocation assay by modifying the Cre-induced readout in host cells from kanamycin resistance to GFP expression. This improvement allowed us to detect translocation of the VirD2 relaxase protein in the absence of transferred DNA, showing that attachment to the transferred DNA is not essential for transport by the VirB/D4 system. We also found another translocated effector protein, namely the VirD5 protein encoded by the tumor-inducing plasmid. According to secondary structure predictions, the C termini of all VirB/D4-translocated proteins identified so far are unstructured; however, they contain a characteristic hydropathic profile. Based on sequence alignments and mutational analysis of VirF, we conclude that the C-terminal transport signal for recruitment and translocation of effector proteins by the A. tumefaciens VirB/D4 system is hydrophilic and has a net positive charge with a consensus motif of R-X(7)-R-X-R-X-R-X-X(n) 〉 .
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    RVK:
    RVK:
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2005
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    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 2
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 188, No. 23 ( 2006-12), p. 8222-8230
    Abstract: Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 3
    In: Infection and Immunity, American Society for Microbiology, Vol. 74, No. 1 ( 2006-01), p. 108-117
    Abstract: Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related α 2 -proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens , suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
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    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
    detail.hit.zdb_id: 1483247-1
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  • 4
    Online Resource
    Online Resource
    Proceedings of the National Academy of Sciences ; 2009
    In:  Proceedings of the National Academy of Sciences Vol. 106, No. 24 ( 2009-06-16), p. 9643-9648
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 106, No. 24 ( 2009-06-16), p. 9643-9648
    Abstract: Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2 82–202 , which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana . These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    RVK:
    RVK:
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2009
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Hindawi Limited ; 2007
    In:  Cellular Microbiology Vol. 9, No. 11 ( 2007-11), p. 2644-2657
    In: Cellular Microbiology, Hindawi Limited, Vol. 9, No. 11 ( 2007-11), p. 2644-2657
    Type of Medium: Online Resource
    ISSN: 1462-5814 , 1462-5822
    URL: Issue
    Language: English
    Publisher: Hindawi Limited
    Publication Date: 2007
    detail.hit.zdb_id: 2019990-9
    SSG: 12
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