Abstract
The Agrobacterium tumefaciens VirB11 ATPase is postulated to assemble with VirB proteins and the VirD4 protein into a transport system which is dedicated to the export of oncogenic nucleoprotein particles to plant cells. To gain genetic evidence for interactions between VirB11 and other subunits of this transport system, we screened a PCR-mutagenized virB11 library for alleles that diminish the virulence of the wild-type strain A348. Two classes of alleles displaying negative dominance were identified. One class failed to complement a delta virB11 mutation, indicating that the corresponding mutant proteins are nonfunctional. The second class complemented the delta virB11 mutation, indicating that the mutant proteins are fully functional in strains devoid of native VirB11. Mutations of both classes of alleles were in codons for residues clustered in two regions of VirB11, both located outside the Walker A nucleotide binding motif. All dominant alleles were suppressed at least to some extent by multicopy expression of the virB9, virB10, and/or virB11 genes. Taken together, results of these investigations indicate that (i) a functional T-complex transporter is composed of more than one VirB11 subunit and (ii) VirB11 undergoes complex formation with VirB9 and VirB10 during transporter biogenesis.
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- Anderson L. B., Hertzel A. V., Das A. Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8889–8894. doi: 10.1073/pnas.93.17.8889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baron C., Thorstenson Y. R., Zambryski P. C. The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens. J Bacteriol. 1997 Feb;179(4):1211–1218. doi: 10.1128/jb.179.4.1211-1218.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beaupré C. E., Bohne J., Dale E. M., Binns A. N. Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells. J Bacteriol. 1997 Jan;179(1):78–89. doi: 10.1128/jb.179.1.78-89.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beijersbergen A., Dulk-Ras A. D., Schilperoort R. A., Hooykaas P. J. Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens. Science. 1992 May 29;256(5061):1324–1327. doi: 10.1126/science.256.5061.1324. [DOI] [PubMed] [Google Scholar]
- Berger B. R., Christie P. J. Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes. J Bacteriol. 1994 Jun;176(12):3646–3660. doi: 10.1128/jb.176.12.3646-3660.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berger B. R., Christie P. J. The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain. J Bacteriol. 1993 Mar;175(6):1723–1734. doi: 10.1128/jb.175.6.1723-1734.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Binns A. N., Beaupré C. E., Dale E. M. Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J Bacteriol. 1995 Sep;177(17):4890–4899. doi: 10.1128/jb.177.17.4890-4899.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bundock P., Hooykaas P. J. Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15272–15275. doi: 10.1073/pnas.93.26.15272. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bundock P., den Dulk-Ras A., Beijersbergen A., Hooykaas P. J. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 1995 Jul 3;14(13):3206–3214. doi: 10.1002/j.1460-2075.1995.tb07323.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Censini S., Lange C., Xiang Z., Crabtree J. E., Ghiara P., Borodovsky M., Rappuoli R., Covacci A. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14648–14653. doi: 10.1073/pnas.93.25.14648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen C. Y., Winans S. C. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J Bacteriol. 1991 Feb;173(3):1139–1144. doi: 10.1128/jb.173.3.1139-1144.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christie P. J. Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J Bacteriol. 1997 May;179(10):3085–3094. doi: 10.1128/jb.179.10.3085-3094.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christie P. J. The cag pathogenicity island: mechanistic insights. Trends Microbiol. 1997 Jul;5(7):264–265. doi: 10.1016/S0966-842X(97)88833-6. [DOI] [PubMed] [Google Scholar]
- Christie P. J., Ward J. E., Jr, Gordon M. P., Nester E. W. A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9677–9681. doi: 10.1073/pnas.86.24.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christie P. J., Ward J. E., Winans S. C., Nester E. W. The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J Bacteriol. 1988 Jun;170(6):2659–2667. doi: 10.1128/jb.170.6.2659-2667.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Covacci A., Falkow S., Berg D. E., Rappuoli R. Did the inheritance of a pathogenicity island modify the virulence of Helicobacter pylori? Trends Microbiol. 1997 May;5(5):205–208. doi: 10.1016/S0966-842X(97)01035-4. [DOI] [PubMed] [Google Scholar]
- Dale E. M., Binns A. N., Ward J. E., Jr Construction and characterization of Tn5virB, a transposon that generates nonpolar mutations, and its use to define virB8 as an essential virulence gene in Agrobacterium tumefaciens. J Bacteriol. 1993 Feb;175(3):887–891. doi: 10.1128/jb.175.3.887-891.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Farizo K. M., Cafarella T. G., Burns D. L. Evidence for a ninth gene, ptlI, in the locus encoding the pertussis toxin secretion system of Bordetella pertussis and formation of a PtlI-PtlF complex. J Biol Chem. 1996 Dec 6;271(49):31643–31649. doi: 10.1074/jbc.271.49.31643. [DOI] [PubMed] [Google Scholar]
- Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fernandez D., Dang T. A., Spudich G. M., Zhou X. R., Berger B. R., Christie P. J. The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface. J Bacteriol. 1996 Jun;178(11):3156–3167. doi: 10.1128/jb.178.11.3156-3167.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fernandez D., Spudich G. M., Zhou X. R., Christie P. J. The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus. J Bacteriol. 1996 Jun;178(11):3168–3176. doi: 10.1128/jb.178.11.3168-3176.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fullner K. J., Nester E. W. Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol. 1996 Mar;178(6):1498–1504. doi: 10.1128/jb.178.6.1498-1504.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fullner K. J., Stephens K. M., Nester E. W. An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA. Mol Gen Genet. 1994 Dec 15;245(6):704–715. doi: 10.1007/BF00297277. [DOI] [PubMed] [Google Scholar]
- Garfinkel D. J., Simpson R. B., Ream L. W., White F. F., Gordon M. P., Nester E. W. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell. 1981 Nov;27(1 Pt 2):143–153. doi: 10.1016/0092-8674(81)90368-8. [DOI] [PubMed] [Google Scholar]
- Garza A. G., Bronstein P. A., Valdez P. A., Harris-Haller L. W., Manson M. D. Extragenic suppression of motA missense mutations of Escherichia coli. J Bacteriol. 1996 Nov;178(21):6116–6122. doi: 10.1128/jb.178.21.6116-6122.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
- Lessl M., Balzer D., Pansegrau W., Lanka E. Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem. 1992 Oct 5;267(28):20471–20480. [PubMed] [Google Scholar]
- Lin T. S., Kado C. I. The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens. Mol Microbiol. 1993 Aug;9(4):803–812. doi: 10.1111/j.1365-2958.1993.tb01739.x. [DOI] [PubMed] [Google Scholar]
- Mecsas J. J., Strauss E. J. Molecular mechanisms of bacterial virulence: type III secretion and pathogenicity islands. Emerg Infect Dis. 1996 Oct-Dec;2(4):270–288. doi: 10.3201/eid0204.960403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miranda A., Janssen G., Hodges L., Peralta E. G., Ream W. Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism. J Bacteriol. 1992 Apr;174(7):2288–2297. doi: 10.1128/jb.174.7.2288-2297.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piers K. L., Heath J. D., Liang X., Stephens K. M., Nester E. W. Agrobacterium tumefaciens-mediated transformation of yeast. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1613–1618. doi: 10.1073/pnas.93.4.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pohlman R. F., Genetti H. D., Winans S. C. Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol. 1994 Nov;14(4):655–668. doi: 10.1111/j.1365-2958.1994.tb01304.x. [DOI] [PubMed] [Google Scholar]
- Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rashkova S., Spudich G. M., Christie P. J. Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase. J Bacteriol. 1997 Feb;179(3):583–591. doi: 10.1128/jb.179.3.583-591.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Regensburg-Tuïnk A. J., Hooykaas P. J. Transgenic N. glauca plants expressing bacterial virulence gene virF are converted into hosts for nopaline strains of A. tumefaciens. Nature. 1993 May 6;363(6424):69–71. doi: 10.1038/363069a0. [DOI] [PubMed] [Google Scholar]
- Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
- Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
- Sheng J., Citovsky V. Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. Plant Cell. 1996 Oct;8(10):1699–1710. doi: 10.1105/tpc.8.10.1699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shirasu K., Koukolíková-Nicola Z., Hohn B., Kado C. I. An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol Microbiol. 1994 Feb;11(3):581–588. doi: 10.1111/j.1365-2958.1994.tb00338.x. [DOI] [PubMed] [Google Scholar]
- Spee J. H., de Vos W. M., Kuipers O. P. Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP. Nucleic Acids Res. 1993 Feb 11;21(3):777–778. doi: 10.1093/nar/21.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spudich G. M., Fernandez D., Zhou X. R., Christie P. J. Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7512–7517. doi: 10.1073/pnas.93.15.7512. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stephens K. M., Roush C., Nester E. Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence. J Bacteriol. 1995 Jan;177(1):27–36. doi: 10.1128/jb.177.1.27-36.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tummuru M. K., Sharma S. A., Blaser M. J. Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol Microbiol. 1995 Dec;18(5):867–876. doi: 10.1111/j.1365-2958.1995.18050867.x. [DOI] [PubMed] [Google Scholar]
- Ueguchi C., Ito K. Multicopy suppression: an approach to understanding intracellular functioning of the protein export system. J Bacteriol. 1992 Mar;174(5):1454–1461. doi: 10.1128/jb.174.5.1454-1461.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ward J. E., Akiyoshi D. E., Regier D., Datta A., Gordon M. P., Nester E. W. Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid. J Biol Chem. 1988 Apr 25;263(12):5804–5814. [PubMed] [Google Scholar]
- Ward J. E., Jr, Dale E. M., Binns A. N. Activity of the Agrobacterium T-DNA transfer machinery is affected by virB gene products. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9350–9354. doi: 10.1073/pnas.88.20.9350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ward J. E., Jr, Dale E. M., Christie P. J., Nester E. W., Binns A. N. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes. J Bacteriol. 1990 Sep;172(9):5187–5199. doi: 10.1128/jb.172.9.5187-5199.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ward J. E., Jr, Dale E. M., Nester E. W., Binns A. N. Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J Bacteriol. 1990 Sep;172(9):5200–5210. doi: 10.1128/jb.172.9.5200-5210.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watson B., Currier T. C., Gordon M. P., Chilton M. D., Nester E. W. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1975 Jul;123(1):255–264. doi: 10.1128/jb.123.1.255-264.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss A. A., Johnson F. D., Burns D. L. Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2970–2974. doi: 10.1073/pnas.90.7.2970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winans S. C., Burns D. L., Christie P. J. Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol. 1996 Feb;4(2):64–68. doi: 10.1016/0966-842X(96)81513-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winans S. C. Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev. 1992 Mar;56(1):12–31. doi: 10.1128/mr.56.1.12-31.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zupan J. R., Zambryski P. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol. 1995 Apr;107(4):1041–1047. doi: 10.1104/pp.107.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
