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. 1996 Feb;178(4):961–970. doi: 10.1128/jb.178.4.961-970.1996

Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens.

S L Doty 1, M C Yu 1, J I Lundin 1, J D Heath 1, E W Nester 1
PMCID: PMC177754  PMID: 8576069

Abstract

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.

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Selected References

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  1. Ankenbauer R. G., Nester E. W. Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides. J Bacteriol. 1990 Nov;172(11):6442–6446. doi: 10.1128/jb.172.11.6442-6446.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banta L. M., Joerger R. D., Howitz V. R., Campbell A. M., Binns A. N. Glu-255 outside the predicted ChvE binding site in VirA is crucial for sugar enhancement of acetosyringone perception by Agrobacterium tumefaciens. J Bacteriol. 1994 Jun;176(11):3242–3249. doi: 10.1128/jb.176.11.3242-3249.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cangelosi G. A., Ankenbauer R. G., Nester E. W. Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6708–6712. doi: 10.1073/pnas.87.17.6708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cangelosi G. A., Best E. A., Martinetti G., Nester E. W. Genetic analysis of Agrobacterium. Methods Enzymol. 1991;204:384–397. doi: 10.1016/0076-6879(91)04020-o. [DOI] [PubMed] [Google Scholar]
  5. Chang C. H., Winans S. C. Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein. J Bacteriol. 1992 Nov;174(21):7033–7039. doi: 10.1128/jb.174.21.7033-7039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Charles T. C., Doty S. L., Nester E. W. Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations. Appl Environ Microbiol. 1994 Nov;60(11):4192–4194. doi: 10.1128/aem.60.11.4192-4194.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charles T. C., Nester E. W. A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1993 Oct;175(20):6614–6625. doi: 10.1128/jb.175.20.6614-6625.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chilton M. D., Currier T. C., Farrand S. K., Bendich A. J., Gordon M. P., Nester E. W. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3672–3676. doi: 10.1073/pnas.71.9.3672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  10. Close T. J., Zaitlin D., Kado C. I. Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58. Plasmid. 1984 Sep;12(2):111–118. doi: 10.1016/0147-619x(84)90057-x. [DOI] [PubMed] [Google Scholar]
  11. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Doty S. L., Chang M., Nester E. W. The chromosomal virulence gene, chvE, of Agrobacterium tumefaciens is regulated by a LysR family member. J Bacteriol. 1993 Dec;175(24):7880–7886. doi: 10.1128/jb.175.24.7880-7886.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garfinkel D. J., Nester E. W. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol. 1980 Nov;144(2):732–743. doi: 10.1128/jb.144.2.732-743.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gutierrez C., Devedjian J. C. A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3999–3999. doi: 10.1093/nar/17.10.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huang M. L., Cangelosi G. A., Halperin W., Nester E. W. A chromosomal Agrobacterium tumefaciens gene required for effective plant signal transduction. J Bacteriol. 1990 Apr;172(4):1814–1822. doi: 10.1128/jb.172.4.1814-1822.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Humphreys G. O., Willshaw G. A., Smith H. R., Anderson E. S. Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids. Mol Gen Genet. 1976 Apr 23;145(1):101–108. doi: 10.1007/BF00331564. [DOI] [PubMed] [Google Scholar]
  17. Iuchi S. Phosphorylation/dephosphorylation of the receiver module at the conserved aspartate residue controls transphosphorylation activity of histidine kinase in sensor protein ArcB of Escherichia coli. J Biol Chem. 1993 Nov 15;268(32):23972–23980. [PubMed] [Google Scholar]
  18. Jin S. G., Prusti R. K., Roitsch T., Ankenbauer R. G., Nester E. W. Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG. J Bacteriol. 1990 Sep;172(9):4945–4950. doi: 10.1128/jb.172.9.4945-4950.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jin S. G., Roitsch T., Christie P. J., Nester E. W. The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes. J Bacteriol. 1990 Feb;172(2):531–537. doi: 10.1128/jb.172.2.531-537.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jin S., Roitsch T., Ankenbauer R. G., Gordon M. P., Nester E. W. The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation. J Bacteriol. 1990 Feb;172(2):525–530. doi: 10.1128/jb.172.2.525-530.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jin S., Song Y., Pan S. Q., Nester E. W. Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium. Mol Microbiol. 1993 Feb;7(4):555–562. doi: 10.1111/j.1365-2958.1993.tb01146.x. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lee K., Dudley M. W., Hess K. M., Lynn D. G., Joerger R. D., Binns A. N. Mechanism of activation of Agrobacterium virulence genes: identification of phenol-binding proteins. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8666–8670. doi: 10.1073/pnas.89.18.8666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leroux B., Yanofsky M. F., Winans S. C., Ward J. E., Ziegler S. F., Nester E. W. Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant. EMBO J. 1987 Apr;6(4):849–856. doi: 10.1002/j.1460-2075.1987.tb04830.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Melchers L. S., Regensburg-Tuïnk T. J., Bourret R. B., Sedee N. J., Schilperoort R. A., Hooykaas P. J. Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens. EMBO J. 1989 Jul;8(7):1919–1925. doi: 10.1002/j.1460-2075.1989.tb03595.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Milburn M. V., Privé G. G., Milligan D. L., Scott W. G., Yeh J., Jancarik J., Koshland D. E., Jr, Kim S. H. Three-dimensional structures of the ligand-binding domain of the bacterial aspartate receptor with and without a ligand. Science. 1991 Nov 29;254(5036):1342–1347. doi: 10.1126/science.1660187. [DOI] [PubMed] [Google Scholar]
  27. Morel P., Powell B. S., Rogowsky P. M., Kado C. I. Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens. Mol Microbiol. 1989 Sep;3(9):1237–1246. doi: 10.1111/j.1365-2958.1989.tb00274.x. [DOI] [PubMed] [Google Scholar]
  28. Pan S. Q., Charles T., Jin S., Wu Z. L., Nester E. W. Preformed dimeric state of the sensor protein VirA is involved in plant--Agrobacterium signal transduction. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9939–9943. doi: 10.1073/pnas.90.21.9939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Parkinson J. S. Signal transduction schemes of bacteria. Cell. 1993 Jun 4;73(5):857–871. doi: 10.1016/0092-8674(93)90267-t. [DOI] [PubMed] [Google Scholar]
  30. Pazour G. J., Ta C. N., Das A. Mutants of Agrobacterium tumefaciens with elevated vir gene expression. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6941–6945. doi: 10.1073/pnas.88.16.6941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Raineri D. M., Boulton M. I., Davies J. W., Nester E. W. VirA, the plant-signal receptor, is responsible for the Ti plasmid-specific transfer of DNA to maize by Agrobacterium. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3549–3553. doi: 10.1073/pnas.90.8.3549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seligman L., Manoil C. An amphipathic sequence determinant of membrane protein topology. J Biol Chem. 1994 Aug 5;269(31):19888–19896. [PubMed] [Google Scholar]
  33. Shimoda N., Toyoda-Yamamoto A., Aoki S., Machida Y. Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium. J Biol Chem. 1993 Dec 15;268(35):26552–26558. [PubMed] [Google Scholar]
  34. Shimoda N., Toyoda-Yamamoto A., Nagamine J., Usami S., Katayama M., Sakagami Y., Machida Y. Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6684–6688. doi: 10.1073/pnas.87.17.6684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  36. Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tokishita S., Kojima A., Mizuno T. Transmembrane signal transduction and osmoregulation in Escherichia coli: functional importance of the transmembrane regions of membrane-located protein kinase, EnvZ. J Biochem. 1992 Jun;111(6):707–713. doi: 10.1093/oxfordjournals.jbchem.a123823. [DOI] [PubMed] [Google Scholar]
  38. Tokishita S., Mizuno T. Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling. Mol Microbiol. 1994 Aug;13(3):435–444. doi: 10.1111/j.1365-2958.1994.tb00438.x. [DOI] [PubMed] [Google Scholar]
  39. Turk S. C., van Lange R. P., Regensburg-Tuïnk T. J., Hooykaas P. J. Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens. Plant Mol Biol. 1994 Aug;25(5):899–907. doi: 10.1007/BF00028884. [DOI] [PubMed] [Google Scholar]
  40. Turk S. C., van Lange R. P., Sonneveld E., Hooykaas P. J. The chimeric VirA-tar receptor protein is locked into a highly responsive state. J Bacteriol. 1993 Sep;175(17):5706–5709. doi: 10.1128/jb.175.17.5706-5709.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Winans S. C., Kerstetter R. A., Ward J. E., Nester E. W. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J Bacteriol. 1989 Mar;171(3):1616–1622. doi: 10.1128/jb.171.3.1616-1622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]

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