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. 1996 Oct;178(19):5706–5711. doi: 10.1128/jb.178.19.5706-5711.1996

VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.

A L Jones 1, E M Lai 1, K Shirasu 1, C I Kado 1
PMCID: PMC178410  PMID: 8824616

Abstract

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli.

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Selected References

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  1. 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]
  2. Covacci A., Rappuoli R. Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon. Mol Microbiol. 1993 May;8(3):429–434. doi: 10.1111/j.1365-2958.1993.tb01587.x. [DOI] [PubMed] [Google Scholar]
  3. Frost L. S., Ippen-Ihler K., Skurray R. A. Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol Rev. 1994 Jun;58(2):162–210. doi: 10.1128/mr.58.2.162-210.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gallie D. R., Novak S., Kado C. I. Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium. Plasmid. 1985 Sep;14(2):171–175. doi: 10.1016/0147-619x(85)90078-2. [DOI] [PubMed] [Google Scholar]
  5. Gay P., Le Coq D., Steinmetz M., Berkelman T., Kado C. I. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985 Nov;164(2):918–921. doi: 10.1128/jb.164.2.918-921.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gelvin S. B., Habeck L. L. vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens. J Bacteriol. 1990 Mar;172(3):1600–1608. doi: 10.1128/jb.172.3.1600-1608.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grant S. G., Jessee J., Bloom F. R., Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645–4649. doi: 10.1073/pnas.87.12.4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jones A. L., Shirasu K., Kado C. I. The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein. J Bacteriol. 1994 Sep;176(17):5255–5261. doi: 10.1128/jb.176.17.5255-5261.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kado C. I. Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis. Mol Microbiol. 1994 Apr;12(1):17–22. doi: 10.1111/j.1365-2958.1994.tb00990.x. [DOI] [PubMed] [Google Scholar]
  10. Kao J. C., Perry K. L., Kado C. I. Indoleacetic acid complementation and its relation to host range specifying genes on the Ti plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1982;188(3):425–432. doi: 10.1007/BF00330044. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lessl M., Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell. 1994 May 6;77(3):321–324. doi: 10.1016/0092-8674(94)90146-5. [DOI] [PubMed] [Google Scholar]
  13. Motallebi-Veshareh M., Balzer D., Lanka E., Jagura-Burdzy G., Thomas C. M. Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication. Mol Microbiol. 1992 Apr;6(7):907–920. doi: 10.1111/j.1365-2958.1992.tb01541.x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Quandt J., Hynes M. F. Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene. 1993 May 15;127(1):15–21. doi: 10.1016/0378-1119(93)90611-6. [DOI] [PubMed] [Google Scholar]
  16. Rogowsky P. M., Powell B. S., Shirasu K., Lin T. S., Morel P., Zyprian E. M., Steck T. R., Kado C. I. Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit. Plasmid. 1990 Mar;23(2):85–106. doi: 10.1016/0147-619x(90)90028-b. [DOI] [PubMed] [Google Scholar]
  17. Shirasu K., Kado C. I. Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens. FEMS Microbiol Lett. 1993 Aug 1;111(2-3):287–294. doi: 10.1111/j.1574-6968.1993.tb06400.x. [DOI] [PubMed] [Google Scholar]
  18. Shirasu K., Kado C. I. The virB operon of the Agrobacterium tumefaciens virulence regulon has sequence similarities to B, C and D open reading frames downstream of the pertussis toxin-operon and to the DNA transfer-operons of broad-host-range conjugative plasmids. Nucleic Acids Res. 1993 Jan 25;21(2):353–354. doi: 10.1093/nar/21.2.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Steck T. R., Kado C. I. Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains. J Bacteriol. 1990 Apr;172(4):2191–2193. doi: 10.1128/jb.172.4.2191-2193.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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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