Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jan;178(2):435–440. doi: 10.1128/jb.178.2.435-440.1996

Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes.

C Fuqua 1, S C Winans 1
PMCID: PMC177675  PMID: 8550463

Abstract

Ti plasmids of Agrobacterium tumefaciens, in addition to transferring oncogenic DNA to the nuclei of infected plant cells, can conjugally transfer between agrobacteria. Conjugation of wide-host-range octopine-type Ti plasmids requires a tumor-released arginine derivative called octopine. Octopine stimulates expression of the traR gene, whose product directly activates other tra genes in the presence of an acylated homoserine lactone called Agrobacterium autoinducer (AAI). We have localized the transcription starts of three tra promoters and find conserved elements (tra boxes) at virtually identical positions upstream of each promoter. Disruption of these tra boxes abolished induction of each promoter. Deletion analysis of the traI promoter indicates that tra boxes are the only upstream elements required for transcriptional activation. Since Ti plasmid donor cells both produce and respond to AAI, we tested whether expression of tra promoters was enhanced by high concentrations of bacteria. Both tra gene expression and conjugation itself were strongly stimulated either by high donor densities or by exogenous AAI.

Full Text

The Full Text of this article is available as a PDF (295.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beck C. F., Warren R. A. Divergent promoters, a common form of gene organization. Microbiol Rev. 1988 Sep;52(3):318–326. doi: 10.1128/mr.52.3.318-326.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Da Re S., Bertagnoli S., Fourment J., Reyrat J. M., Kahn D. Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory domain. Nucleic Acids Res. 1994 May 11;22(9):1555–1561. doi: 10.1093/nar/22.9.1555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devine J. H., Shadel G. S., Baldwin T. O. Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC7744. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5688–5692. doi: 10.1073/pnas.86.15.5688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dunny G. M., Leonard B. A., Hedberg P. J. Pheromone-inducible conjugation in Enterococcus faecalis: interbacterial and host-parasite chemical communication. J Bacteriol. 1995 Feb;177(4):871–876. doi: 10.1128/jb.177.4.871-876.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engebrecht J., Nealson K., Silverman M. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell. 1983 Mar;32(3):773–781. doi: 10.1016/0092-8674(83)90063-6. [DOI] [PubMed] [Google Scholar]
  10. Engebrecht J., Silverman M. Nucleotide sequence of the regulatory locus controlling expression of bacterial genes for bioluminescence. Nucleic Acids Res. 1987 Dec 23;15(24):10455–10467. doi: 10.1093/nar/15.24.10455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fuqua C., Burbea M., Winans S. C. Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene. J Bacteriol. 1995 Mar;177(5):1367–1373. doi: 10.1128/jb.177.5.1367-1373.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fuqua W. C., Winans S. C. A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. J Bacteriol. 1994 May;176(10):2796–2806. doi: 10.1128/jb.176.10.2796-2806.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuqua W. C., Winans S. C., Greenberg E. P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol. 1994 Jan;176(2):269–275. doi: 10.1128/jb.176.2.269-275.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galinier A., Garnerone A. M., Reyrat J. M., Kahn D., Batut J., Boistard P. Phosphorylation of the Rhizobium meliloti FixJ protein induces its binding to a compound regulatory region at the fixK promoter. J Biol Chem. 1994 Sep 23;269(38):23784–23789. [PubMed] [Google Scholar]
  15. Gambello M. J., Iglewski B. H. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol. 1991 May;173(9):3000–3009. doi: 10.1128/jb.173.9.3000-3009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Genetello C., Van Larebeke N., Holsters M., De Picker A., Van Montagu M., Schell J. Ti plasmids of Agrobacterium as conjugative plasmids. Nature. 1977 Feb 10;265(5594):561–563. doi: 10.1038/265561a0. [DOI] [PubMed] [Google Scholar]
  17. Gray K. M., Passador L., Iglewski B. H., Greenberg E. P. Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. J Bacteriol. 1994 May;176(10):3076–3080. doi: 10.1128/jb.176.10.3076-3080.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Habeeb L. F., Wang L., Winans S. C. Transcription of the octopine catabolism operon of the Agrobacterium tumor-inducing plasmid pTiA6 is activated by a LysR-type regulatory protein. Mol Plant Microbe Interact. 1991 Jul-Aug;4(4):379–385. doi: 10.1094/mpmi-4-379. [DOI] [PubMed] [Google Scholar]
  19. Hwang I., Cook D. M., Farrand S. K. A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer. J Bacteriol. 1995 Jan;177(2):449–458. doi: 10.1128/jb.177.2.449-458.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hwang I., Li P. L., Zhang L., Piper K. R., Cook D. M., Tate M. E., Farrand S. K. TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4639–4643. doi: 10.1073/pnas.91.11.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ishihama A. Protein-protein communication within the transcription apparatus. J Bacteriol. 1993 May;175(9):2483–2489. doi: 10.1128/jb.175.9.2483-2489.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jagura-Burdzy G., Thomas C. M. KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10571–10575. doi: 10.1073/pnas.91.22.10571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones S., Yu B., Bainton N. J., Birdsall M., Bycroft B. W., Chhabra S. R., Cox A. J., Golby P., Reeves P. J., Stephens S. The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. EMBO J. 1993 Jun;12(6):2477–2482. doi: 10.1002/j.1460-2075.1993.tb05902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  25. Li J., Kustu S., Stewart V. In vitro interaction of nitrate-responsive regulatory protein NarL with DNA target sequences in the fdnG, narG, narK and frdA operon control regions of Escherichia coli K-12. J Mol Biol. 1994 Aug 12;241(2):150–165. doi: 10.1006/jmbi.1994.1485. [DOI] [PubMed] [Google Scholar]
  26. McGowan S., Sebaihia M., Jones S., Yu B., Bainton N., Chan P. F., Bycroft B., Stewart G. S., Williams P., Salmond G. P. Carbapenem antibiotic production in Erwinia carotovora is regulated by CarR, a homologue of the LuxR transcriptional activator. Microbiology. 1995 Mar;141(Pt 3):541–550. doi: 10.1099/13500872-141-3-541. [DOI] [PubMed] [Google Scholar]
  27. Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science. 1993 May 21;260(5111):1127–1130. doi: 10.1126/science.8493556. [DOI] [PubMed] [Google Scholar]
  28. Pierson L. S., 3rd, Keppenne V. D., Wood D. W. Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density. J Bacteriol. 1994 Jul;176(13):3966–3974. doi: 10.1128/jb.176.13.3966-3974.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Piper K. R., Beck von Bodman S., Farrand S. K. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature. 1993 Apr 1;362(6419):448–450. doi: 10.1038/362448a0. [DOI] [PubMed] [Google Scholar]
  30. Roggiani M., Dubnau D. ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. J Bacteriol. 1993 May;175(10):3182–3187. doi: 10.1128/jb.175.10.3182-3187.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shadel G. S., Baldwin T. O. Identification of a distantly located regulatory element in the luxD gene required for negative autoregulation of the Vibrio fischeri luxR gene. J Biol Chem. 1992 Apr 15;267(11):7690–7695. [PubMed] [Google Scholar]
  32. Shadel G. S., Baldwin T. O. Positive autoregulation of the Vibrio fischeri luxR gene. LuxR and autoinducer activate cAMP-catabolite gene activator protein complex-independent and -dependent luxR transcription. J Biol Chem. 1992 Apr 15;267(11):7696–7702. [PubMed] [Google Scholar]
  33. Shadel G. S., Baldwin T. O. The Vibrio fischeri LuxR protein is capable of bidirectional stimulation of transcription and both positive and negative regulation of the luxR gene. J Bacteriol. 1991 Jan;173(2):568–574. doi: 10.1128/jb.173.2.568-574.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stevens A. M., Dolan K. M., Greenberg E. P. Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12619–12623. doi: 10.1073/pnas.91.26.12619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Swift S., Winson M. K., Chan P. F., Bainton N. J., Birdsall M., Reeves P. J., Rees C. E., Chhabra S. R., Hill P. J., Throup J. P. A novel strategy for the isolation of luxI homologues: evidence for the widespread distribution of a LuxR:LuxI superfamily in enteric bacteria. Mol Microbiol. 1993 Nov;10(3):511–520. doi: 10.1111/j.1365-2958.1993.tb00923.x. [DOI] [PubMed] [Google Scholar]
  36. Tabata S., Hooykaas P. J., Oka A. Sequence determination and characterization of the replicator region in the tumor-inducing plasmid pTiB6S3. J Bacteriol. 1989 Mar;171(3):1665–1672. doi: 10.1128/jb.171.3.1665-1672.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tempé J., Petit A., Holsters M., Montagu M., Schell J. Thermosensitive step associated with transfer of the Ti plasmid during conjugation: Possible relation to transformation in crown gall. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2848–2849. doi: 10.1073/pnas.74.7.2848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wang L., Helmann J. D., Winans S. C. The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite. Cell. 1992 May 15;69(4):659–667. doi: 10.1016/0092-8674(92)90229-6. [DOI] [PubMed] [Google Scholar]
  39. Zhang L. H., Kerr A. A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens. J Bacteriol. 1991 Mar;173(6):1867–1872. doi: 10.1128/jb.173.6.1867-1872.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhang L., Murphy P. J., Kerr A., Tate M. E. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature. 1993 Apr 1;362(6419):446–448. doi: 10.1038/362446a0. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES