Applied Microbiology and Biotechnology

, Volume 102, Issue 4, pp 1823–1836 | Cite as

Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system

  • Shinji YamamotoEmail author
  • Ayako Sakai
  • Vita Agustina
  • Kazuki Moriguchi
  • Katsunori Suzuki
Applied genetics and molecular biotechnology


Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new “Ti/Ri eviction plasmids,” each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.


Ti plasmid Plasmid curing Agrobacterium Plasmid incompatibility Luciferase reporter 



This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 25850003, 24580115, and 15H04479. We thank Dr. E. Szegedi for providing bacterial strains and Dr. T. Oyama (Kyoto University) for the luxAB plasmid. Pathogenic strains isolated outside of Japan were imported with permissions issued by the Japanese Plant Quarantine Office and were handled under directions given by the same organization.


This study was funded by the JSPS (25850003, 24580115, and 15H04479).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Austin S, Nordström K (1990) Partition-mediated incompatibility of bacterial plasmids. Cell 60(3):351–354. PubMedCrossRefGoogle Scholar
  2. Baldwin TO, Berends T, Bunch TA, Holzman TF, Rausch SK, Shamansky L, Treat ML, Ziegler MM (1984) Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry 23(16):3663–3667. PubMedCrossRefGoogle Scholar
  3. Barton BM, Harding GP, Zuccarelli AJ (1995) A general method for detecting and sizing large plasmids. Anal Biochem 226(2):235–240. PubMedCrossRefGoogle Scholar
  4. Boivin R, Chalifour FP, Dion P (1988) Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium. Mol Gen Genet 213(1):50–55. PubMedCrossRefGoogle Scholar
  5. Broothaerts W, Mitchell HJ, Weir B, Kaines S, Smith LM, Yang W, Mayer JE, Roa-Rodriguez C, Jefferson RA (2005) Gene transfer to plants by diverse species of bacteria. Nature 433(7026):629–633. PubMedCrossRefGoogle Scholar
  6. Cervantes-Rivera R, Romero-López C, Berzal-Herranz A, Cevallos MA (2010) Analysis of the mechanism of action of the antisense RNA that controls the replication of the repABC plasmid p42d. J Bacteriol 192(13):3268–3278. PubMedPubMedCentralCrossRefGoogle Scholar
  7. Cervantes-Rivera R, Pedraza-López F, Pérez-Segura G, Cevallos MA (2011) The replication origin of a repABC plasmid. BMC Microbiol 11(1):1–14. CrossRefGoogle Scholar
  8. Cevallos MA, Cervantes-Rivera R, Gutiérrez-Ríos RM (2008) The repABC plasmid family. Plasmid 60(1):19–37. PubMedCrossRefGoogle Scholar
  9. Chai Y, Winans SC (2005) A small antisense RNA downregulates expression of an essential replicase protein of an Agrobacterium tumefaciens Ti plasmid. Mol Microbiol 56(6):1574–1585. PubMedCrossRefGoogle Scholar
  10. Chilton M-D, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci U S A 71(9):3672–3676. PubMedPubMedCentralCrossRefGoogle Scholar
  11. Christie PJ, Gordon JE (2014) The Agrobacterium Ti plasmids. Microbiol Spectr 2(6).
  12. Gérard JC, Canaday J, Szegedi E, de la Salle H, Otten L (1992) Physical map of the vitopine Ti plasmid pTiS4. Plasmid 28(2):146–156. PubMedCrossRefGoogle Scholar
  13. Gonzalez V, Santamaria RI, Bustos P, Hernandez-Gonzalez I, Medrano-Soto A, Moreno-Hagelsieb G, Janga SC, Ramirez MA, Jimenez-Jacinto V, Collado-Vides J, Davila G (2006) The partitioned Rhizobium etli genome: genetic and metabolic redundancy in seven interacting replicons. Proc Natl Acad Sci U S A 103(10):3834–3839. PubMedPubMedCentralCrossRefGoogle Scholar
  14. Goodner B, Hinkle G, Gattung S, Miller N, Blanchard M, Qurollo B, Goldman BS, Cao Y, Askenazi M, Halling C, Mullin L, Houmiel K, Gordon J, Vaudin M, Iartchouk O, Epp A, Liu F, Wollam C, Allinger M, Doughty D, Scott C, Lappas C, Markelz B, Flanagan C, Crowell C, Gurson J, Lomo C, Sear C, Strub G, Cielo C, Slater S (2001) Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294(5550):2323–2328. PubMedCrossRefGoogle Scholar
  15. Hamilton RH, Fall MZ (1971) The loss of tumor-initiating ability in Agrobacterium tumefaciens by incubation at high temperature. Experientia 27(2):229–230. PubMedCrossRefGoogle Scholar
  16. Hattori Y, Iwata K, Suzuki K, Uraji M, Ohta N, Katoh A, Yoshida K (2001) Sequence characterization of the vir region of a nopaline type Ti plasmid, pTi-SAKURA. Genes Genet Syst 76(2):121–130. PubMedCrossRefGoogle Scholar
  17. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168(3):1291–1301. PubMedPubMedCentralCrossRefGoogle Scholar
  18. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2(4):208–218. CrossRefGoogle Scholar
  19. Hooykaas PJ, den Dulk-Ras H, Ooms G, Schilperoort RA (1980) Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens. J Bacteriol 143(3):1295–1306PubMedPubMedCentralGoogle Scholar
  20. Jouanin L, Tourneur J, Tourneur C, Casse-Delbart F (1986) Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4. Plasmid 16(2):124–134. PubMedCrossRefGoogle Scholar
  21. Khan SR, Gaines J, Roop RM, Farrand SK (2008) Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ Microbiol 74(16):5053–5062. PubMedPubMedCentralCrossRefGoogle Scholar
  22. Kiyokawa K, Yamamoto S, Sakuma K, Tanaka K, Moriguchi K, Suzuki K (2009) Construction of disarmed Ti plasmids transferable between Escherichia coli and Agrobacterium species. Appl Environ Microbiol 75(7):1845–1851. PubMedPubMedCentralCrossRefGoogle Scholar
  23. Kiyokawa K, Yamamoto S, Sato Y, Momota N, Tanaka K, Moriguchi K, Suzuki K (2012) Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid. Genes Cells 17(7):597–610. PubMedCrossRefGoogle Scholar
  24. Knauf VC, Nester EW (1982) Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid 8(1):45–54. PubMedCrossRefGoogle Scholar
  25. Kovacs LG, Pueppke SG (1993) The chromosomal background of Agrobacterium tumefaciens Chry5 conditions high virulence on soybean. Mol Plant-Microbe Interact 6(5):601–608. CrossRefGoogle Scholar
  26. Lacroix B, Tzfira T, Vainstein A, Citovsky V (2006) A case of promiscuity: Agrobacterium’s endless hunt for new partners. Trends Genet 22(1):29–37. PubMedCrossRefGoogle Scholar
  27. Maas R, Saadi S, Maas WK (1989) Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86. Mol Gen Genet MGG 218(2):190–198. PubMedCrossRefGoogle Scholar
  28. Moore L, Warren G, Strobel G (1979) Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes. Plasmid 2(4):617–626. PubMedCrossRefGoogle Scholar
  29. Morton ER, Merritt PM, Bever JD, Fuqua C (2013) Large deletions in the pAtC58 megaplasmid of Agrobacterium tumefaciens can confer reduced carriage cost and increased expression of virulence genes. Genome Biol Evol 5:1353–1364.
  30. Nair GR, Liu Z, Binns AN (2003) Reexamining the role of the accessory plasmid pAtC58 in the virulence of Agrobacterium tumefaciens strain C58. Plant Physiol 133(3):989–999. PubMedPubMedCentralCrossRefGoogle Scholar
  31. Nester EW, Kosuge T (1981) Plasmids specifying plant hyperplasias. Annu Rev Microbiol 35(1):531–565. PubMedCrossRefGoogle Scholar
  32. Nishikawa M, Suzuki K, Yoshida K (1990) Structural and functional stability of IncP plasmids during stepwise transmission by trans-kingdom mating: promiscuous conjugation of Escherichia coli and Saccharomyces cerevisiae. Japanese. J Genet 65:323–334Google Scholar
  33. Ono NN, Tian L (2011) The multiplicity of hairy root cultures: prolific possibilities. Plant Sci 180(3):439–446. PubMedCrossRefGoogle Scholar
  34. Otten L, Burr T, Szegedi E (2008) Agrobacterium: a disease-causing bacterium. In: Tzfira T, Citovsky V (eds) Agrobacterium: from biology to biotechnology. Springer New York, New York, pp 1–46. Google Scholar
  35. Pappas KM, Cevallos MA (2011) Plasmids of the Rhizobiaceae and their role in interbacterial and transkingdom interactions. In: Biocommunication in soil microorganisms. Springer Berlin Heidelberg, Germany, pp 295–337. CrossRefGoogle Scholar
  36. Pappas KM, Winans SC (2003) The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon. Mol Microbiol 49(2):441–455. PubMedCrossRefGoogle Scholar
  37. Pérez-Oseguera Á, Cevallos MA (2013) RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon. Plasmid 70(3):362–376. PubMedCrossRefGoogle Scholar
  38. Pinto UM, Pappas KM, Winans SC (2012) The ABCs of plasmid replication and segregation. Nat Rev Microbiol 10:755–765PubMedCrossRefGoogle Scholar
  39. Ramírez-Romero MA, Soberón N, Pérez-Oseguera A, Téllez-Sosa J, Cevallos MA (2000) Structural elements required for replication and incompatibility of the Rhizobium etli symbiotic plasmid. J Bacteriol 182(11):3117–3124. PubMedPubMedCentralCrossRefGoogle Scholar
  40. Ramírez-Romero MA, Tellez-Sosa J, Barrios H, Perez-Oseguera A, Rosas V, Cevallos MA (2001) RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon. Mol Microbiol 42(1):195–204PubMedCrossRefGoogle Scholar
  41. Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145(1):69–73. PubMedCrossRefGoogle Scholar
  42. Slater SC, Goodner BW, Setubal JC, Goldman BS, Wood DW, Nester EW (2008) The Agrobacterium tumefaciens C58 genome. In: Tzfira T, Citovsky V (eds) Agrobacterium: from biology to biotechnology. Springer New York, New York, pp 149–181. CrossRefGoogle Scholar
  43. Soberón N, Venkova-Canova T, Ramírez-Romero MA, Téllez-Sosa J, Cevallos MA (2004) Incompatibility and the partitioning site of the repABC basic replicon of the symbiotic plasmid from Rhizobium etli. Plasmid 51(3):203–216. PubMedCrossRefGoogle Scholar
  44. Suzuki K, Hattori Y, Uraji M, Ohta N, Iwata K, Murata K, Kato A, Yoshida K (2000) Complete nucleotide sequence of a plant tumor-inducing Ti plasmid. Gene 242(1-2):331–336. PubMedCrossRefGoogle Scholar
  45. Suzuki K, Tanaka K, Yamamoto S, Kiyokawa K, Moriguchi K, Yoshida K (2009) Ti and Ri plasmids. In: Schwartz E (ed) Microbial megaplasmids. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 133–147. CrossRefGoogle Scholar
  46. Szegedi E, Czakó M, Otten L, Koncz CS (1988) Opines in crown gall tumours induced by biotype 3 isolates of Agrobacterium tumefaciens. Physiol Mol Plant Pathol 32(2):237–247. CrossRefGoogle Scholar
  47. Szegedi E, Czako M, Otten L (1996) Further evidence that the vitopine-type pTi’s of Agrobacterium vitis represent a novel group of Ti plasmids. Mol Plant-Microbe Interact 9(2):139–143. CrossRefGoogle Scholar
  48. Szittner R, Meighen E (1990) Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium. J Biol Chem 265(27):16581–16587PubMedGoogle Scholar
  49. Tanaka K, Arafat HH, Urbanczyk H, Yamamoto S, Moriguchi K, Sawada H, Suzuki K (2009) Ability of Agrobacterium tumefaciens and A. rhizogenes strains, inability of A. vitis and A. rubi strains to adapt to salt-insufficient environment, and taxonomic significance of a simple salt requirement test in the pathogenic Agrobacterium species. J Gen Appl Microbiol 55(1):35–41. PubMedCrossRefGoogle Scholar
  50. Tzfira T, Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 17(2):147–154. PubMedCrossRefGoogle Scholar
  51. Uraji M, Suzuki K, Yoshida K (2002) A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens. Genes Genet Syst 77(1):1–9. PubMedCrossRefGoogle Scholar
  52. Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen I, Schilperoort RA, Schell J (1974) Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252(5479):169–170. PubMedCrossRefGoogle Scholar
  53. Venkova-Canova T, Soberón NE, Ramírez-Romero MA, Cevallos MA (2004) Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem-loop structure. Mol Microbiol 54(5):1431–1444. PubMedCrossRefGoogle Scholar
  54. Wendt T, Doohan F, Winckelmann D, Mullins E (2011) Gene transfer into Solanum tuberosum via Rhizobium spp. Transgenic Res 20(2):377–386. PubMedCrossRefGoogle Scholar
  55. White FF, Nester EW (1980) Relationship of plasmids responsible for hairy root and crown gall tumorigenicity. J Bacteriol 144(2):710–720PubMedPubMedCentralGoogle Scholar
  56. Wilms I, Overloper A, Nowrousian M, Sharma CM, Narberhaus F (2012) Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens. RNA Biol 9(4):446–457. PubMedPubMedCentralCrossRefGoogle Scholar
  57. Wise AA, Liu Z, Binns AN (2006) Culture and maintenance of Agrobacterium strains. In: Wang K (ed) Agrobacterium protocols. Humana Press, Totowa, pp 3–14.
  58. Yamamoto S, Suzuki K (2012) Development of a reinforced Ti-eviction plasmid useful for construction of Ti plasmid-free Agrobacterium strains. J Microbiol Methods 89(1):53–56. PubMedCrossRefGoogle Scholar
  59. Yamamoto S, Uraji M, Tanaka K, Moriguchi K, Suzuki K (2007) Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability. Genes Genet Syst 82(3):197–206. PubMedCrossRefGoogle Scholar
  60. Yamamoto S, Kiyokawa K, Tanaka K, Moriguchi K, Suzuki K (2009) Novel toxin-antitoxin system composed of serine protease and AAA-ATPase homologues determines the high level of stability and incompatibility of the tumor-inducing plasmid pTiC58. J Bacteriol 191(14):4656–4666. PubMedPubMedCentralCrossRefGoogle Scholar
  61. Yamamoto S, Agustina V, Sakai A, Moriguchi K, Suzuki K (2017) An extra repABC locus in the incRh2 Ti plasmid pTiBo542 exerts incompatibility toward an incRh1 plasmid. Plasmid 90:20–29. PubMedCrossRefGoogle Scholar
  62. Young JPW, Crossman LC, Johnston AWB, Thomson NR, Ghazoui ZF, Hull KH, Wexler M, Curson ARJ, Todd JD, Poole PS, Mauchline TH, East AK, Quail MA, Churcher C, Arrowsmith C, Cherevach I, Chillingworth T, Clarke K, Cronin A, Davis P, Fraser A, Hance Z, Hauser H, Jagels K, Moule S, Mungall K, Norbertczak H, Rabbinowitsch E, Sanders M, Simmonds M, Whitehead S, Parkhill J (2006) The genome of Rhizobium leguminosarum has recognizable core and accessory components. Genome Biol 7(4):R34. PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Shinji Yamamoto
    • 1
    Email author
  • Ayako Sakai
    • 1
  • Vita Agustina
    • 1
  • Kazuki Moriguchi
    • 1
  • Katsunori Suzuki
    • 1
  1. 1.Department of Biological Science, Graduate School of ScienceHiroshima UniversityHiroshimaJapan

Personalised recommendations