Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Sep;179(18):5768–5776. doi: 10.1128/jb.179.18.5768-5776.1997

Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT.

E S Paterson 1, V N Iyer 1
PMCID: PMC179465  PMID: 9294433

Abstract

The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.

Full Text

The Full Text of this article is available as a PDF (204.9 KB).

Selected References

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

  1. Barlett M. M., Erickson M. J., Meyer R. J. Recombination between directly repeated origins of conjugative transfer cloned in M13 bacteriophage DNA models ligation of the transferred plasmid strand. Nucleic Acids Res. 1990 Jun 25;18(12):3579–3586. doi: 10.1093/nar/18.12.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhattacharjee M. K., Meyer R. J. Specific binding of MobA, a plasmid-encoded protein involved in the initiation and termination of conjugal DNA transfer, to single-stranded oriT DNA. Nucleic Acids Res. 1993 Sep 25;21(19):4563–4568. doi: 10.1093/nar/21.19.4563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhattacharjee M., Rao X. M., Meyer R. J. Role of the origin of transfer in termination of strand transfer during bacterial conjugation. J Bacteriol. 1992 Oct;174(20):6659–6665. doi: 10.1128/jb.174.20.6659-6665.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brasch M. A., Meyer R. J. A 38 base-pair segment of DNA is required in cis for conjugative mobilization of broad host-range plasmid R1162. J Mol Biol. 1987 Dec 5;198(3):361–369. doi: 10.1016/0022-2836(87)90286-5. [DOI] [PubMed] [Google Scholar]
  5. Coupland G. M., Brown A. M., Willetts N. S. The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing. Mol Gen Genet. 1987 Jun;208(1-2):219–225. doi: 10.1007/BF00330445. [DOI] [PubMed] [Google Scholar]
  6. Farrand S. K., Hwang I., Cook D. M. The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F. J Bacteriol. 1996 Jul;178(14):4233–4247. doi: 10.1128/jb.178.14.4233-4247.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Furuya N., Komano T. Specific binding of the NikA protein to one arm of 17-base-pair inverted repeat sequences within the oriT region of plasmid R64. J Bacteriol. 1995 Jan;177(1):46–51. doi: 10.1128/jb.177.1.46-51.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fürste J. P., Pansegrau W., Ziegelin G., Kröger M., Lanka E. Conjugative transfer of promiscuous IncP plasmids: interaction of plasmid-encoded products with the transfer origin. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1771–1775. doi: 10.1073/pnas.86.6.1771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gao Q., Luo Y., Deonier R. C. Initiation and termination of DNA transfer at F plasmid oriT. Mol Microbiol. 1994 Feb;11(3):449–458. doi: 10.1111/j.1365-2958.1994.tb00326.x. [DOI] [PubMed] [Google Scholar]
  10. Hengen P. N., Iyer V. N. DNA cassettes containing the origin of transfer (oriT) of two broad-host-range transfer systems. Biotechniques. 1992 Jul;13(1):56-8, 60, 62. [PubMed] [Google Scholar]
  11. Inamoto S., Yoshioka Y., Ohtsubo E. Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100. J Biol Chem. 1991 Jun 5;266(16):10086–10092. [PubMed] [Google Scholar]
  12. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  13. Lahue E. E., Matson S. W. Purified Escherichia coli F-factor TraY protein binds oriT. J Bacteriol. 1990 Mar;172(3):1385–1391. doi: 10.1128/jb.172.3.1385-1391.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lanka E., Wilkins B. M. DNA processing reactions in bacterial conjugation. Annu Rev Biochem. 1995;64:141–169. doi: 10.1146/annurev.bi.64.070195.001041. [DOI] [PubMed] [Google Scholar]
  15. Llosa M., Bolland S., Grandoso G., de la Cruz F. Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC. J Bacteriol. 1994 Jun;176(11):3210–3217. doi: 10.1128/jb.176.11.3210-3217.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Llosa M., Bolland S., de la Cruz F. Genetic organization of the conjugal DNA processing region of the IncW plasmid R388. J Mol Biol. 1994 Jan 14;235(2):448–464. doi: 10.1006/jmbi.1994.1005. [DOI] [PubMed] [Google Scholar]
  17. Llosa M., Grandoso G., de la Cruz F. Nicking activity of TrwC directed against the origin of transfer of the IncW plasmid R388. J Mol Biol. 1995 Feb 10;246(1):54–62. doi: 10.1006/jmbi.1994.0065. [DOI] [PubMed] [Google Scholar]
  18. Loewen P. C., Switala J. Template secondary structure can increase the error frequency of the DNA polymerase from Thermus aquaticus. Gene. 1995 Oct 16;164(1):59–63. doi: 10.1016/0378-1119(95)00383-h. [DOI] [PubMed] [Google Scholar]
  19. Luo Y., Gao Q., Deonier R. C. Boundaries of the nicking region for the F plasmid transfer origin, oriT. Mol Microbiol. 1995 Mar;15(5):829–837. doi: 10.1111/j.1365-2958.1995.tb02353.x. [DOI] [PubMed] [Google Scholar]
  20. Luo Y., Gao Q., Deonier R. C. Mutational and physical analysis of F plasmid traY protein binding to oriT. Mol Microbiol. 1994 Feb;11(3):459–469. doi: 10.1111/j.1365-2958.1994.tb00327.x. [DOI] [PubMed] [Google Scholar]
  21. Matson S. W., Nelson W. C., Morton B. S. Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I. J Bacteriol. 1993 May;175(9):2599–2606. doi: 10.1128/jb.175.9.2599-2606.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nelson W. C., Morton B. S., Lahue E. E., Matson S. W. Characterization of the Escherichia coli F factor traY gene product and its binding sites. J Bacteriol. 1993 Apr;175(8):2221–2228. doi: 10.1128/jb.175.8.2221-2228.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  24. Pansegrau W., Lanka E. Mechanisms of initiation and termination reactions in conjugative DNA processing. Independence of tight substrate binding and catalytic activity of relaxase (TraI) of IncPalpha plasmid RP4. J Biol Chem. 1996 May 31;271(22):13068–13076. doi: 10.1074/jbc.271.22.13068. [DOI] [PubMed] [Google Scholar]
  25. Pansegrau W., Schröder W., Lanka E. Relaxase (TraI) of IncP alpha plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2925–2929. doi: 10.1073/pnas.90.7.2925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pansegrau W., Ziegelin G., Lanka E. Covalent association of the traI gene product of plasmid RP4 with the 5'-terminal nucleotide at the relaxation nick site. J Biol Chem. 1990 Jun 25;265(18):10637–10644. [PubMed] [Google Scholar]
  27. Paterson E. S., Iyer V. N. The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids. J Bacteriol. 1992 Jan;174(2):499–507. doi: 10.1128/jb.174.2.499-507.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sanjanwala B., Ganesan A. T. Genetic structure and domains of DNA polymerase III of Bacillus subtilis. Mol Gen Genet. 1991 May;226(3):467–472. doi: 10.1007/BF00260660. [DOI] [PubMed] [Google Scholar]
  29. Scherzinger E., Lurz R., Otto S., Dobrinski B. In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins. Nucleic Acids Res. 1992 Jan 11;20(1):41–48. doi: 10.1093/nar/20.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thatte V., Iyer V. N. Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli. Gene. 1983 Mar;21(3):227–236. doi: 10.1016/0378-1119(83)90006-9. [DOI] [PubMed] [Google Scholar]
  31. Thompson T. L., Centola M. B., Deonier R. C. Location of the nick at oriT of the F plasmid. J Mol Biol. 1989 Jun 5;207(3):505–512. doi: 10.1016/0022-2836(89)90460-9. [DOI] [PubMed] [Google Scholar]
  32. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  33. Waters V. L., Hirata K. H., Pansegrau W., Lanka E., Guiney D. G. Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1456–1460. doi: 10.1073/pnas.88.4.1456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Willetts N., Maule J. Specificities of IncF plasmid conjugation genes. Genet Res. 1986 Feb;47(1):1–11. doi: 10.1017/s0016672300024447. [DOI] [PubMed] [Google Scholar]
  35. Zhang S., Meyer R. J. Localized denaturation of oriT DNA within relaxosomes of the broad-host-range plasmid R1162. Mol Microbiol. 1995 Aug;17(4):727–735. doi: 10.1111/j.1365-2958.1995.mmi_17040727.x. [DOI] [PubMed] [Google Scholar]
  36. Ziegelin G., Fürste J. P., Lanka E. TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin. J Biol Chem. 1989 Jul 15;264(20):11989–11994. [PubMed] [Google Scholar]

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

RESOURCES