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. 1996 Dec;178(23):6888–6894. doi: 10.1128/jb.178.23.6888-6894.1996

The primase of broad-host-range plasmid R1162 is active in conjugal transfer.

D Henderson 1, R J Meyer 1
PMCID: PMC178590  PMID: 8955311

Abstract

The broad-host-range plasmid R1162 is conjugally mobilized at high frequency by the IncP-1 plasmid R751 but is poorly mobilized by pOX38, a derivative of the F factor. In both cases, the origin of transfer (oriT) and the Mob proteins of R1162 are required, indicating that these plasmids are mobilized by similar mechanisms. R1162 encodes a primase, essential for vegetative replication of the plasmid, that is made both as a separate protein and as the carboxy-terminal domain of MobA, one of the R1162 mobilization proteins (P. Scholz, V. Haring, B. Wittman-Liebold, K. Ashman, M. Bagdasarian, and E. Scherzinger, Gene 75:271-288, 1989). When R751 is the mobilizing vector, the primase is not required for mobilization of plasmids containing cloned mob-oriT R1162 DNA. However, detectable mobilization of such plasmids by pOX38 requires both the primase and its cognate initiation site, oriented for synthesis of the complement to the transferred strand. The long form of the primase is required for optimal transfer: R1162 replicons lacking this form also are not transferred detectably by pOX38 and are less well mobilized by R751. The distance between oriT and the primase initiation site affects the frequency of mobilization, and this effect is polar in the direction of transfer. Our results indicate that the R1162 primase is active in mobilization of R1162 and suggest that the MobA-linked form is an adaptation increasing its effectiveness during transfer.

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Selected References

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

  1. Bhattacharjee M. K., Meyer R. J. A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase. Nucleic Acids Res. 1991 Mar 11;19(5):1129–1137. doi: 10.1093/nar/19.5.1129. [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. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [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. Brasch M. A., Meyer R. J. Genetic organization of plasmid R1162 DNA involved in conjugative mobilization. J Bacteriol. 1986 Aug;167(2):703–710. doi: 10.1128/jb.167.2.703-710.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chandler M., Galas D. J. Cointegrate formation mediated by Tn9. II. Activity of IS1 is modulated by external DNA sequences. J Mol Biol. 1983 Oct 15;170(1):61–91. doi: 10.1016/s0022-2836(83)80227-7. [DOI] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Derbyshire K. M., Hatfull G., Willetts N. Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region. Mol Gen Genet. 1987 Jan;206(1):161–168. doi: 10.1007/BF00326552. [DOI] [PubMed] [Google Scholar]
  9. Figurski D., Meyer R., Miller D. S., Helinski D. R. Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease. Gene. 1976;1(1):107–119. doi: 10.1016/0378-1119(76)90010-x. [DOI] [PubMed] [Google Scholar]
  10. Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
  11. Hershfield V., Boyer H. W., Yanofsky C., Lovett M. A., Helinski D. R. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3455–3459. doi: 10.1073/pnas.71.9.3455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honda Y., Sakai H., Komano T. Two single-strand DNA initiation signals located in the oriV region of plasmid RSF1010. Gene. 1988 Sep 7;68(2):221–228. doi: 10.1016/0378-1119(88)90024-8. [DOI] [PubMed] [Google Scholar]
  13. Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim K., Meyer R. J. Unidirectional transfer of broad host-range plasmid R1162 during conjugative mobilization. Evidence for genetically distinct events at oriT. J Mol Biol. 1989 Aug 5;208(3):501–505. doi: 10.1016/0022-2836(89)90513-5. [DOI] [PubMed] [Google Scholar]
  15. Lin L. S., Meyer R. J. DNA synthesis is initiated at two positions within the origin of replication of plasmid R1162. Nucleic Acids Res. 1987 Oct 26;15(20):8319–8331. doi: 10.1093/nar/15.20.8319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mead G. M., Thompson J., Sweetenham J. W., Buchanan R. B., Whitehouse J. M., Williams C. J. Extensive stage small cell carcinoma of the bronchus. A randomised study of etoposide given orally by one-day or five-day schedule together with intravenous adriamycin and cyclophosphamide. Cancer Chemother Pharmacol. 1987;19(2):172–174. doi: 10.1007/BF00254574. [DOI] [PubMed] [Google Scholar]
  17. Merryweather A., Rees C. E., Smith N. M., Wilkins B. M. Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria. EMBO J. 1986 Nov;5(11):3007–3012. doi: 10.1002/j.1460-2075.1986.tb04599.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rao X. M., Meyer R. J. Conjugal mobilization of plasmid DNA: termination frequency at the origin of transfer of plasmid R1162. J Bacteriol. 1994 Oct;176(19):5958–5961. doi: 10.1128/jb.176.19.5958-5961.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scherzinger E., Bagdasarian M. M., Scholz P., Lurz R., Rückert B., Bagdasarian M. Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins. Proc Natl Acad Sci U S A. 1984 Feb;81(3):654–658. doi: 10.1073/pnas.81.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Scholz P., Haring V., Wittmann-Liebold B., Ashman K., Bagdasarian M., Scherzinger E. Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene. 1989 Feb 20;75(2):271–288. doi: 10.1016/0378-1119(89)90273-4. [DOI] [PubMed] [Google Scholar]
  22. Weiss R. B., Dunn D. M., Atkins J. F., Gesteland R. F. Slippery runs, shifty stops, backward steps, and forward hops: -2, -1, +1, +2, +5, and +6 ribosomal frameshifting. Cold Spring Harb Symp Quant Biol. 1987;52:687–693. doi: 10.1101/sqb.1987.052.01.078. [DOI] [PubMed] [Google Scholar]
  23. Willetts N., Crowther C. Mobilization of the non-conjugative IncQ plasmid RSF1010. Genet Res. 1981 Jun;37(3):311–316. doi: 10.1017/s0016672300020310. [DOI] [PubMed] [Google Scholar]
  24. Wong E. M., Muesing M. A., Polisky B. Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3570–3574. doi: 10.1073/pnas.79.11.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Zhou H. S., Meyer R. J. Deletion of sites for initiation of DNA synthesis in the origin of broad host-range plasmid R1162. J Mol Biol. 1990 Aug 5;214(3):685–697. doi: 10.1016/0022-2836(90)90286-u. [DOI] [PubMed] [Google Scholar]

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