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. 1985 Oct;164(1):446–455. doi: 10.1128/jb.164.1.446-455.1985

Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.

T J Schmidhauser, D R Helinski
PMCID: PMC214264  PMID: 4044529

Abstract

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.

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Selected References

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  1. Barth P. T., Ellis K., Bechhofer D. H., Figurski D. H. Involvement of kil and kor genes in the phenotype of a host-range mutant of RP4. Mol Gen Genet. 1984;197(2):236–243. doi: 10.1007/BF00330969. [DOI] [PubMed] [Google Scholar]
  2. Bechhofer D. H., Figurski D. H. Map location and nucleotide sequence of korA, a key regulatory gene of promiscuous plasmid RK2. Nucleic Acids Res. 1983 Nov 11;11(21):7453–7469. doi: 10.1093/nar/11.21.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burkardt H. J., Riess G., Pühler A. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol. 1979 Oct;114(2):341–348. doi: 10.1099/00221287-114-2-341. [DOI] [PubMed] [Google Scholar]
  5. Chikami G. K., Guiney D. G., Schmidhauser T. J., Helinski D. R. Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication. J Bacteriol. 1985 May;162(2):656–660. doi: 10.1128/jb.162.2.656-660.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Corbin D., Ditta G., Helinski D. R. Clustering of nitrogen fixation (nif) genes in Rhizobium meliloti. J Bacteriol. 1982 Jan;149(1):221–228. doi: 10.1128/jb.149.1.221-228.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  8. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Figurski D. H., Pohlman R. F., Bechhofer D. H., Prince A. S., Kelton C. A. Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1935–1939. doi: 10.1073/pnas.79.6.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Figurski D. H., Young C., Schreiner H. C., Pohlman R. F., Bechhofer D. H., Prince A. S., D'Amico T. F. Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon. Basic Life Sci. 1985;30:227–241. doi: 10.1007/978-1-4613-2447-8_19. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Fornari C. S., Kaplan S. Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA. J Bacteriol. 1982 Oct;152(1):89–97. doi: 10.1128/jb.152.1.89-97.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guiney D. G. Host range of conjugation and replication functions of the Escherichia coli sex plasmid Flac. Comparison with the broad host-range plasmid RK2. J Mol Biol. 1982 Dec 15;162(3):699–703. doi: 10.1016/0022-2836(82)90397-7. [DOI] [PubMed] [Google Scholar]
  15. Guiney D. G., Yakobson E. Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3595–3598. doi: 10.1073/pnas.80.12.3595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karoui H., Bex F., Drèze P., Couturier M. Ham22, a mini-F mutation which is lethal to host cell and promotes recA-dependent induction of lambdoid prophage. EMBO J. 1983;2(11):1863–1868. doi: 10.1002/j.1460-2075.1983.tb01672.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kornacki J. A., West A. H., Firshein W. Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2. Plasmid. 1984 Jan;11(1):48–57. doi: 10.1016/0147-619x(84)90006-4. [DOI] [PubMed] [Google Scholar]
  18. Lanka E., Barth P. T. Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance. J Bacteriol. 1981 Dec;148(3):769–781. doi: 10.1128/jb.148.3.769-781.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meyer R. J., Helinski D. R. Unidirectional replication of the P-group plasmid RK2. Biochim Biophys Acta. 1977 Sep 6;478(1):109–113. doi: 10.1016/0005-2787(77)90249-0. [DOI] [PubMed] [Google Scholar]
  20. Meyer R., Figurski D., Helinski D. R. Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2. Mol Gen Genet. 1977 Apr 29;152(3):129–135. doi: 10.1007/BF00268809. [DOI] [PubMed] [Google Scholar]
  21. Meyer R., Hinds M. Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2. J Bacteriol. 1982 Dec;152(3):1078–1090. doi: 10.1128/jb.152.3.1078-1090.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'neill E. A., Berlinberg C., Bender R. A. Activity of Plasmid Replicons in CAULOBACTER CRESCENTUS : Rp4 and Cole1. Genetics. 1983 Apr;103(4):593–604. doi: 10.1093/genetics/103.4.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ogura T., Hiraga S. Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4784–4788. doi: 10.1073/pnas.80.15.4784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pohlman R. F., Figurski D. H. Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA. J Bacteriol. 1983 Nov;156(2):584–591. doi: 10.1128/jb.156.2.584-591.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schmidhauser T. J., Filutowicz M., Helinski D. R. Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria. Plasmid. 1983 May;9(3):325–330. doi: 10.1016/0147-619x(83)90010-0. [DOI] [PubMed] [Google Scholar]
  26. Schwab H., Saurugger P. N., Lafferty R. M. Occurrence of deletion plasmids at high rates after conjugative transfer of the plasmids RP4 and RK2 from Escherichia coli to Alcaligenes eutrophus H16. Arch Microbiol. 1983 Nov;136(2):140–146. doi: 10.1007/BF00404789. [DOI] [PubMed] [Google Scholar]
  27. Selvaraj G., Iyer V. N. Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. J Bacteriol. 1983 Dec;156(3):1292–1300. doi: 10.1128/jb.156.3.1292-1300.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shinger V., Thomas C. M. Transcription in the trfA region of broad host range plasmid RK2 is regulated by trfB and korB. Mol Gen Genet. 1984;195(3):523–529. doi: 10.1007/BF00341457. [DOI] [PubMed] [Google Scholar]
  29. Shingler V., Thomas C. M. Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products. J Mol Biol. 1984 May 25;175(3):229–249. doi: 10.1016/0022-2836(84)90346-2. [DOI] [PubMed] [Google Scholar]
  30. Smith C. A., Shingler V., Thomas C. M. The trfA and trfB promoter regions of broad host range plasmid RK2 share common potential regulatory sequences. Nucleic Acids Res. 1984 Apr 25;12(8):3619–3630. doi: 10.1093/nar/12.8.3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smith C. A., Thomas C. M. Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2. Mol Gen Genet. 1983;190(2):245–254. doi: 10.1007/BF00330647. [DOI] [PubMed] [Google Scholar]
  32. Smith C. A., Thomas C. M. Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2. J Gen Microbiol. 1984 Jul;130(7):1651–1663. doi: 10.1099/00221287-130-7-1651. [DOI] [PubMed] [Google Scholar]
  33. Smith C. A., Thomas C. M. Nucleotide sequence of the trfA gene of broad host-range plasmid RK2. J Mol Biol. 1984 May 25;175(3):251–262. doi: 10.1016/0022-2836(84)90347-4. [DOI] [PubMed] [Google Scholar]
  34. Stokes H. W., Moore R. J., Krishnapillai V. Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18. Plasmid. 1981 Mar;5(2):202–212. doi: 10.1016/0147-619x(81)90021-4. [DOI] [PubMed] [Google Scholar]
  35. Thomas C. M. Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1. Plasmid. 1981 May;5(3):277–291. doi: 10.1016/0147-619x(81)90005-6. [DOI] [PubMed] [Google Scholar]
  36. Thomas C. M., Hussain A. A., Smith C. A. Maintenance of broad host range plasmid RK2 replicons in Pseudomonas aeruginosa. Nature. 1982 Aug 12;298(5875):674–676. doi: 10.1038/298674a0. [DOI] [PubMed] [Google Scholar]
  37. Thomas C. M., Hussain A. A. The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression. EMBO J. 1984 Jul;3(7):1513–1519. doi: 10.1002/j.1460-2075.1984.tb02004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thomas C. M., Meyer R., Helinski D. R. Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol. 1980 Jan;141(1):213–222. doi: 10.1128/jb.141.1.213-222.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Thomas C. M., Smith C. A., Shingler V., Cross M. A., Hussain A. A., Pinkney M. Regulation of replication and maintenance functions of broad host-range plasmid RK2. Basic Life Sci. 1985;30:261–276. doi: 10.1007/978-1-4613-2447-8_21. [DOI] [PubMed] [Google Scholar]
  40. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  41. Watson B., Currier T. C., Gordon M. P., Chilton M. D., Nester E. W. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1975 Jul;123(1):255–264. doi: 10.1128/jb.123.1.255-264.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Young C., Bechhofer D. H., Figurski D. H. Gene regulation in plasmid RK2: positive control by korA in the expression of korC. J Bacteriol. 1984 Jan;157(1):247–252. doi: 10.1128/jb.157.1.247-252.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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