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. 1996 Jul;62(7):2621–2628. doi: 10.1128/aem.62.7.2621-2628.1996

Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR.

A Götz 1, R Pukall 1, E Smit 1, E Tietze 1, R Prager 1, H Tschäpe 1, J D van Elsas 1, K Smalla 1
PMCID: PMC168041  PMID: 8779598

Abstract

Primer systems for PCR amplification of different replicon-specific DNA regions were designed on the basis of published sequences for plasmids belonging to the incompatibility (Inc) groups IncP, IncN, IncW, and IncQ. The specificities of these primer systems for the respective Inc groups were tested with a collection of reference plasmids belonging to 21 different Inc groups. Almost all primer systems were found to be highly specific for the reference plasmid for which they were designed. In addition, the primers were tested with plasmids which had previously been grouped by traditional incompatibility testing to the IncN, IncW, IncP, or IncQ group. All IncQ plasmids gave PCR products with the IncQ primer systems tested. However, PCR products were obtained for only some of the IncN, IncP, and IncW group plasmids. Dot blot and Southern blot analyses of the plasmids revealed that PCR-negative plasmids also failed to hybridize with probes derived from the reference plasmids. The results indicated that plasmids assigned to the same Inc group by traditional methods might be partially or completely different from their respective reference plasmids at the DNA level. With a few exceptions, all plasmids related to the reference plasmid at the DNA level also reacted with the primer systems tested. PCR amplification of total DNA extracted directly from different soil and manure slurry samples revealed the prevalence of IncQ- and IncP-specific sequences in several of these samples. In contrast, IncN- and IncW-specific sequences were detected mainly in DNA obtained from manure slurries.

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Selected References

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  1. Bale M. J., Day M. J., Fry J. C. Novel method for studying plasmid transfer in undisturbed river epilithon. Appl Environ Microbiol. 1988 Nov;54(11):2756–2758. doi: 10.1128/aem.54.11.2756-2758.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Couturier M., Bex F., Bergquist P. L., Maas W. K. Identification and classification of bacterial plasmids. Microbiol Rev. 1988 Sep;52(3):375–395. doi: 10.1128/mr.52.3.375-395.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dahlberg C., Hermansson M. Abundance of Tn3, Tn21, and Tn501 transposase (tnpA) sequences in bacterial community DNA from marine environments. Appl Environ Microbiol. 1995 Aug;61(8):3051–3056. doi: 10.1128/aem.61.8.3051-3056.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Don R. H., Pemberton J. M. Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol. 1981 Feb;145(2):681–686. doi: 10.1128/jb.145.2.681-686.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Frey J., Bagdasarian M. M., Bagdasarian M. Replication and copy number control of the broad-host-range plasmid RSF1010. Gene. 1992 Apr 1;113(1):101–106. doi: 10.1016/0378-1119(92)90675-f. [DOI] [PubMed] [Google Scholar]
  8. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jagura-Burdzy G., Thomas C. M. Purification of KorA protein from broad host range plasmid RK2: definition of a hierarchy of KorA operators. J Mol Biol. 1995 Oct 13;253(1):39–50. doi: 10.1006/jmbi.1995.0534. [DOI] [PubMed] [Google Scholar]
  10. Jovanovic O. S., Ayres E. K., Figurski D. H. Host-inhibitory functions encoded by promiscuous plasmids. Transient arrest of Escherichia coli segregants that fail to inherit plasmid RK2. J Mol Biol. 1994 Mar 18;237(1):52–64. doi: 10.1006/jmbi.1994.1208. [DOI] [PubMed] [Google Scholar]
  11. Krishnan B. R., Fobert P. R., Seitzer U., Iyer V. N. Mutations within the replicon of the IncN plasmid pCU1 that affect its Escherichia coli polA-independence but not its autonomous replication ability. Gene. 1990 Jul 2;91(1):1–7. doi: 10.1016/0378-1119(90)90155-k. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Lessl M., Balzer D., Pansegrau W., Lanka E. Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem. 1992 Oct 5;267(28):20471–20480. [PubMed] [Google Scholar]
  14. Lessl M., Balzer D., Weyrauch K., Lanka E. The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation. J Bacteriol. 1993 Oct;175(20):6415–6425. doi: 10.1128/jb.175.20.6415-6425.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Llosa M., Bolland S., de la Cruz F. Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46. Mol Gen Genet. 1991 May;226(3):473–483. doi: 10.1007/BF00260661. [DOI] [PubMed] [Google Scholar]
  17. Neilson J. W., Josephson K. L., Pepper I. L., Arnold R. B., Di Giovanni G. D., Sinclair N. A. Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil. Appl Environ Microbiol. 1994 Nov;60(11):4053–4058. doi: 10.1128/aem.60.11.4053-4058.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Okumura M. S., Kado C. I. The region essential for efficient autonomous replication of pSa in Escherichia coli. Mol Gen Genet. 1992 Oct;235(1):55–63. doi: 10.1007/BF00286181. [DOI] [PubMed] [Google Scholar]
  19. Pansegrau W., Lanka E., Barth P. T., Figurski D. H., Guiney D. G., Haas D., Helinski D. R., Schwab H., Stanisich V. A., Thomas C. M. Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. J Mol Biol. 1994 Jun 24;239(5):623–663. doi: 10.1006/jmbi.1994.1404. [DOI] [PubMed] [Google Scholar]
  20. Roberts R. C., Ström A. R., Helinski D. R. The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss. J Mol Biol. 1994 Mar 18;237(1):35–51. doi: 10.1006/jmbi.1994.1207. [DOI] [PubMed] [Google Scholar]
  21. Scherzinger E., Haring V., Lurz R., Otto S. Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins. Nucleic Acids Res. 1991 Mar 25;19(6):1203–1211. doi: 10.1093/nar/19.6.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Sundin G. W., Bender C. L. Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae. Appl Environ Microbiol. 1993 Apr;59(4):1018–1024. doi: 10.1128/aem.59.4.1018-1024.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sundin G. W., Demezas D. H., Bender C. L. Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides. Appl Environ Microbiol. 1994 Dec;60(12):4421–4431. doi: 10.1128/aem.60.12.4421-4431.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thomas C. M., Thorsted P. PCR probes for promiscuous plasmids. Microbiology. 1994 Jan;140(Pt 1):1–1. doi: 10.1099/13500872-140-1-1. [DOI] [PubMed] [Google Scholar]
  27. Top E. M., Holben W. E., Forney L. J. Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation. Appl Environ Microbiol. 1995 May;61(5):1691–1698. doi: 10.1128/aem.61.5.1691-1698.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tschäpe H., Tietze E. Characterization of conjugative plasmids belonging to a new incompatibility group (IncZ). Z Allg Mikrobiol. 1983;23(6):393–401. doi: 10.1002/jobm.3630230610. [DOI] [PubMed] [Google Scholar]
  29. Tschäpe H., Tietze E., Koch C. Characterization of conjugative R plasmids belonging to the new incompatibility group IncU. J Gen Microbiol. 1981 Nov;127(1):155–160. doi: 10.1099/00221287-127-1-155. [DOI] [PubMed] [Google Scholar]
  30. Zatyka M., Jagura-Burdzy G., Thomas C. M. Regulation of transfer genes of promiscuous IncP alpha plasmid RK2: repression of Tra1 region transcription both by relaxosome proteins and by the Tra2 regulator TrbA. Microbiology. 1994 Nov;140(Pt 11):2981–2990. doi: 10.1099/13500872-140-11-2981. [DOI] [PubMed] [Google Scholar]
  31. Ziegelin G., Pansegrau W., Strack B., Balzer D., Kröger M., Kruft V., Lanka E. Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4. DNA Seq. 1991;1(5):303–327. doi: 10.3109/10425179109020786. [DOI] [PubMed] [Google Scholar]

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