Journal of Genetics

, 65:103 | Cite as

Genetic analysis of bacterial plasmid promiscuity

  • Viji Krishnapillai


The genetic basis of the promiscuous behaviour of bacterial plasmids has been investigated by study of the incompatibility P-1 group of conjugative plasmids of gram-negative bacteria. Both transposon mutagenesis and the construction of minireplicons linking varying combinations of the plasmid genome have shown that specific genomic regions control the conjugational transfer and vegetative replication of the plasmid in specific bacterial hosts. These include the plasmid DNA primase gene, the origin of plasmid transfer, a region near the origin of transfer, the origin of plasmid vegetative replication, thetrans- acting gene essential for the initiation of plasmid replication and a region involved in its regulation. DNA sequence analysis has identified the requirement of specific direct repeats within the origin of replication for plasmid replication in some but not in other hosts. The cloning of some of the trans-acting genes onto multicopy cloning vectors and complementation tests have shown that the requirements of these gene products vary in different hosts and that the plasmid has evolved genetic strategies for their optimal expression.


Promiscuous plasmids genetic analysis transposon mutagenesis minireplicons DNA sequence analysis 


  1. Bagdasarian M and Timmis K N 1982 Host-vector systems for gene cloning inPseudomonas.Curr. Top Microbiol. Immunol. 96: 47–67PubMedGoogle Scholar
  2. Barth P T 1979 RP4 and R300B as wide host-range plasmid cloning vehicles. InPlasmids of medical, environmental and commercial importance (eds) K N Timmis and A Pühler (Amsterdam: Elsevier /North Holland Biomedical Press) pp. 399–410Google Scholar
  3. Barth P T, Ellis K, Bechhofer D H and Figurski D H 1984 Involvement ofkit andkor genes in the phenotype of a host-range mutant of RP4.Mol. Gen, Genet. 197: 236–243CrossRefGoogle Scholar
  4. Barth P T, Grinter N J and Bradley D E 1978 Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.J. Bacterial. 133: 43–52Google Scholar
  5. Barth P T, Tobin L and Sharpe G S 1981 Development of broad host-range plasmid vectors. InMolecular biology, pathogenicity and ecology of bacterial plasmids (eds) S B Levy, R C Clowes and E L Koenig (New York: Plenum Press) pp. 439–448Google Scholar
  6. Boulnois G J, Varley J M, Sharpe G S and Franklin FCH 1985 Transposon donor plasmids, based on ColIb-P9, for use inPseudomonas putida and a variety of other gram negative bacteria.Mol. Gen. Genet. 200: 65–67PubMedCrossRefGoogle Scholar
  7. Burkardt H, Reiss G and Pühler A 1979 Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical.J. Gen. Microbiol. 114: 341–348PubMedGoogle Scholar
  8. Chatfield L K and Wilkins B M 1984 Conjugative transfer of IncII plasmid DNA primase.Mol. Gen. Genet. 197: 461–466PubMedCrossRefGoogle Scholar
  9. Cowan P and Krishnapillai V 1982 Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18.Plasmid 8: 164–174PubMedCrossRefGoogle Scholar
  10. Cross M A, Warne S R and Thomas C M 1986 Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis.Plasmid 15: 132–146PubMedCrossRefGoogle Scholar
  11. Datta N, Hedges R W, Shaw E J, Sykes R B and Richmond M H 1971 Properties of an R factor fromPseudomonas aeruginosa.J. Bacterial. 108: 1244–1249Google Scholar
  12. Dean H F and Morgan A F 1983 Integration of R91-5::Tn501 into thePseudomonas putida PPN chromosome and genetic circularity of the chromosomal map.J. Bacterial. 153: 485–497Google Scholar
  13. Engel H W B, Soedirman N, Rost J A, van Leeuwen W J and van Embden J D A 1980 Transferability of macrolide, lincomycin, and streptogramin resistances between group A, B and D Streptococci,Streptococcus pneumoniae, andStaphylococcus aureus. J. Bacterial. 142: 407–413Google Scholar
  14. Figurski D H, Young C, Schreiner H C, Pohlman R F, Bechhofer D H, Prince A S and D’Amico T F 1984 Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon. InPlasmids in bacteria (eds) D R Helinski, S N Cohen and D B Clewell (New York: Plenum) pp. 227–242Google Scholar
  15. Filutowicz M, McEachern M, Greener A, Mukhopadhyay P, Uhlenhopp E, Durland R and Helinski D 1984 Role of the π initiation protein and direct nucleotide sequence repeats in the regulation of plasmid R6K replication. InPlasmids in bacteria (eds) D R Helinski, S N Cohen and D B Clewell (New York: Plenum) pp. 125–140Google Scholar
  16. Frey J and Krisch H M 1985 Ω mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species.Gene 36: 143–150PubMedCrossRefGoogle Scholar
  17. Früh R, Watson J M and Haas D 1983 Construction of recombination-deficient strains ofPseudomonas aeruginosa.Mol. Gen. Genet. 191: 334–337PubMedCrossRefGoogle Scholar
  18. Grinter N J 1981 Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage λ carrying ISI.Plasmid 5: 267–276PubMedCrossRefGoogle Scholar
  19. Guiney D G 1982 Host range of conjugation and replication functions ofEscherichia coli sex plasmid Flac. Comparison with the broad host range plasmid RK2.J. Mol. Biol. 162: 699–703PubMedCrossRefGoogle Scholar
  20. Guiney D G, Chikami G, Deiss C and Yakobson E 1984 The origin of plasmid DNA transfer during bacterial conjugation. InPlasmids in bacteria (eds) D R Helinski, S N Cohen and D B Clewell (New York: Plenum) pp. 521–534Google Scholar
  21. Haas D 1983 Genetic aspects of biodegradation by pseudomonads.Experientia (Basel) 39: 1199–1213Google Scholar
  22. Holloway B W, Crowther C, Dean H, Hagedorn J, Holmes N and Morgan A F 1982 Integration of plasmids into thePseudomonas chromosome. InDrug resistance in bacteria (eds) S Mitsuhashi (Tokyo: Japan Scientific Societies Press) pp. 231–242Google Scholar
  23. Jacoby G A 1985 Resistance plasmids ofPseudomonas. InThe biology of Pseudomonas (ed.) J R Sokatch (New York: Academic Press) (in press)Google Scholar
  24. Jacoby G A and Shapiro J A 1977 Plasmids studied inPseudomonas aeruginosa and other pseudomonads. InDNA insertion elements, plasmids and episomes (eds) A I Bukhari, J A Shapiro and S L Adhya (New York: Cold Spring Harbor Lab.) pp. 639–656Google Scholar
  25. Kornacki J A, West A H and Firshein W 1984 Proteins encoded by thetrans-acting replication and maintenance regions of broad host range plasmid RK2.Plasmid 11: 48–57PubMedCrossRefGoogle Scholar
  26. Krishnapillai V 1979 DNA insertion mutagenesis in aPseudomonas aeruginosa R plasmid.Plasmid 2: 237–246PubMedCrossRefGoogle Scholar
  27. Krishnapillai V, Nash J and Lanka E 1984 Insertion mutations in the promiscuous IncP-1 plasmid R18 which affect its host range betweenPseudomonas species.Plasmid 12: 170–180PubMedCrossRefGoogle Scholar
  28. Krishnapillai V, Pühler A and Lanka E 1986 Molecular cloning into Tn5 and integration in thePseudomonas aeruginosa chromosome: a tool for heterologous gene expression.J. Gen. Microbiol. 32: 707–715Google Scholar
  29. Krishnapillai V, Wexler M, Nash J and Figurski D 1986 A Tn7 insertion in thetrfA operon of the promiscuous plasmid R18 which affects its host range (in preparation)Google Scholar
  30. Lanka E and Barth P T 1981 Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance.J. Bacterial 148: 769–781Google Scholar
  31. Lanka E, Lurz R, Kröger M and Fürste J P 1984 Plasmid RP4 encodes two forms of a DNA primase.Mol. Gen. Genet. 194: 65–72PubMedCrossRefGoogle Scholar
  32. Lichstenstein C and Brenner S 1982 Unique insertion site of Tn7 in theE. coli chromosome.Nature (London) 297: 601–603CrossRefGoogle Scholar
  33. Mermod N, Lehrbach P R, Reineke and Timmis K N 1984 Transcription of the TOL plasmid toluate catabolic pathway operon ofPseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters.EMBO J. 3: 2461–2466PubMedGoogle Scholar
  34. Merryweather A, Barth P T and Wilkins B M 1986 Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.J. Bacterial. (in press)Google Scholar
  35. Meyer R and Helinski D R 1977 Unidirectional replication of the P-group plasmid RK2.Biochim. Biophys. Acta 478: 109–113PubMedGoogle Scholar
  36. Meyer R and Hinds M 1982 Multiple mechanisms for expression of incompatibility by broad host range plasmid RK2.J. Bacteriol. 152: 1078–1090PubMedGoogle Scholar
  37. Minton N P and Clarke L E 1985 Identification of the promoter of thePseudomonas gene coding for carboxypeptidase G2.Mol. Appl. Genet. 3: 26–35Google Scholar
  38. Moore D D, Denniston K J and Blattner F R 1981 Sequence organization of the origins of DNA replication in lambdoid coliphages.Gene 14: 91–101PubMedCrossRefGoogle Scholar
  39. Murooka Y, Takizawa N and Harada T 1981 Introduction of bacteriophage Mu into bacteria of various genera and intergeneric gene transfer by RP4: Mu.J. Bacteriol. 145: 358–368Google Scholar
  40. Nano F E, Shepherd W D, Watkins M M, Kuhl S A and Kaplan S 1984 Broad host range plasmid vector for thein vitro construction of transcriptional/translationallac fusions.Gene 34: 219–226CrossRefGoogle Scholar
  41. Nash J and Krishnapillai V 1986 DNA sequence analysis of Tn7 insertion mutations inoriV of the promiscuous plasmid R18 which affects its host range (in preparation)Google Scholar
  42. Nash J, Lanka E and Krishnapillai V 1986 Role of IncP-1 plasmid primase in promiscuous conjugation (in preparation)Google Scholar
  43. Pohlman R F and Figurski D H 1983 Conditional lethal mutants of thekil B determinant of broad host range plasmid RK2.Plasmid 10: 82–95PubMedCrossRefGoogle Scholar
  44. Royle P L and Holloway B W 1981 New prime plasmids forPseudomonas aeruginosa.Genet. Res. 37: 265–274PubMedCrossRefGoogle Scholar
  45. Schilf W and Krishnapillai V 1986 Genetic analysis of insertion mutations of the promiscuous IncP-1 plasmid Rl8 mapping nearoriT which affect its host range.Plasmid 15: 48–56PubMedCrossRefGoogle Scholar
  46. Schmidhauser T J and Helinski D R 1985 Regions of broad host range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.J. Bacteriol. 164: 446–455PubMedGoogle Scholar
  47. Schreiner H C, Bechoffer D H, Pohlman R F, Young C, Borden P A and Figurski D H 1985 Replication control in promiscuous plasmid RK2:kil andkor functions affect expression of the essential replication genetrfA. J. Bacteriol. 163: 228–237Google Scholar
  48. Scott J R 1984 Regulation of plasmid replication.Microbiol. Rev. 48: 1–23PubMedGoogle Scholar
  49. Sharpe G S 1984 Broad host range cloning vectors for gram-negative bacteria.Gene 29: 93–102PubMedCrossRefGoogle Scholar
  50. Shingler V and Thomas C M 1984a Analysis of thetrfA region of broad host range plasmid RK2 by transposon mutagenesis and identification of polypeptide products.J. Mol. Biol. 175: 229–249PubMedCrossRefGoogle Scholar
  51. Shingler V and Thomas C M 1984b Transcription in thetrfA region of broad host range plasmid RK2 is regulated bytrfB andkorB. Mol. Gen. Genet. 195: 523–529CrossRefGoogle Scholar
  52. Simon R, Priefer U and Pühler A 1983 A broad host range mobilization system forin vivo genetic engineering: transposon mutagenesis in gram-negative bacteria.Bio. Technology 1: 784–791Google Scholar
  53. Smith C A and Thomas C M 1984 Nucleotide sequence of thetrfA gene of broad host range plasmid RK2.J. Mol. Biol. 75: 251–262CrossRefGoogle Scholar
  54. Stalker D M, Thomas C M and Helinski D R 1981 Nucleotide sequence of thetrfA gene of broad host range plasmid RK2.Gen. Genet. 181: 8–12CrossRefGoogle Scholar
  55. Stokes H W, Moore R J and Krishnapillai V 1981 Complementation analysis inPseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18.Plasmid 5: 202–212PubMedCrossRefGoogle Scholar
  56. Tardif G and Grant R B 1983 Transfer of plasmids fromEscherichia coli toPseudomonas aeruginosa: characterization of aPseudomonas aeruginosa mutant with enhanced recipient ability for enterobacterial plasmids.Antimicrob. Agents Chemother. 24: 201–208PubMedGoogle Scholar
  57. Tatra P K and Goodwin P M 1983 R-plasmid mediated chromosome mobilization in the facultative methylotrophPseudomonas AMI,J. Gen. Microbiol. 129: 2629–2632Google Scholar
  58. Thomas C M, Cross M A, Hussain A A K and Smith C A 1984a Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2,EMBO J. 3: 57–63PubMedGoogle Scholar
  59. Thomas C M and Hussain A A K 1984 ThekorB gene of broad host range plasmid RK2 is a major copy number control element which may act together withtrfB by limitingIrfA expression.EMBO J. 3: 1513–1519PubMedGoogle Scholar
  60. Thomas C M, Smith C A, Shingler V, Cross M A, Hussain A A K and Pinkney M 1984b Regulation of replication and maintenance functions of broad host range plasmid RK2. InPlasmids in bacteria (eds) D R Helinski, S N Cohen and D B Clewell (New York: Plenum) pp. 261–276Google Scholar
  61. Vapnek D, Lipman M B and Rupp W D 1971 Physical properties and mechanism of transfer of R factors inE. coli. J. Bacterial. 108: 508–514Google Scholar
  62. Vapnek D and Rupp W D 1970 Asymmetric segregation of the complementary sex-factor DNA strands during conjugation inE. coli. J. Mol. Biol. 53: 287–303Google Scholar
  63. Waters S H, Rogowsky P, Grinsted J, Altenbuchner J and Schmitt R 1983 The tetracycline resistance determinants of RP1 and Tn/72/: nucleotide sequence analysis.Nucleic Acids Res. 11: 6089–6105PubMedCrossRefGoogle Scholar
  64. Whitta S, Sinclair M I and Holloway B W 1985 Transposes mutagenesis inMethylobacterium AMI (Pseudomonas AMI).J. Gen. Microbiol. 131: 1547–1549Google Scholar
  65. Wolk C P, Vonshak A, Kehoe P and Elhai J 1984 Construction of shuttle vectors capable of conjugative transfer fromEscherichia coli to nitrogen-fixing filamentous cyanobacteria.Proc. Natl. Acad. Sci. USA 81: 1561–1565PubMedCrossRefGoogle Scholar
  66. Yanisch-Perron C, Vieira J and Messing J 1985 Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors.Gene 33: 103–119PubMedCrossRefGoogle Scholar
  67. Young C, Burlage R S and Figurski D H 1986 Regulatory cooperation in broad host range plasmid RK2:kor B and a new gene (kor E) helpkorA controlkit A. J. Bacteriol. (in press)Google Scholar

Copyright information

© Indian Academy of Sciences 1986

Authors and Affiliations

  • Viji Krishnapillai
    • 1
  1. 1.Department of GeneticsMonash UniversityClaytonAustralia

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