Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Jul;172(7):3849–3858. doi: 10.1128/jb.172.7.3849-3858.1990

Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number.

R H Durland 1, D R Helinski 1
PMCID: PMC213366  PMID: 2193920

Abstract

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were o overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 +/- 0.6 ng of TrfA-32 and 1.8 +/- 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean +/- standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.

Full text

PDF
3853

Images in this article

3852Image
on p.3852
3854Image
on p.3854
3854Image
on p.3854

Selected References

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

  1. Abeles A. L. P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA. J Biol Chem. 1986 Mar 15;261(8):3548–3555. [PubMed] [Google Scholar]
  2. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  3. Bazaral M., Helinski D. R. Replication of a bacterial plasmid and an episome in Escherichia coli. Biochemistry. 1970 Jan 20;9(2):399–406. doi: 10.1021/bi00804a029. [DOI] [PubMed] [Google Scholar]
  4. Bechhofer D. H., Kornacki J. A., Firshein W., Figurski D. H. Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB. Proc Natl Acad Sci U S A. 1986 Jan;83(2):394–398. doi: 10.1073/pnas.83.2.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  6. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  7. Chattoraj D. K., Snyder K. M., Abeles A. L. P1 plasmid replication: multiple functions of RepA protein at the origin. Proc Natl Acad Sci U S A. 1985 May;82(9):2588–2592. doi: 10.1073/pnas.82.9.2588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Datta N., Hedges R. W. Host ranges of R factors. J Gen Microbiol. 1972 May;70(3):453–460. doi: 10.1099/00221287-70-3-453. [DOI] [PubMed] [Google Scholar]
  10. Ditta G., Virts E., Palomares A., Kim C. H. The nifA gene of Rhizobium meliloti is oxygen regulated. J Bacteriol. 1987 Jul;169(7):3217–3223. doi: 10.1128/jb.169.7.3217-3223.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Durland R. H., Helinski D. R. The sequence encoding the 43-kilodalton trfA protein is required for efficient replication or maintenance of minimal RK2 replicons in Pseudomonas aeruginosa. Plasmid. 1987 Sep;18(2):164–169. doi: 10.1016/0147-619x(87)90044-8. [DOI] [PubMed] [Google Scholar]
  12. Durland R. H., Toukdarian A., Fang F., Helinski D. R. Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers. J Bacteriol. 1990 Jul;172(7):3859–3867. doi: 10.1128/jb.172.7.3859-3867.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Figurski D. H., Meyer R. J., Helinski D. R. Suppression of Co1E1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro. J Mol Biol. 1979 Sep 25;133(3):295–318. doi: 10.1016/0022-2836(79)90395-4. [DOI] [PubMed] [Google Scholar]
  15. Filutowicz M., McEachern M. J., Helinski D. R. Positive and negative roles of an initiator protein at an origin of replication. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9645–9649. doi: 10.1073/pnas.83.24.9645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Filutowicz M., Uhlenhopp E., Helinski D. R. Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K. J Mol Biol. 1986 Jan 20;187(2):225–239. doi: 10.1016/0022-2836(86)90230-5. [DOI] [PubMed] [Google Scholar]
  17. Germino J., Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell. 1983 Aug;34(1):125–134. doi: 10.1016/0092-8674(83)90142-3. [DOI] [PubMed] [Google Scholar]
  18. Germino J., Bastia D. The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding. Cell. 1983 Jan;32(1):131–140. doi: 10.1016/0092-8674(83)90503-2. [DOI] [PubMed] [Google Scholar]
  19. Givskov M., Stougaard P., Light J., Molin S. Identification and characterization of mutations responsible for a runaway replication phenotype of plasmid R1. Gene. 1987;57(2-3):203–211. doi: 10.1016/0378-1119(87)90123-5. [DOI] [PubMed] [Google Scholar]
  20. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hancock K., Tsang V. C. India ink staining of proteins on nitrocellulose paper. Anal Biochem. 1983 Aug;133(1):157–162. doi: 10.1016/0003-2697(83)90237-3. [DOI] [PubMed] [Google Scholar]
  22. Ingram L. C., Richmond M. H., Sykes R. B. Molecular characterization of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns. Antimicrob Agents Chemother. 1973 Feb;3(2):279–288. doi: 10.1128/aac.3.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lanka E., Lurz R., Fürste J. P. Molecular cloning and mapping of SphI restriction fragments of plasmid RP4. Plasmid. 1983 Nov;10(3):303–307. doi: 10.1016/0147-619x(83)90047-1. [DOI] [PubMed] [Google Scholar]
  26. Masson L., Ray D. S. Mechanism of autonomous control of the Escherichia coli F plasmid: purification and characterization of the repE gene product. Nucleic Acids Res. 1988 Jan 25;16(2):413–424. doi: 10.1093/nar/16.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Olsen R. H., Shipley P. Host range and properties of the Pseudomonas aeruginosa R factor R1822. J Bacteriol. 1973 Feb;113(2):772–780. doi: 10.1128/jb.113.2.772-780.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pinkney M., Diaz R., Lanka E., Thomas C. M. Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III. J Mol Biol. 1988 Oct 20;203(4):927–938. doi: 10.1016/0022-2836(88)90118-0. [DOI] [PubMed] [Google Scholar]
  30. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  31. Rokeach L. A., Søgaard-Andersen L., Molin S. Two functions of the E protein are key elements in the plasmid F replication control system. J Bacteriol. 1985 Dec;164(3):1262–1270. doi: 10.1128/jb.164.3.1262-1270.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sadler J. R., Tecklenburg M., Betz J. L. Plasmids containing many tandem copies of a synthetic lactose operator. Gene. 1980 Feb;8(3):279–300. doi: 10.1016/0378-1119(80)90005-0. [DOI] [PubMed] [Google Scholar]
  34. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Schmidhauser T. J., Helinski D. R. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. doi: 10.1128/jb.164.1.446-455.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schreiner H. C., Bechhofer D. H., Pohlman R. F., Young C., Borden P. A., Figurski D. H. Replication control in promiscuous plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA. J Bacteriol. 1985 Jul;163(1):228–237. doi: 10.1128/jb.163.1.228-237.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Shingler V., Thomas C. M. Analysis of nonpolar insertion mutations in the trfA gene of IncP plasmid RK2 which affect its broad-host-range property. Biochim Biophys Acta. 1989 Apr 12;1007(3):301–308. doi: 10.1016/0167-4781(89)90152-8. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. 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]
  43. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  44. Stalker D. M., Kolter R., Helinski D. R. Plasmid R6K DNA replication. I. Complete nucleotide sequence of an autonomously replicating segment. J Mol Biol. 1982 Oct 15;161(1):33–43. doi: 10.1016/0022-2836(82)90276-5. [DOI] [PubMed] [Google Scholar]
  45. Stalker D. M., Thomas C. M., Helinski D. R. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):8–12. doi: 10.1007/BF00338997. [DOI] [PubMed] [Google Scholar]
  46. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  47. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  48. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Theophilus B. D., Cross M. A., Smith C. A., Thomas C. M. Regulation of the trfA and trfB promoters of broad host range plasmid RK2: identification of sequences essential for regulation by trfB/korA/korD. Nucleic Acids Res. 1985 Nov 25;13(22):8129–8142. doi: 10.1093/nar/13.22.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Thomas C. M., Cross M. A., Hussain A. A., Smith C. A. Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2. EMBO J. 1984 Jan;3(1):57–63. doi: 10.1002/j.1460-2075.1984.tb01761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Thomas C. M. Evidence for the involvement of the incC locus of broad host range plasmid RK2 in plasmid maintenance. Plasmid. 1986 Jul;16(1):15–29. doi: 10.1016/0147-619x(86)90075-2. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Thomas C. M., Stalker D. M., Helinski D. R. Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):1–7. doi: 10.1007/BF00338996. [DOI] [PubMed] [Google Scholar]
  56. Tokino T., Murotsu T., Matsubara K. Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4109–4113. doi: 10.1073/pnas.83.12.4109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tolun A., Helinski D. R. Separation of the minimal replication region of the F plasmid into a replication origin segment and a trans-acting segment. Mol Gen Genet. 1982;186(3):372–377. doi: 10.1007/BF00729456. [DOI] [PubMed] [Google Scholar]
  58. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. Young C., Prince A. S., Figurski D. H. korA function of promiscuous plasmid RK2: an autorepressor that inhibits expression of host-lethal gene kilA and replication gene trfA. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7374–7378. doi: 10.1073/pnas.82.21.7374. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES