1887

Abstract

Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of . Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional cells. Analysis of the dynamics of TrfA– complex formation during the cell cycle revealed that TrfA binds primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the cell cycle. Both and analysis of RK2 replication initiation in cells demonstrated that it is independent of the DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.065490-0
2013-06-01
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/6/1010.html?itemId=/content/journal/micro/10.1099/mic.0.065490-0&mimeType=html&fmt=ahah

References

  1. Blasina A., Kittell B. L., Toukdarian A. E., Helinski D. R.. ( 1996;). Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins. . Proc Natl Acad Sci U S A 93:, 3559–3564. [CrossRef][PubMed]
    [Google Scholar]
  2. Caspi R., Helinski D. R., Pacek M., Konieczny I.. ( 2000;). Interactions of DnaA proteins from distantly related bacteria with the replication origin of the broad host range plasmid RK2. . J Biol Chem 275:, 18454–18461. [CrossRef][PubMed]
    [Google Scholar]
  3. Caspi R., Pacek M., Consiglieri G., Helinski D. R., Toukdarian A., Konieczny I.. ( 2001;). A broad host range replicon with different requirements for replication initiation in three bacterial species. . EMBO J 20:, 3262–3271. [CrossRef][PubMed]
    [Google Scholar]
  4. Castaing J. P., Bouet J. Y., Lane D.. ( 2008;). F plasmid partition depends on interaction of SopA with non-specific DNA. . Mol Microbiol 70:, 1000–1011.[PubMed]
    [Google Scholar]
  5. Cheng L., Keiler K. C.. ( 2009;). Correct timing of dnaA transcription and initiation of DNA replication requires trans translation. . J Bacteriol 191:, 4268–4275. [CrossRef][PubMed]
    [Google Scholar]
  6. Collier J., Murray S. R., Shapiro L.. ( 2006;). DnaA couples DNA replication and the expression of two cell cycle master regulators. . EMBO J 25:, 346–356. [CrossRef][PubMed]
    [Google Scholar]
  7. Das N., Chattoraj D. K.. ( 2004;). Origin pairing (‘handcuffing’) and unpairing in the control of P1 plasmid replication. . Mol Microbiol 54:, 836–849. [CrossRef][PubMed]
    [Google Scholar]
  8. Dingwall A., Shapiro L.. ( 1989;). Rate, origin, and bidirectionality of Caulobacter chromosome replication as determined by pulsed-field gel electrophoresis. . Proc Natl Acad Sci U S A 86:, 119–123. [CrossRef][PubMed]
    [Google Scholar]
  9. Doran K. S., Konieczny I., Helinski D. R.. ( 1998;). Replication origin of the broad host range plasmid RK2. Positioning of various motifs is critical for initiation of replication. . J Biol Chem 273:, 8447–8453. [CrossRef][PubMed]
    [Google Scholar]
  10. Durland R. H., Toukdarian A., Fang F., Helinski D. R.. ( 1990;). Mutations in trfA replication gene of broad-host-range plasmid RK2 result in elevated plasmid copy numbers. . J Bacteriol 172:, 3859–3867. [CrossRef][PubMed]
    [Google Scholar]
  11. Durland R. H., Helinski D. R.. ( 1987;). The sequence encoding the 43-kilodalton trfA protein is required for efficient replication or maintenance of minimal RK2 replicons in Pseudomonas aeruginosa. . Plasmid 18:, 164–169. [CrossRef][PubMed]
    [Google Scholar]
  12. Easter C. L., Sobecky P. A., Helinski D. R.. ( 1997;). Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2. . J Bacteriol 179:, 6472–6479.[PubMed]
    [Google Scholar]
  13. Ely B.. ( 1991;). Genetics of Caulobacter crescentus. . Methods Enzymol 204:, 372–384. [CrossRef][PubMed]
    [Google Scholar]
  14. Evinger M., Agabian N.. ( 1977;). Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. . J Bacteriol 132:, 294–301.[PubMed]
    [Google Scholar]
  15. Fang F. C., Helinski D. R.. ( 1991;). Broad-host-range properties of plasmid RK2: importance of overlapping genes encoding the plasmid replication initiation protein TrfA. . J Bacteriol 173:, 5861–5868.[PubMed]
    [Google Scholar]
  16. Figurski D. H., Helinski D. R.. ( 1979;). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. . Proc Natl Acad Sci U S A 76:, 1648–1652. [CrossRef][PubMed]
    [Google Scholar]
  17. Gasset-Rosa F., Díaz-López T., Lurz R., Prieto A., Fernández-Tresguerres M. E., Giraldo R.. ( 2008;). Negative regulation of pPS10 plasmid replication: origin pairing by zipping-up DNA-bound RepA monomers. . Mol Microbiol 68:, 560–572. [CrossRef][PubMed]
    [Google Scholar]
  18. Gilchrist A., Smit J.. ( 1991;). Transformation of freshwater and marine caulobacters by electroporation. . J Bacteriol 173:, 921–925.[PubMed]
    [Google Scholar]
  19. Havey J. C., Vecchiarelli A. G., Funnell B. E.. ( 2012;). ATP-regulated interactions between P1 ParA, ParB and non-specific DNA that are stabilized by the plasmid partition site, parS.. Nucleic Acids Res 40:, 801–812. [CrossRef][PubMed]
    [Google Scholar]
  20. Herrero M., de Lorenzo V., Timmis K. N.. ( 1990;). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. . J Bacteriol 172:, 6557–6567.[PubMed]
    [Google Scholar]
  21. Ho T. Q., Zhong Z., Aung S., Pogliano J.. ( 2002;). Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli. . EMBO J 21:, 1864–1872. [CrossRef][PubMed]
    [Google Scholar]
  22. Hottes A. K., Shapiro L., McAdams H. H.. ( 2005;). DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus. . Mol Microbiol 58:, 1340–1353. [CrossRef][PubMed]
    [Google Scholar]
  23. Isaac J. H., Holloway B. W.. ( 1968;). Control of pyrimidine biosynthesis in Pseudomonas aeruginosa.. J Bacteriol 96:, 1732–1741.[PubMed]
    [Google Scholar]
  24. Jacobs C., Hung D., Shapiro L.. ( 2001;). Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle. . Proc Natl Acad Sci U S A 98:, 4095–4100. [CrossRef][PubMed]
    [Google Scholar]
  25. Jensen R. B.. ( 2006;). Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus. . J Bacteriol 188:, 2244–2253. [CrossRef][PubMed]
    [Google Scholar]
  26. Jensen R. B., Shapiro L.. ( 1999;). Chromosome segregation during the prokaryotic cell division cycle. . Curr Opin Cell Biol 11:, 726–731. [CrossRef][PubMed]
    [Google Scholar]
  27. Jensen R. B., Wang S. C., Shapiro L.. ( 2001;). A moving DNA replication factory in Caulobacter crescentus. . EMBO J 20:, 4952–4963. [CrossRef][PubMed]
    [Google Scholar]
  28. Jiang Y., Pacek M., Helinski D. R., Konieczny I., Toukdarian A.. ( 2003;). A multifunctional plasmid-encoded replication initiation protein both recruits and positions an active helicase at the replication origin. . Proc Natl Acad Sci U S A 100:, 8692–8697. [CrossRef][PubMed]
    [Google Scholar]
  29. Jonas K., Chen Y. E., Laub M. T.. ( 2011;). Modularity of the bacterial cell cycle enables independent spatial and temporal control of DNA replication. . Curr Biol 21:, 1092–1101. [CrossRef][PubMed]
    [Google Scholar]
  30. Judd E. M., Ryan K. R., Moerner W. E., Shapiro L., McAdams H. H.. ( 2003;). Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter. . Proc Natl Acad Sci U S A 100:, 8235–8240. [CrossRef][PubMed]
    [Google Scholar]
  31. Judd E. M., Comolli L. R., Chen J. C., Downing K. H., Moerner W. E., McAdams H. H.. ( 2005;). Distinct constrictive processes, separated in time and space, divide Caulobacter inner and outer membranes. . J Bacteriol 187:, 6874–6882. [CrossRef][PubMed]
    [Google Scholar]
  32. Kolatka K., Witosinska M., Pierechod M., Konieczny I.. ( 2008;). Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2. . Microbiology 154:, 2847–2856. [CrossRef][PubMed]
    [Google Scholar]
  33. Kolatka K., Kubik S., Rajewska M., Konieczny I.. ( 2010;). Replication and partitioning of the broad-host-range plasmid RK2. . Plasmid 64:, 119–134. [CrossRef][PubMed]
    [Google Scholar]
  34. Konieczny I.. ( 2003;). Strategies for helicase recruitment and loading in bacteria. . EMBO Rep 4:, 37–41. [CrossRef][PubMed]
    [Google Scholar]
  35. Konieczny I., Helinski D. R.. ( 1997;). Helicase delivery and activation by DnaA and TrfA proteins during the initiation of replication of the broad host range plasmid RK2. . J Biol Chem 272:, 33312–33318. [CrossRef][PubMed]
    [Google Scholar]
  36. Konieczny I., Doran K. S., Helinski D. R., Blasina A.. ( 1997;). Role of TrfA and DnaA proteins in origin opening during initiation of DNA replication of the broad host range plasmid RK2. . J Biol Chem 272:, 20173–20178. [CrossRef][PubMed]
    [Google Scholar]
  37. Kowalczyk L., Rajewska M., Konieczny I.. ( 2005;). Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin. . Mol Microbiol 57:, 1439–1449. [CrossRef][PubMed]
    [Google Scholar]
  38. Lesley J. A., Shapiro L.. ( 2008;). SpoT regulates DnaA stability and initiation of DNA replication in carbon-starved Caulobacter crescentus. . J Bacteriol 190:, 6867–6880. [CrossRef][PubMed]
    [Google Scholar]
  39. Marczynski G. T., Shapiro L.. ( 2002;). Control of chromosome replication in Caulobacter crescentus. . Annu Rev Microbiol 56:, 625–656. [CrossRef][PubMed]
    [Google Scholar]
  40. Marczynski G. T., Dingwall A., Shapiro L.. ( 1990;). Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle. . J Mol Biol 212:, 709–722. [CrossRef][PubMed]
    [Google Scholar]
  41. Meyer R., Figurski D., Helinski D. R.. ( 1975;). Molecular vehicle properties of the broad host range plasmid RK2. . Science 190:, 1226–1228. [CrossRef][PubMed]
    [Google Scholar]
  42. Morrison P. F., Chattoraj D. K.. ( 2004;). Replication of a unit-copy plasmid F in the bacterial cell cycle: a replication rate function analysis. . Plasmid 52:, 13–30. [CrossRef][PubMed]
    [Google Scholar]
  43. Newton A.. ( 1972;). Role of transcription in the temporal control of development in Caulobacter crescentus (stalk–rifampin–RNA synthesis–DNA synthesis–motility). . Proc Natl Acad Sci U S A 69:, 447–451. [CrossRef][PubMed]
    [Google Scholar]
  44. Park K., Han E., Paulsson J., Chattoraj D. K.. ( 2001;). Origin pairing (‘handcuffing’) as a mode of negative control of P1 plasmid copy number. . EMBO J 20:, 7323–7332. [CrossRef][PubMed]
    [Google Scholar]
  45. Pinkney M., Diaz R., Lanka E., Thomas C. M.. ( 1988;). 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 203:, 927–938. [CrossRef][PubMed]
    [Google Scholar]
  46. Pogliano J., Ho T. Q., Zhong Z., Helinski D. R.. ( 2001;). Multicopy plasmids are clustered and localized in Escherichia coli. . Proc Natl Acad Sci U S A 98:, 4486–4491. [CrossRef][PubMed]
    [Google Scholar]
  47. Purdy Drew K. R., Pogliano J.. ( 2011;). Dynamic instability-driven centering/segregating mechanism in bacteria. . Proc Natl Acad Sci U S A 108:, 11075–11080. [CrossRef][PubMed]
    [Google Scholar]
  48. Quon K. C., Yang B., Domian I. J., Shapiro L., Marczynski G. T.. ( 1998;). Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin. . Proc Natl Acad Sci U S A 95:, 120–125. [CrossRef][PubMed]
    [Google Scholar]
  49. Ruiz-Barba J. L., Maldonado A., Jiménez-Díaz R.. ( 2005;). Small-scale total DNA extraction from bacteria and yeast for PCR applications. . Anal Biochem 347:, 333–335. [CrossRef][PubMed]
    [Google Scholar]
  50. Schmidhauser T. J., 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:, 446–455.[PubMed]
    [Google Scholar]
  51. Shingler V., Thomas C. M.. ( 1984;). Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products. . J Mol Biol 175:, 229–249. [CrossRef][PubMed]
    [Google Scholar]
  52. Sia E. A., Roberts R. C., Easter C., Helinski D. R., Figurski D. H.. ( 1995;). Different relative importances of the . par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. . J Bacteriol 177:, 2789–2797.[PubMed]
    [Google Scholar]
  53. Smith C. A., Shingler V., Thomas C. M.. ( 1984;). The trfA and trfB promoter regions of broad host range plasmid RK2 share common potential regulatory sequences. . Nucleic Acids Res 12:, 3619–3630. [CrossRef][PubMed]
    [Google Scholar]
  54. Szardenings F., Guymer D., Gerdes K.. ( 2011;). ParA ATPases can move and position DNA and subcellular structures. . Curr Opin Microbiol 14:, 712–718. [CrossRef][PubMed]
    [Google Scholar]
  55. Tal S., Paulsson J.. ( 2012;). Evaluating quantitative methods for measuring plasmid copy numbers in single cells. . Plasmid 67:, 167–173. [CrossRef][PubMed]
    [Google Scholar]
  56. Taylor J. A., Ouimet M. C., Wargachuk R., Marczynski G. T.. ( 2011;). The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites. . Mol Microbiol 82:, 312–326. [CrossRef][PubMed]
    [Google Scholar]
  57. Terrana B., Newton A.. ( 1975;). Pattern of unequal cell division and development in Caulobacter crescentus. . Dev Biol 44:, 380–385. [CrossRef][PubMed]
    [Google Scholar]
  58. Toukdarian A. E., Helinski D. R.. ( 1998;). TrfA dimers play a role in copy-number control of RK2 replication. . Gene 223:, 205–211. [CrossRef][PubMed]
    [Google Scholar]
  59. Tsai J. W., Alley M. R.. ( 2001;). Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway. . J Bacteriol 183:, 5001–5007. [CrossRef][PubMed]
    [Google Scholar]
  60. Wegrzyn A., Wegrzyn G., Herman A., Taylor K.. ( 1996;). Protein inheritance: λ plasmid replication perpetuated by the heritable replication complex. . Genes Cells 1:, 953–963. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.065490-0
Loading
/content/journal/micro/10.1099/mic.0.065490-0
Loading

Data & Media loading...

Supplements

Supplementary material 

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error