1887

Abstract

We have investigated the replication patterns of the two chromosomes of the bacterium grown in four different media. By combining flow cytometry and quantitative real-time PCR with computer simulations, we show that in rich media, cells grow with overlapping replication cycles of both the large chromosome (ChrI) and the small chromosome (ChrII). In Luria–Bertani (LB) medium, initiation occurs at four copies of the ChrI origin and two copies of the ChrII origin. Replication of ChrII was found to occur at the end of the ChrI replication period in all four growth conditions. Novel cell-sorting experiments with marker frequency analysis support these conclusions. Incubation with protein synthesis inhibitors indicated that the potential for initiation of replication of ChrII was present at the same time as that of ChrI, but was actively delayed until much of ChrI was replicated. Investigations of the localization of SeqA bound to new DNA at replication forks indicated that the forks were co-localized in pairs when cells grew without overlapping replication cycles and in higher-order structures during more rapid growth. The increased degree of fork organization during rapid growth may be a means by which correct segregation of daughter molecules is facilitated.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.045112-0
2011-03-01
2019-12-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/3/695.html?itemId=/content/journal/micro/10.1099/mic.0.045112-0&mimeType=html&fmt=ahah

References

  1. Adachi, S., Fukushima, T. & Hiraga, S. ( 2008; ). Dynamic events of sister chromosomes in the cell cycle of Escherichia coli. Genes Cells 13, 181–197,[CrossRef]
    [Google Scholar]
  2. Allardet-Servent, A., Michaux-Charachon, S., Jumas-Bilak, E., Karayan, L. & Ramuz, M. ( 1993; ). Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome. J Bacteriol 175, 7869–7874.
    [Google Scholar]
  3. Boye, E. & Løbner-Olesen, A. ( 1991; ). Bacterial growth control studied by flow cytometry. Res Microbiol 142, 131–135,[CrossRef]
    [Google Scholar]
  4. Bremer, H. & Churchward, G. ( 1977; ). An examination of the Cooper–Helmstetter theory of DNA replication in bacteria and its underlying assumptions. J Theor Biol 69, 645–654,[CrossRef]
    [Google Scholar]
  5. Brendler, T., Abeles, A. & Austin, S. ( 1995; ). A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene. EMBO J 14, 4083–4089.
    [Google Scholar]
  6. Brendler, T., Sawitzke, J., Sergueev, K. & Austin, S. ( 2000; ). A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation. EMBO J 19, 6249–6258,[CrossRef]
    [Google Scholar]
  7. Campbell, J. L. & Kleckner, N. ( 1990; ). E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork. Cell 62, 967–979,[CrossRef]
    [Google Scholar]
  8. Clark, D. J. & Maaloe, O. ( 1967; ). DNA replication and division cycle in Escherichia coli. J Mol Biol 23, 99–112,[CrossRef]
    [Google Scholar]
  9. Cooper, S. & Helmstetter, C. E. ( 1968; ). Chromosome replication and the division cycle of Escherichia coli B/r. J Mol Biol 31, 519–540,[CrossRef]
    [Google Scholar]
  10. Demarre, G. & Chattoraj, D. K. ( 2010; ). DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle. PLoS Genet 6, e1000939,[CrossRef]
    [Google Scholar]
  11. Dryselius, R., Izutsu, K., Honda, T. & Iida, T. ( 2008; ). Differential replication dynamics for large and small Vibrio chromosomes affect gene dosage, expression and location. BMC Genomics 9, 559,[CrossRef]
    [Google Scholar]
  12. Duigou, S., Knudsen, K. G., Skovgaard, O., Egan, E. S., Løbner-Olesen, A. & Waldor, M. K. ( 2006; ). Independent control of replication initiation of the two Vibrio cholerae chromosomes by DnaA and RctB. J Bacteriol 188, 6419–6424,[CrossRef]
    [Google Scholar]
  13. Duigou, S., Yamaichi, Y. & Waldor, M. K. ( 2008; ). ATP negatively regulates the initiator protein of Vibrio cholerae chromosome II replication. Proc Natl Acad Sci U S A 105, 10577–10582,[CrossRef]
    [Google Scholar]
  14. Egan, E. S. & Waldor, M. K. ( 2003; ). Distinct replication requirements for the two Vibrio cholerae chromosomes. Cell 114, 521–530,[CrossRef]
    [Google Scholar]
  15. Egan, E. S., Løbner-Olesen, A. & Waldor, M. K. ( 2004; ). Synchronous replication initiation of the two Vibrio cholerae chromosomes. Curr Biol 14, R501–R502,[CrossRef]
    [Google Scholar]
  16. Egan, E. S., Fogel, M. A. & Waldor, M. K. ( 2005; ). Divided genomes: negotiating the cell cycle in prokaryotes with multiple chromosomes. Mol Microbiol 56, 1129–1138,[CrossRef]
    [Google Scholar]
  17. Fiebig, A., Keren, K. & Theriot, J. A. ( 2006; ). Fine-scale time-lapse analysis of the biphasic, dynamic behaviour of the two Vibrio cholerae chromosomes. Mol Microbiol 60, 1164–1178,[CrossRef]
    [Google Scholar]
  18. Fogel, M. A. & Waldor, M. K. ( 2005; ). Distinct segregation dynamics of the two Vibrio cholerae chromosomes. Mol Microbiol 55, 125–136,
    [Google Scholar]
  19. Fogel, M. A. & Waldor, M. K. ( 2006; ). A dynamic, mitotic-like mechanism for bacterial chromosome segregation. Genes Dev 20, 3269–3282,[CrossRef]
    [Google Scholar]
  20. Fossum, S., Crooke, E. & Skarstad, K. ( 2007; ). Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli. EMBO J 26, 4514–4522,[CrossRef]
    [Google Scholar]
  21. Gilbert, D. M. ( 2001; ). Making sense of eukaryotic DNA replication origins. Science 294, 96–100,[CrossRef]
    [Google Scholar]
  22. Heidelberg, J. F., Eisen, J. A., Nelson, W. C., Clayton, R. A., Gwinn, M. L., Dodson, R. J., Haft, D. H., Hickey, E. K., Peterson, J. D. & other authors ( 2000; ). DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406, 477–483,[CrossRef]
    [Google Scholar]
  23. Hiraga, S., Ichinose, C., Niki, H. & Yamazoe, M. ( 1998; ). Cell cycle-dependent duplication and bidirectional migration of SeqA-associated DNA–-protein complexes in E. coli. Mol Cell 1, 381–387,[CrossRef]
    [Google Scholar]
  24. Hiraga, S., Ichinose, C., Onogi, T., Niki, H. & Yamazoe, M. ( 2000; ). Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli. Genes Cells 5, 327–341,[CrossRef]
    [Google Scholar]
  25. Katayama, T., Fujimitsu, K. & Ogawa, T. ( 2001; ). Multiple pathways regulating DnaA function in Escherichia coli: distinct roles for DnaA titration by the datA locus and the regulatory inactivation of DnaA. Biochimie 83, 13–17,[CrossRef]
    [Google Scholar]
  26. Koch, B., Ma, X. F. & Løbner-Olesen, A. ( 2010; ). Replication of Vibrio cholerae chromosome I in Escherichia coli: dependence on Dam methylation. J Bacteriol 192, 3903–3914,[CrossRef]
    [Google Scholar]
  27. Løbner-Olesen, A., Skovgaard, O. & Marinus, M. G. ( 2005; ). Dam methylation: coordinating cellular processes. Curr Opin Microbiol 8, 154–160,[CrossRef]
    [Google Scholar]
  28. Lu, M., Campbell, J. L., Boye, E. & Kleckner, N. ( 1994; ). SeqA: a negative modulator of replication initiation in E. coli. Cell 77, 413–426,[CrossRef]
    [Google Scholar]
  29. Molina, F. & Skarstad, K. ( 2004; ). Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism. Mol Microbiol 52, 1597–1612,[CrossRef]
    [Google Scholar]
  30. Morigen, Odsbu, I. & Skarstad, K. ( 2009; ). Growth rate dependent numbers of SeqA structures organize the multiple replication forks in rapidly growing Escherichia coli. Genes Cells 14, 643–657,[CrossRef]
    [Google Scholar]
  31. Nielsen, H. J., Youngren, B., Hansen, F. G. & Austin, S. ( 2007; ). Dynamics of Escherichia coli chromosome segregation during multifork replication. J Bacteriol 189, 8660–8666,[CrossRef]
    [Google Scholar]
  32. Odsbu, I., Morigen & Skarstad, K. ( 2009; ). A reduction in ribonucleotide reductase activity slows down the chromosome replication fork but does not change its localization. PLoS ONE 4, e7617,[CrossRef]
    [Google Scholar]
  33. Okada, K., Iida, T., Kita-Tsukamoto, K. & Honda, T. ( 2005; ). Vibrios commonly possess two chromosomes. J Bacteriol 187, 752–757,[CrossRef]
    [Google Scholar]
  34. Onogi, T., Niki, H., Yamazoe, M. & Hiraga, S. ( 1999; ). The assembly and migration of SeqA–Gfp fusion in living cells of Escherichia coli. Mol Microbiol 31, 1775–1782,[CrossRef]
    [Google Scholar]
  35. Pearson, G. D. N., Woods, A., Chiang, S. L. & Mekalanos, J. J. ( 1993; ). CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor. Proc Natl Acad Sci U S A 90, 3750–3754,[CrossRef]
    [Google Scholar]
  36. Rasmussen, T., Jensen, R. B. & Skovgaard, O. ( 2007; ). The two chromosomes of Vibrio cholerae are initiated at different time points in the cell cycle. EMBO J 26, 3124–3131,[CrossRef]
    [Google Scholar]
  37. Saint-Dic, D., Kehrl, J., Frushour, B. & Kahng, L. S. ( 2008; ). Excess SeqA leads to replication arrest and a cell division defect in Vibrio cholerae. J Bacteriol 190, 5870–5878,[CrossRef]
    [Google Scholar]
  38. Skarstad, K., Steen, H. B. & Boye, E. ( 1985; ). Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations. J Bacteriol 163, 661–668.
    [Google Scholar]
  39. Skarstad, K., Boye, E. & Steen, H. B. ( 1986; ). Timing of initiation of chromosome replication in individual Escherichia coli cells. EMBO J 5, 1711–1717.
    [Google Scholar]
  40. Slater, S., Wold, S., Lu, M., Boye, E., Skarstad, K. & Kleckner, N. ( 1995; ). E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. Cell 82, 927–936,[CrossRef]
    [Google Scholar]
  41. Srivastava, P. & Chattoraj, D. K. ( 2007; ). Selective chromosome amplification in Vibrio cholerae. Mol Microbiol 66, 1016–1028,[CrossRef]
    [Google Scholar]
  42. Srivastava, P., Fekete, R. A. & Chattoraj, D. K. ( 2006; ). Segregation of the replication terminus of the two Vibrio cholerae chromosomes. J Bacteriol 188, 1060–1070,[CrossRef]
    [Google Scholar]
  43. Suwanto, A. & Kaplan, S. ( 1989; ). Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. J Bacteriol 171, 5850–5859.
    [Google Scholar]
  44. Torheim, N. K., Boye, E., Løbner-Olesen, A., Stokke, T. & Skarstad, K. ( 2000; ). The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein. Mol Microbiol 37, 629–638,
    [Google Scholar]
  45. Trucksis, M., Michalski, J., Deng, Y. K. & Kaper, J. B. ( 1998; ). The Vibrio cholerae genome contains two unique circular chromosomes. Proc Natl Acad Sci U S A 95, 14464–14469,[CrossRef]
    [Google Scholar]
  46. Vesth, T., Wassenaar, T. M., Hallin, P. F., Snipen, L., Lagesen, K. & Ussery, D. W. ( 2010; ). On the origins of a Vibrio species. Microb Ecol 59, 1–13,
    [Google Scholar]
  47. von Freiesleben, U., Rasmussen, K. V. & Schaechter, M. ( 1994; ). SeqA limits DnaA activity in replication from oriC in Escherichia coli. Mol Microbiol 14, 763–772,[CrossRef]
    [Google Scholar]
  48. Waldminghaus, T. & Skarstad, K. ( 2009; ). The Escherichia coli SeqA protein. Plasmid 61, 141–150,[CrossRef]
    [Google Scholar]
  49. Waldminghaus, T. & Skarstad, K. ( 2010; ). ChIP on Chip: suprising results are ofen artifacts. BMC Genomics 11, 414.[CrossRef]
    [Google Scholar]
  50. Weitao, T., Nordström, K. & Dasgupta, S. ( 2000; ). Escherichia coli cell cycle control genes affect chromosome superhelicity. EMBO Rep 1, 494–499.[CrossRef]
    [Google Scholar]
  51. Wigley, P. & Burton, N. F. ( 2000; ). Multiple chromosomes in Burkholderia cepacia and B. gladioli and their distribution in clinical and environmental strains of B. cepacia. J Appl Microbiol 88, 914–918,[CrossRef]
    [Google Scholar]
  52. Woodfine, K., Fiegler, H., Beare, D. M., Collins, J. E., McCann, O. T., Young, B. D., Debernardi, S., Mott, R., Dunham, I. & Carter, N. P. ( 2004; ). Replication timing of the human genome. Hum Mol Genet 13, 191–202,
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.045112-0
Loading
/content/journal/micro/10.1099/mic.0.045112-0
Loading

Data & Media loading...

Supplements

Oligonucleotides used in this study [PDF](72 KB)

PDF

[PDF](27 KB)

PDF

[PDF](8652 KB)

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