1887

Abstract

The minimal length of integrated homologous donor DNA tracks in transformation and factors influencing the location and length of tracks were determined. Donor DNA contained the gene region (kanamycin resistance, Km). This region carried nine approximately evenly spaced silent nucleotide sequence tags and was embedded in heterologous DNA. Recipient cells carried the normal gene with a central 10 bp deletion (kanamycin-sensitive). The Km transformants obtained had donor DNA tracks integrated covering on average only 4.6 (2–7) of the nine tags, corresponding to about 60 % of the 959 nt homologous donor DNA segment. The track positions were biased towards the 3′ end of . While the replication direction of recipient DNA did not affect track positions, inhibited transcription (by rifampicin) shifted the beginning of tracks towards the promoter. Absence of the RecJ DNase decreased the length of tracks. Absence of SbcCD DNase increased the integration frequency of the 5′ part of , which can form hairpin structures of 43–75 nt, suggesting that SbcCD DNase interferes with hairpins in transforming DNA. In homology-facilitated illegitimate recombination events during transformation (in which a homologous DNA segment serves as a recombinational anchor to facilitate illegitimate recombination in neighbouring heterologous DNA), on average only about half of the approximately 800 nt long tagged anchor sequences were integrated. From donor DNA with an approximately 5000 nt long homologous segment having the gene in the middle, most transformants (74 %) had only a part of the donor integrated, showing that short track integration occurs frequently also from large homologous DNA. It is discussed how short track integration steps can also accomplish incorporation of large DNA molecules.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/021378-0
2008-12-01
2019-10-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/12/3676.html?itemId=/content/journal/micro/10.1099/mic.0.2008/021378-0&mimeType=html&fmt=ahah

References

  1. Barbe, V., Vallenet, D., Fonknechten, N., Kreimeyer, A., Oztas, S., Labarre, L., Cruveiller, S., Robert, C., Duprat, S. & other authors ( 2004; ). Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res 32, 5766–5779.[CrossRef]
    [Google Scholar]
  2. Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B. & Schaller, H. ( 1982; ). Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19, 327–336.[CrossRef]
    [Google Scholar]
  3. Bodmer, W. F. ( 1966; ). Integration of deoxyribonuclease-treated DNA in Bacillus subtilis transformation. J Gen Physiol 49, 233–258.[CrossRef]
    [Google Scholar]
  4. Bodmer, W. F. & Ganesan, A. T. ( 1964; ). Biochemical and genetic studies of integration and recombination in Bacillus subtilis transformation. Genetics 50, 717–738.
    [Google Scholar]
  5. Chalker, A. F., Leach, D. R. & Lloyd, R. G. ( 1988; ). Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome. Gene 71, 201–205.[CrossRef]
    [Google Scholar]
  6. Chen, I. & Dubnau, D. ( 2004; ). DNA uptake during bacterial transformation. Nat Rev Microbiol 2, 241–249.[CrossRef]
    [Google Scholar]
  7. Connelly, J. C., de Leau, E. S. & Leach, D. R. ( 1999; ). DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli. Nucleic Acids Res 27, 1039–1046.[CrossRef]
    [Google Scholar]
  8. de Vries, J. & Wackernagel, W. ( 2002; ). Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination. Proc Natl Acad Sci U S A 99, 2094–2099.[CrossRef]
    [Google Scholar]
  9. de Vries, J. & Wackernagel, W. ( 2005; ). Microbial horizontal gene transfer and the DNA release from transgenic crop plants. Plant Soil 266, 91–104.[CrossRef]
    [Google Scholar]
  10. de Vries, J., Heine, M. & Wackernagel, W. ( 2003; ). Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp. Appl Environ Microbiol 69, 4455–4462.[CrossRef]
    [Google Scholar]
  11. Dower, W. J., Miller, J. F. & Ragsdale, C. W. ( 1988; ). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16, 6127–6145.[CrossRef]
    [Google Scholar]
  12. Dubnau, D. & Cirigliano, C. ( 1972; ). Fate of transforming DNA following uptake by competent Bacillus subtilis. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA. J Mol Biol 64, 9–29.[CrossRef]
    [Google Scholar]
  13. Eggleston, A. K. & West, S. C. ( 2000; ). Cleavage of Holliday junctions by the Escherichia coli RuvABC complex. J Biol Chem 275, 26467–26476.[CrossRef]
    [Google Scholar]
  14. Ephrussi-Taylor, H. & Gray, T. C. ( 1966; ). Genetic studies of recombining DNA in pneumococcal transformation. J Gen Physiol 49, 211–231.[CrossRef]
    [Google Scholar]
  15. Fornili, S. L. & Fox, M. S. ( 1977; ). Electron microscope visualization of the products of Bacillus subtilis transformation. J Mol Biol 113, 181–191.[CrossRef]
    [Google Scholar]
  16. Friedman-Ohana, R. & Cohen, A. ( 1998; ). Heteroduplex joint formation in Escherichia coli recombination is initiated by pairing of a 3′-ending strand. Proc Natl Acad Sci U S A 95, 6909–6914.[CrossRef]
    [Google Scholar]
  17. Gerischer, U. & Ornston, L. N. ( 2001; ). Dependence of linkage of alleles on their physical distance in natural transformation of Acinetobacter sp. strain ADP1. Arch Microbiol 176, 465–469.[CrossRef]
    [Google Scholar]
  18. Gurney, T., Jr & Fox, M. S. ( 1968; ). Physical and genetic hybrids formed in bacterial transformation. J Mol Biol 32, 83–100.[CrossRef]
    [Google Scholar]
  19. Hanahan, D. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[CrossRef]
    [Google Scholar]
  20. Harms, K. & Wackernagel, W. ( 2008; ). The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi. Microbiology 154, 2437–2445.[CrossRef]
    [Google Scholar]
  21. Harms, K., Schön, V., Kickstein, E. & Wackernagel, W. ( 2007; ). The RecJ DNase strongly suppresses genomic integration of short but not long foreign DNA fragments by homology-facilitated illegitimate recombination during transformation of Acinetobacter baylyi. Mol Microbiol 64, 691–702.[CrossRef]
    [Google Scholar]
  22. Honigberg, S. M. & Radding, C. M. ( 1988; ). The mechanics of winding and unwinding helices in recombination: torsional stress associated with strand transfer promoted by RecA protein. Cell 54, 525–532.[CrossRef]
    [Google Scholar]
  23. Hülter, N. & Wackernagel, W. ( 2008; ). Double illegitimate recombination events integrate DNA segments through two different mechanisms during natural transformation of Acinetobacter baylyi. Mol Microbiol 67, 984–995.[CrossRef]
    [Google Scholar]
  24. Juni, E. ( 1972; ). Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J Bacteriol 112, 917–931.
    [Google Scholar]
  25. Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. ( 1988; ). Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 191–197.[CrossRef]
    [Google Scholar]
  26. Kickstein, E., Harms, K. & Wackernagel, W. ( 2007; ). Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi. Microbiology 153, 2259–2270.[CrossRef]
    [Google Scholar]
  27. Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., Lauder, S. D. & Rehrauer, W. M. ( 1994; ). Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 58, 401–465.
    [Google Scholar]
  28. Lacks, S. A. ( 1988; ). Mechanisms of genetic recombination in Gram-positive bacteria. In Genetic Recombination, pp. 43–86. Edited by R. Kucherlapati & G. R. Smith. Washington, DC: American Society for Microbiology.
  29. Lacks, S. A. ( 2000; ). DNA uptake by transformable bacteria. In Transport of Molecules Across Microbial Membranes, pp. 138–168. Edited by J. K. Broome-Smith, S. Baumberg, C. J. Stirling & F. B. Ward. Cambridge: Cambridge University Press.
  30. Leach, D. R., Okely, E. A. & Pinder, D. J. ( 1997; ). Repair by recombination of DNA containing a palindromic sequence. Mol Microbiol 26, 597–606.[CrossRef]
    [Google Scholar]
  31. Liu, L. F. & Wang, J. C. ( 1987; ). Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A 84, 7024–7027.[CrossRef]
    [Google Scholar]
  32. Lorenz, M. G. & Wackernagel, W. ( 1994; ). Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 58, 563–602.
    [Google Scholar]
  33. Meier, P. & Wackernagel, W. ( 2003; ). Mechanisms of homology-facilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri. Mol Microbiol 48, 1107–1118.[CrossRef]
    [Google Scholar]
  34. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  35. Modrich, P. & Lahue, R. ( 1996; ). Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem 65, 101–133.[CrossRef]
    [Google Scholar]
  36. Morrison, D. A. & Mannarelli, B. ( 1979; ). Transformation in pneumococcus: nuclease resistance of deoxyribonucleic acid in the eclipse complex. J Bacteriol 140, 655–665.
    [Google Scholar]
  37. Morrison, D. A., Mortier-Barriere, I., Attaiech, L. & Claverys, J. P. ( 2007; ). Identification of the major protein component of the pneumococcal eclipse complex. J Bacteriol 189, 6497–6500.[CrossRef]
    [Google Scholar]
  38. Mortier-Barriere, I., Velten, M., Dupaigne, P., Mirouze, N., Pietrement, O., McGovern, S., Fichant, B., Martin, P., Noirot, P. & other authors ( 2007; ). A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell 130, 824–836.[CrossRef]
    [Google Scholar]
  39. Notani, N. & Goodgal, S. H. ( 1966; ). On the nature of recombinants formed during transformation in Haemophilus influenzae. J Gen Physiol 49, 197–209.[CrossRef]
    [Google Scholar]
  40. Pasta, F. & Sicard, M. A. ( 1996; ). Exclusion of long heterologous insertions and deletions from the pairing synapsis in pneumococcal transformation. Microbiology 142, 695–705.[CrossRef]
    [Google Scholar]
  41. Pasta, F. & Sicard, M. A. ( 1999; ). Polarity of recombination in transformation of Streptococcus pneumoniae. Proc Natl Acad Sci U S A 96, 2943–2948.[CrossRef]
    [Google Scholar]
  42. 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]
    [Google Scholar]
  43. Prudhomme, M., Libante, V. & Claverys, J. P. ( 2002; ). Homologous recombination at the border: insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae. Proc Natl Acad Sci U S A 99, 2100–2105.[CrossRef]
    [Google Scholar]
  44. Rahmouni, A. R. & Wells, R. D. ( 1992; ). Direct evidence for the effect of transcription on local DNA supercoiling in vivo. J Mol Biol 223, 131–144.[CrossRef]
    [Google Scholar]
  45. Razavy, H., Szigety, S. K. & Rosenberg, S. M. ( 1996; ). Evidence for both 3′ and 5′ single-strand DNA ends in intermediates in chi-stimulated recombination in vivo. Genetics 142, 333–339.
    [Google Scholar]
  46. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  47. Stubbings, W., Bostock, J., Ingham, E. & Chopra, I. ( 2006; ). Mechanisms of the post-antibiotic effects induced by rifampicin and gentamicin in Escherichia coli. J Antimicrob Chemother 58, 444–448.[CrossRef]
    [Google Scholar]
  48. Thomas, C. M. & Nielsen, K. M. ( 2005; ). Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat Rev Microbiol 3, 711–721.[CrossRef]
    [Google Scholar]
  49. Viswanathan, M. & Lovett, S. T. ( 1998; ). Single-strand DNA-specific exonucleases in Escherichia coli. Roles in repair and mutation avoidance. Genetics 149, 7–16.
    [Google Scholar]
  50. Wang, J. C. ( 1996; ). DNA topoisomerases. Annu Rev Biochem 65, 635–692.[CrossRef]
    [Google Scholar]
  51. Warren, G. J. & Green, R. L. ( 1985; ). Comparison of physical and genetic properties of palindromic DNA sequences. J Bacteriol 161, 1103–1111.
    [Google Scholar]
  52. Yoshimura, H., Yoshino, M., Hirose, T., Nakamura, Y., Higashi, M., Hase, T., Yamaguchi, K., Hirokawa, H. & Masamune, Y. ( 1986; ). Biological characteristics of palindromic DNA (II). J Gen Appl Microbiol 32, 393–404.[CrossRef]
    [Google Scholar]
  53. Zuker, M. ( 2003; ). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406–3415.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/021378-0
Loading
/content/journal/micro/10.1099/mic.0.2008/021378-0
Loading

Data & Media loading...

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