Current Microbiology

, Volume 20, Issue 6, pp 373–379 | Cite as

Stabilization of a plasmid-encoded LacZ phenotype inBacillus subtilis

  • Yuval Shoham
  • Arnold L. Demain


Recombinant plasmid pCED3 was structurally unstable inBacillus subtilis cultures grown in the presence of kanamycin to eliminate the effects of segregational instability. Analysis of 96 modified plasmids indicated that deletions in the plasmid occur at many different sites. The presence of plasmid pCED3 slowed the growth rate of theB. subtilis host. Cells that contained modified plasmids grew faster than the parental cells and took over the population. Two different methodologies were developed to reduce the cultural instability of the plasmid-directed LacZ+ phenotype. By growing the cells in a medium that supports a low growth rate, the growth rate ratio between modified and parental cells was reduced, resulting in a partial stabilization (40 generations) of the LacZ+ phenotype in the population [35]. Removal of a 4.77 kbEcoRI fragment (which consists primarily of the pBR322 replicon) from plasmid pCED3 produced a more stable plasmid derivative, designated pYS1. Cells harboring plasmid pYS1 grew faster than pCED3-bearing cells, although the level of activity of β-galactosidase was similar in both strains. By combining the two approaches (i.e., growth of pYS1-bearing cells in a medium that supports low growth rate), the LacZ+ phenotype was stably maintained in the cell population for over 170 generations. Under these conditions, there was no detectable difference between the growth rates of cells bearing the pYS1 plasmid and further modified plasmids.


Growth Rate Cell Population Kanamycin Recombinant Plasmid Rate Ratio 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Albertini AM, Hofer M, Calos MP, Miller JH (1982) On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell 29:319–328PubMedGoogle Scholar
  2. 2.
    Ballester S, Lopez P, Espinosa M, Alonso JC, Lacks AS (1989) Plasmid structural instability associated with pC194 replication functions. J Bacteriol 171:2271–2277PubMedGoogle Scholar
  3. 3.
    Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254PubMedGoogle Scholar
  4. 4.
    Bron S, Luxen E, Swart P (1988) Instability of recombinant pUB110 plasmids inBacillus subtilis: plasmid encoded stability function and effects of DNA inserts. Plasmid 19:231–241PubMedGoogle Scholar
  5. 5.
    Contente S, Dubnau D (1979) Marker rescue transformation by linear plasmid inBacillus subtilis. Plasmid 2:555–571PubMedGoogle Scholar
  6. 6.
    Donnelly EC, Sonenshein AL (1982) Genetic fusion ofEscherichia coli lac genes to aBacillus subtilis promoter. In: Ganesan AT, Chang S, Hoch J (eds) Molecular cloning and gene regulation in bacilli, New York: Academic Press, pp 63–72Google Scholar
  7. 7.
    Edlund T, Normark S (1981) Recombination between short DNA homologies causes tandem duplication. Nature 292:269–271PubMedGoogle Scholar
  8. 8.
    Ehrlich SD, Niaudet B, Michel B (1982) Use of plasmids fromStaphylococcus aureus for cloning of DNA inBacillus subtilis. Curr Top Microbiol Immunol 96:19–29PubMedGoogle Scholar
  9. 9.
    Ehrlich SD, Noirot PH, Petit MA, Janniere L, Michel B, Riele HT (1986) Structural instability ofBacillus subtilis plasmids. In: Setlow JK, Hollaender A (eds) Genetic Engineering, vol. 8. New York: Plenum, pp 71–83Google Scholar
  10. 10.
    Fang A, Demain AL (1989) A new chemically-defined medium for RAC-certified and other strains ofBacillus subtilis. Appl Microbiol Biotechnol 30:144–147Google Scholar
  11. 11.
    Farabaugh PJ, Schmeissner U, Hofer M, Miller JH (1978) Genetic studies of thelac repressor. VII. On the molecular nature of spontaneous hotspots in thelacI gene ofE. coli. J Mol Biol 126:847–857PubMedGoogle Scholar
  12. 12.
    Ghosal D, Saedler H (1979) IS2-61 and IS-611 arise by illegitimate recombination from IS2-6. Mol Gen Genet 176:233–238PubMedGoogle Scholar
  13. 13.
    Goebel W, Kreft J, Burger KJ (1979) Molecular cloning inBacillus subtilis. In: Timmis KN, Puhler A (eds) Plasmids of medical, environmental and commercial importance. Amsterdam: Elsevier/North Holland Biomedical Press, pp 471–480Google Scholar
  14. 14.
    Grcyzan TJ, Contente S, Dubnau D (1978) Characterization ofStaphylococcus aureus plasmids introduced by transformation intoBacillus subtilis. J Bacteriol 134:318–329PubMedGoogle Scholar
  15. 15.
    Gruss AD, Ehrlich SD (1988) Insertion of foreign DNA into plasmids from gram-positive bacteria induces formation of high molecular weight plasmid multimers. J Bacteriol 170:1183–1190PubMedGoogle Scholar
  16. 16.
    Gruss AD, Ross HF, Novick RP (1987) Functional analysis of palindromic sequences required for normal replication of several staphylococcal plasmids. Proc Natl Acad Sci USA 84:2165–2169PubMedGoogle Scholar
  17. 17.
    Hahn J, Dubnau D (1985) Analysis of plasmid deletional instability inBacillus subtilis. J Bacteriol 162:1014–1023PubMedGoogle Scholar
  18. 18.
    Ikeda H, Aoki K, Naito A (1982) Illegitimate recombination mediatedin vitro by DNA gyrase ofEscherichia coli: structure of recombinant DNA molecule. Proc Natl Acad Sci USA 79:3724–3728PubMedGoogle Scholar
  19. 19.
    Ikeda H, Kawasaki I, Gellert M (1984) Mechanism of illegitimate recombination. Common sites for recombination and cleavage mediated byEscherichia coli DNA gyrase. Molec Gen Genet 196:546–549PubMedGoogle Scholar
  20. 20.
    Jones IM, Primrose SB, Ehrlich SD (1982) Replication between short direct repeats in a RecA host. Mol Gen Genet 188:486–489PubMedGoogle Scholar
  21. 21.
    Katz E, Demain AL (1977) The peptide antibiotics ofBacillus: chemistry, biogenesis and possible functions. Bacteriol Rev 41:449–474PubMedGoogle Scholar
  22. 22.
    Kreft J, Hughes C (1982) Cloning vectors derived from plasmids and phage ofBacillus. Curr Top Microbiol Immunol 96:1–17Google Scholar
  23. 23.
    Keft J, Parrisius J, Burger KJ, Goebel W (1982) Expression and instability of heterologous genes inB. subtilis. In: Ganesan AT, Chang S, Hoch J (eds) Molecular cloning and gene regulation in bacilli. New York: Academic Press, pp 145–157Google Scholar
  24. 24.
    Lopez P, Espinosa M, Greenberg B, Lacks SA (1984) Generation of deletions in pneumococcalmal genes inBacillus subtilis. Proc Natl Acad Sci USA 81:5189–5193PubMedGoogle Scholar
  25. 25.
    Lusky M, Botchan M (1981) Inhibitory effect of specific pBR322 DNA sequences upon SV40 replication in simian cells. Nature 293:79–81PubMedGoogle Scholar
  26. 26.
    Michel B, Ehrlich DS (1986) Illegitimate recombination at the origin of the plasmid pC194. EMBO J 5:3691–3696PubMedGoogle Scholar
  27. 27.
    Miller J (1972) Experiments in molecular genetics: Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, pp 352–355Google Scholar
  28. 28.
    Nakano MM, Ogawara H, Sekiya T (1984) Recombination between short direct repeats inStreptomyces lavendulae plasmid. J Bacteriol 157:658–660PubMedGoogle Scholar
  29. 29.
    Peijnenburg AACM, Bron S, Venema G (1988) Plasmid deletion formation inBacillus subtilis. Plasmid 20:23–32PubMedGoogle Scholar
  30. 30.
    Piggot PJ, Hoch JH (1985) Revised genetic linkage map ofBacillus subtilis. Microbiol Rev 49:158–179PubMedGoogle Scholar
  31. 31.
    Post LE, Arfsten AE, Davis GR, Nomura M (1980) DNA sequences of the promoter region for the alpha ribosomal protein operon inEscherichia coli. J. Biol. Chem. 255:4653–4659PubMedGoogle Scholar
  32. 32.
    Priest FG (1977) Extracellular enzyme synthesis in the genusBacillus. Bacteriol Rev 41:711–753PubMedGoogle Scholar
  33. 33.
    Sadaie Y, Kenneth BC, Doi RH (1980) Purification and characterization of a kanamycin nucleotidyltransferase from plasmid pUB110-carrying cells ofBacillus subtilis. J Bacteriol 141:1178–1182PubMedGoogle Scholar
  34. 34.
    Semon O, Raomovva N, Smith TF, Alama ME, Cohen S (1987) Plasmid-determined bleomycin resistance inStaphylococcus aureus. Plasmid 17:46–53PubMedGoogle Scholar
  35. 35.
    Shoham Y, Demain AL (1990) Effect of medium composition on the maintenance of a recombinant plasmid inBacillus subtilis. Enzyme Microb Technol, in pressGoogle Scholar
  36. 36.
    Spizizen J (1958) Transformation of biochemically deficient strains ofBacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 44:1072–1078Google Scholar
  37. 37.
    Viret J-F, Olonso JC (1987) Generation of linear multigenome length plasmid molecules inBacillus subtilis. Nucleic Acids Res 15:6349–6367PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 1990

Authors and Affiliations

  • Yuval Shoham
    • 1
  • Arnold L. Demain
    • 1
  1. 1.Fermentation Microbiology Laboratory, Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA

Personalised recommendations