Archives of Microbiology

, Volume 136, Issue 4, pp 275–280 | Cite as

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA

  • S. Sadashiva Karnik
  • K. P. Gopinathan
Original Papers


Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl2, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From 32P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).

Key words

Transfection Mycobacterium smegmatis Mycobacteriophage I3 Calcium sensitization Glycine sensitization Protamine sulfate Exogenous RNA DNA uptake 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bailey AD, Fry BA (1968) Effect of ribonucleic acid on the infectivity of phage DNA. Biochim Biophys Acta 166:726–728Google Scholar
  2. Barksdale L, Kim KS (1977) Mycobacterium. Bacteriol Rev 41:217–372Google Scholar
  3. Boling ME, Setlow JK, Allison DP (1972) Bacteriophage of Haemophilus influenzae. I. Differences between infection by whole phage, extracted DNA and prophage DNA extracted from lysogenic cells. J Mol Biol 63:335–348Google Scholar
  4. Chater KF, Hopwood DA, Kieser T, Thompson CJ (1982) Gene cloning in Streptomyces. In: Hofschneider PH, Goebel W (eds) Current topics in microbiol immunol, vol 96: Gene cloning in organisms other than E. coli. Springer, Berlin Heidelberg New York, pp 69–95Google Scholar
  5. Friedman EA, Smith HO (1972) An adenosine triphosphate-dependent deoxy-ribonuclease from Haemophilus influenzae Rd. J Biol Chem 247:2859–2865Google Scholar
  6. Grange JM (1975) The genetics of mycobacteria and mycobacteriophages. Tubercle 56:227–238Google Scholar
  7. Green DM (1966) Intracellular inactivation of infective SP82 bacteriophage DNA. J Mol Biol 22:1–13Google Scholar
  8. Hotz G, Mauser R (1969) Infections DNA from coliphage T1. I. Some properties of spheroplast assay system. Molec Gen Genetic 104:178–194Google Scholar
  9. Karnik SS, Gopinathan KP (1980) Possible involvement of a calciumstimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process. J Virol 33:969–975Google Scholar
  10. Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162Google Scholar
  11. Nakamura RM (1970) Transfection of Mycobacterium smegmatis in an acid medium. In: Juhasz SE, Plummer G (eds) Hostvirus relationships in Mycobacterium, Nocardia, and Actinomyces. Charles C. Thomas, Springfield, Ill., pp 166–178Google Scholar
  12. Norgard MV, Imaeda T (1978) Physiological factors involved in the transfection of Mycobacterium smegmatis. J Bacteriol 133:1254–1262Google Scholar
  13. Notani NK, Setlow JK (1974) Mechanism of Bacterial transformation and transfection. Progress in nucleic acid research and molecular biology, vol 14. Academic Press, New York, pp 39–100Google Scholar
  14. Notani NK, Setlow JK, Allison DP (1973) Intracellular events during Infection by Haemophilus influenzae phage and transfection by its DNA. J Mol Biol 75:581–599Google Scholar
  15. Rieber M, Imaeda T (1970) The uptake of mycobacteriohpage DNA and other polynucleotides by mycobacteria. In: Juhasz SE, Plummer G (eds) Host-virus relationships in Mycobacterium, Nocardia and Actinomyces. Charles C. Thomas, Springfield, Ill., pp 144–151Google Scholar
  16. Sadhu C, Gopinathan KP (1982) A rapid procedure for the isolation of spheroplasts from Mycobacterium smegmatis. FEMS Microbiol Letters 15:19–22Google Scholar
  17. Smull CE, Ludwig EH (1962) Enhancement of plaque forming capacity of poliovirus ribonucleic acid with basic proteins. J Bacteriol 84:1035–1040Google Scholar
  18. Spatz M Ch, Trautner TA (1971) The role of recombination in transfection of B. subtilis. Moles Gen Genetics 113:174–194Google Scholar
  19. Tokunaga T, Sellers MI (1964) Infection of Mycobacterium smegmatis with D29 phage DNA. J Exp Med 119:139–149Google Scholar
  20. Tokunaga T, Sellers MI (1970) Transfection of amino acid-sensitized mycobacteria. In: Juhasz SE, Plummer G (eds) Host-virus relationships in Mycobacterium, Nocardia and Actinomyces. Charles C. Thomas, Springfield, Ill, pp 152–165Google Scholar
  21. Wackernagel W (1972) An improved spheroplast assay for λ DNA and the influence of the bacterial genotype on the transfection rate. Virology 48:94–103Google Scholar
  22. Winder FG, Coughlan MP (1969) A nucleoside triphosphate-dependent deoxyribonucleic acid-breakdown system in Mycobacterium smegmatis, and the effect of ion limitation on the activity of this system. Biochem J 111:679–687Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • S. Sadashiva Karnik
    • 1
  • K. P. Gopinathan
    • 1
  1. 1.Microbiology and Cell Biology LaboratoryIndian Institute of ScienceBangaloreIndia

Personalised recommendations