Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Mar;61(3):985–991. doi: 10.1128/aem.61.3.985-991.1995

Development and testing of improved suicide functions for biological containment of bacteria.

S Knudsen 1, P Saadbye 1, L H Hansen 1, A Collier 1, B L Jacobsen 1, J Schlundt 1, O H Karlström 1
PMCID: PMC167358  PMID: 7793926

Abstract

We have developed very efficient suicide functions for biological containment based on the lethal Escherichia coli relF gene. The suicide functions are placed in duplicate within a plasmid and arranged to prevent inactivation by deletion, recombination, and insertional inactivation. The efficiency of this concept was tested in a plasmid containment system that prevents transfer of plasmids to wild-type bacteria. Protection against plasmid transfer was assayed in test tubes and in rat intestine. Protection was efficient and refractory to inactivation by mutation and transposons. The efficiency of the suicide system was also tested in soil and seawater. We show that unprecedented suicide efficiency can be achieved in soil and seawater after suicide induction by IPTG (isopropyl-beta-D-thiogalactopyranoside). More than 7 orders of magnitude reduction in suicide bacteria was achieved.

Full Text

The Full Text of this article is available as a PDF (246.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bech F. W., Jørgensen S. T., Diderichsen B., Karlström O. H. Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene. EMBO J. 1985 Apr;4(4):1059–1066. doi: 10.1002/j.1460-2075.1985.tb03739.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bej A. K., Perlin M. H., Atlas R. M. Model suicide vector for containment of genetically engineered microorganisms. Appl Environ Microbiol. 1988 Oct;54(10):2472–2477. doi: 10.1128/aem.54.10.2472-2477.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Contreras A., Molin S., Ramos J. L. Conditional-suicide containment system for bacteria which mineralize aromatics. Appl Environ Microbiol. 1991 May;57(5):1504–1508. doi: 10.1128/aem.57.5.1504-1508.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Covarrubias L., Cervantes L., Covarrubias A., Soberón X., Vichido I., Blanco A., Kupersztoch-Portnoy Y. M., Bolivar F. Construction and characterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328. Gene. 1981 Jan-Feb;13(1):25–35. doi: 10.1016/0378-1119(81)90040-8. [DOI] [PubMed] [Google Scholar]
  8. Gerdes K., Bech F. W., Jørgensen S. T., Løbner-Olesen A., Rasmussen P. B., Atlung T., Boe L., Karlstrom O., Molin S., von Meyenburg K. Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. EMBO J. 1986 Aug;5(8):2023–2029. doi: 10.1002/j.1460-2075.1986.tb04459.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jensen L. B., Ramos J. L., Kaneva Z., Molin S. A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene. Appl Environ Microbiol. 1993 Nov;59(11):3713–3717. doi: 10.1128/aem.59.11.3713-3717.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kilbane J. J., Malamy M. H. F factor mobilization of non-conjugative chimeric plasmids in Escherichia coli: general mechanisms and a role for site-specific recA-independent recombination at orV1. J Mol Biol. 1980 Oct 15;143(1):73–93. doi: 10.1016/0022-2836(80)90125-4. [DOI] [PubMed] [Google Scholar]
  11. Knudsen S. M., Karlström O. H. Development of efficient suicide mechanisms for biological containment of bacteria. Appl Environ Microbiol. 1991 Jan;57(1):85–92. doi: 10.1128/aem.57.1.85-92.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lanzer M., Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973–8977. doi: 10.1073/pnas.85.23.8973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Miller J. H., Ganem D., Lu P., Schmitz A. Genetic studies of the lac repressor. I. Correlation of mutational sites with specific amino acid residues: construction of a colinear gene-protein map. J Mol Biol. 1977 Jan 15;109(2):275–298. doi: 10.1016/s0022-2836(77)80034-x. [DOI] [PubMed] [Google Scholar]
  15. Molin S., Boe L., Jensen L. B., Kristensen C. S., Givskov M., Ramos J. L., Bej A. K. Suicidal genetic elements and their use in biological containment of bacteria. Annu Rev Microbiol. 1993;47:139–166. doi: 10.1146/annurev.mi.47.100193.001035. [DOI] [PubMed] [Google Scholar]
  16. Recorbet G., Robert C., Givaudan A., Kudla B., Normand P., Faurie G. Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene. Appl Environ Microbiol. 1993 May;59(5):1361–1366. doi: 10.1128/aem.59.5.1361-1366.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stotzky G., Babich H. Fate of genetically-engineered microbes in natural environments. Recomb DNA Tech Bull. 1984 Dec;7(4):163–188. [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES