| Abstract: | The sacB gene from Bacillus subtilis confers sucrose sensitivity upon gram-negative bacteria. The gene was investigated for use as a potential conditional suicide system for Escherichia coli released into soil. To ensure against the loss of the cell death function encoded under nonselective conditions, the nptI-sacR-B suicide cassette was inserted into the E. coli chromosome by using a circular nonreplicative integration vector. Stability studies yielded no loss of the suicide cassette in the integrated E. coli EL1026 strain. sacB induction in the absence of a selective pressure resulted in a lysis efficiency of up to 99.9%. The microcosm experiments confirmed the ability of the suicide cassette to limit the growth and reduce the survival of E. coli strains released into soil. Sucrose addition to sterile soil resulted in a 10(-3)-fold reduction of the final E. coli population density. sacB induction prevented the proliferation and triggered the rapid disappearance of E. coli from natural soil. Mutation to sucrose tolerance occurred at a frequency of 10(-5), making E. coli EL1026 a potential counterselectable donor strain for gene transfer studies. Specificity and potential adaptability to a wide range of gram-negative bacteria are additional conveniences of this conditional suicide system for the containment and counterselection of engineered microorganisms. |
