This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jensen, L B Articles by Molin, S Search for Related Content PubMed PubMed Citation Articles by Jensen, L B Articles by Molin, S Agricola Articles by Jensen, L B Articles by Molin, S

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1993 November; 59(11): 3713-3717

A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene.

Department of Microbiology, Technical University of Denmark, Lyngby.

ABSTRACT

A model substrate-dependent suicide system to biologically contain Pseudomonas putida KT2440 is reported. The system consists of two elements. One element carries a fusion between a synthetic lac promoter (PA1-04/03) and the gef gene, which encodes a killing function. This element is contained within a transposaseless mini-Tn5 transposon so that it can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to control the first and bears a fusion between the promoter of the P. putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene, encoding the Lac repressor, plus xylS2, coding for a positive regulator of Pm. In liquid culture under optimal growth conditions and in sterile and nonsterile soil microcosms, P. putida KT2440 (pWWO) bearing the containment system behaves as designed. In the presence of a XylS effector, such as m-methylbenzoate, the LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the PA1-04/03::gef cassette is no longer prevented and a high rate of cell killing is observed. Fluctuation test analyses revealed that mutants resistant to cell killing arise at a frequency of around 10(-5) to 10(-6) per cell per generation. Mutations are linked to the killing element rather than to the regulatory one. In bacteria bearing two copies of the killing cassette, the rate of appearance of mutants resistant to killing decreased to as low as 10(-8) per cell per generation.

This article has been cited by other articles:

• Torres, B., Jaenecke, S., Timmis, K. N., Garcia, J. L., Diaz, E. (2003). A dual lethal system to enhance containment of recombinant micro-organisms. Microbiology 149: 3595-3601 [Abstract] [Full Text]  
• Haidinger, W., Mayr, U. B., Szostak, M. P., Resch, S., Lubitz, W. (2003). Escherichia coli Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease. Appl. Environ. Microbiol. 69: 6106-6113 [Abstract] [Full Text]  
• Westwater, C., Kasman, L. M., Schofield, D. A., Werner, P. A., Dolan, J. W., Schmidt, M. G., Norris, J. S. (2003). Use of Genetically Engineered Phage To Deliver Antimicrobial Agents to Bacteria: an Alternative Therapy for Treatment of Bacterial Infections. Antimicrob. Agents Chemother. 47: 1301-1307 [Abstract] [Full Text]  
• Ronchel, M. C., Molina, L., Witte, A., Lutbiz, W., Molin, S., Ramos, J. L., Ramos, C. (1998). Characterization of Cell Lysis in Pseudomonas putida Induced upon Expression of Heterologous Killing Genes. Appl. Environ. Microbiol. 64: 4904-4911 [Abstract] [Full Text]  
• Berlyn, M. K. B. (1998). Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map. Microbiol. Mol. Biol. Rev. 62: 814-984 [Abstract] [Full Text]  
• Molina, L., Ramos, C., Ronchel, M.-C., Molin, S., Ramos, J. L. (1998). Construction of an Efficient Biologically Contained Pseudomonas putida Strain and Its Survival in Outdoor Assays. Appl. Environ. Microbiol. 64: 2072-2078 [Abstract] [Full Text]  
• Szafranski, P., Mello, C. M., Sano, T., Smith, C. L., Kaplan, D. L., Cantor, C. R. (1997). A new approach for containment of microorganisms: Dual control of streptavidin expression by antisense RNA and the T7 transcription system. Proc. Natl. Acad. Sci. USA 94: 1059-1063 [Abstract] [Full Text]