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. 1991 May;57(5):1504–1508. doi: 10.1128/aem.57.5.1504-1508.1991

Conditional-Suicide Containment System for Bacteria Which Mineralize Aromatics

Asunción Contreras 1,†, Soren Molin 1, Juan-Luis Ramos 1,*
PMCID: PMC182976  PMID: 16348490

Abstract

A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the Pseudomonas putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene encoding Lac repressor plus xylS, coding for the positive regulator of Pm. The other element carries a fusion between the Ptac promoter and the gef gene, which encodes a killing function. In the presence of XylS effectors, LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the Ptac::gef cassette is no longer prevented and a high rate of cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant XylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl-, and methoxybenzoates; salicylate; and methyl- and chlorosalicylates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown.

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Selected References

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

  1. Abril M. A., Michan C., Timmis K. N., Ramos J. L. Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol. 1989 Dec;171(12):6782–6790. doi: 10.1128/jb.171.12.6782-6790.1989. [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. Gerdes K., Rasmussen P. B., Molin S. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3116–3120. doi: 10.1073/pnas.83.10.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Lehrbach P. R., Zeyer J., Reineke W., Knackmuss H. J., Timmis K. N. Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13. J Bacteriol. 1984 Jun;158(3):1025–1032. doi: 10.1128/jb.158.3.1025-1032.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Mermod N., Ramos J. L., Bairoch A., Timmis K. N. The xylS gene positive regulator of TOL plasmid pWWO: identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon. Mol Gen Genet. 1987 May;207(2-3):349–354. doi: 10.1007/BF00331600. [DOI] [PubMed] [Google Scholar]
  7. Poulsen L. K., Larsen N. W., Molin S., Andersson P. A family of genes encoding a cell-killing function may be conserved in all gram-negative bacteria. Mol Microbiol. 1989 Nov;3(11):1463–1472. doi: 10.1111/j.1365-2958.1989.tb00131.x. [DOI] [PubMed] [Google Scholar]
  8. Ramos J. L., Mermod N., Timmis K. N. Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas. Mol Microbiol. 1987 Nov;1(3):293–300. doi: 10.1111/j.1365-2958.1987.tb01935.x. [DOI] [PubMed] [Google Scholar]
  9. Ramos J. L., Michan C., Rojo F., Dwyer D., Timmis K. Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon. J Mol Biol. 1990 Jan 20;211(2):373–382. doi: 10.1016/0022-2836(90)90358-S. [DOI] [PubMed] [Google Scholar]
  10. Ramos J. L., Stolz A., Reineke W., Timmis K. N. Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8467–8471. doi: 10.1073/pnas.83.22.8467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ramos J. L., Timmis K. N. Experimental evolution of catabolic pathways of bacteria. Microbiol Sci. 1987 Aug;4(8):228–237. [PubMed] [Google Scholar]
  12. Ramos J. L., Wasserfallen A., Rose K., Timmis K. N. Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkylbenzoates. Science. 1987 Jan 30;235(4788):593–596. doi: 10.1126/science.3468623. [DOI] [PubMed] [Google Scholar]
  13. Reineke W., Knackmuss H. J. Construction of haloaromatics utilising bacteria. Nature. 1979 Feb 1;277(5695):385–386. doi: 10.1038/277385a0. [DOI] [PubMed] [Google Scholar]
  14. Rojo F., Pieper D. H., Engesser K. H., Knackmuss H. J., Timmis K. N. Assemblage of ortho cleavage route for simultaneous degradation of chloro- and methylaromatics. Science. 1987 Dec 4;238(4832):1395–1398. doi: 10.1126/science.3479842. [DOI] [PubMed] [Google Scholar]

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