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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Safer host-vector systems for the deliberate release of plant-beneficial Pseudomonas

Background and objectives

Certain bacteria possess properties which make them beneficial to agriculture, such as antagonism of phytopathogenic fungi and bacteria, antibiotic production, plant growth hormone production, nitrogen fixation. Others, particularly the Pseudomonads are particularly adept at the degradation of man-made xenobiotic substances which pollute the environment, either accidentally (e.g. polychlorinated aromatics or oil spills) or deliberately (pesticides and herbicides). Genetic manipulation of such bacteria may render them better able to perform these tasks or to catalyse new reactions not previously possible. The purpose of this project was to stabilise the recombinant genes present in such bacteria, to prevent horizontal transfer to other bacteria, and to eliminate undesirable selective markers such as antibiotic resistance genes.

We aimed to construct safer recombinant DNA vectors for those bacteria (e.g. Pseudomonas, Rhizobium) which might be considered desirable to release into the environment for agricultural purposes, or for pollution bioremediation.


Approach and methodology

Recombinant DNA vectors are usually self-replicating plasmids. Unfortunately these vectors can often be horizontally transferred to other soil bacteria by conjugation, transformation or transduction. These properties make them unsuitable for experiments involving the deliberate release of recombinant bacteria into the environment. Furthermore, they are usually equipped with antibiotic resistance genes, a feature which is also undesirable for deliberate environmental release. For these reasons it was decided to construct vectors which are based on transposons, which have reduced horizontal transmission, being unable to self replicate. The horizontal transmission could be reduced still further by removal of the transposase gene. The antibiotic resistance genes were replaced by siderophore receptors, which act as excellent selective markers, without being of medical importance.
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Main findings and outcome

The project was successful and achieved the desired aim of constructing improved vector systems for the deliberate release of recombinant micro-organisms into the environment. Transposon vectors were modified to contain a maximum number of useful restriction sites to facilitate cloning and gene manipulation. These transposon vectors were carried on pBR322 based plasmids which had also been modified to eliminate the corresponding useful restriction sites, thus facilitating cloning into the transposon part of the DNA molecule. This pBR322-based delivery vehicle was further equipped with the mobilization genes of plasmid RP4 so that it could be transferred at high frequency from E. coli to Pseudomonas, by conjugation in the presence of a conjugative RP4 plasmid. Once inside the Pseudomonas cell, the pBR322 plasmid replicon can no longer function and the plasmid acts as a suicide vector and is rapidly lost from the cell. Thus, the transposon, including the recombinant genes that it may carry, can only survive if it transposes (jumps and integrates) to the host chromosome. In this location it has greater stability and a much reduced rate of horizontal transfer to other bacteria.

In a second stage, the undesirable antibiotic resistance genes of the transposon vectors were replaced with the siderophore receptor pupA from Pseudomonas putida WCS358. The presence of this receptor permits the recipient bacteria to use the Pseudomonas WCS358 pseudobactin-358 siderophores as a source of iron. Thus, in the presence of both iron deprivation and pseudobactin-358, bacteria carrying the transposon-borne siderophore receptor are able to grow, while all others cannot. Thus, this siderophore receptor marker provides a safer selection marker than the classical antibiotic resistance and is able to permit the specific re-isolation of the original bacteria introduced into the soil. Even in the case of horizontal transfer to other bacteria, the transposon would confer little or no selective advantage since selection depends on both iron deprivation and on the simultaneous presence of the pseudobactin-358 siderophores.
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Conclusions

This project showed the feasibility of constructing stable recombinant DNA transposon vectors carrying selective markers other than antibiotic resistance (which is undesirable for bacteria destined for large-scale environmental release). These transposon based vectors were preferable to the plasmid vectors in common use, since they show greatly reduced horizontal transfer and thus reduce the possibility of escape of the recombinant genes to other bacteria by horizontal transfer.

 

Major publications

Davison J., Brunel F., Kaniga K. and Chevalier N., “Recombinant DNA vectors for Pseudomonas”, in Pseudomonas: Biotransformations, Pathogenesis and Evolving Biotechnology, S. Silver, A. Chakrabarty and B. Iglewski (eds.).
American Society for Microbiology, 1990, pp. 242-251.

Davison J., Brunel F., and Phanopoulos A. and Kaniga K., “Engineering bacteria for environmental pollution control and agriculture”, in Biotechnology and Biodegradation, A. Chakrabarty and D. Kamely (eds.), Portfolio Publishing Company, 1990, pp. 83-104.

Kaniga K. and Davison J., “Transposon vectors for the expression and stable integration of cloned genes in rhizosphere bacteria”.
Gene, 100, 1991, pp. 201-205.

Leong J., Bitter W., Koster M., Venturi V. and Weisbeek P., “Molecular analysis of iron transport in plant growth promoting Pseudomonas putida WCS358”.
Biology of Metals, 4, 1991, pp. 36-40.

Raaijmakers J.M., Bitter W., Punte H.l., Bakker P.A., Weisbeek P.J. and Schippers B., “Siderophore receptor pupA as a marker to monitor wild type Pseudomonas putida WCS358 in natural environments”.
Applied and Environmental Microbiology, 60, 1994, pp. 1184-1190.
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imageResearch project
 
Contract number
BAP-0358/0406/0415

Period
January 1989 – December 1990

Coordinator
J. Davison
INRA
Versailles (FR)
 
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Partners


P. Weisbeek,
B. Schippers

Utrecht University (NL)

 
 
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