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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Experimental and modelling studies on the fate in soil of introduced biologically-contained bacteria

Background and objectives

Successful and effective biological control of plant pathogens often requires that genetically modified micro-organisms (GMMs) be introduced into the environment (i.e. soil). This practice carries with it several biosafety issues, and the implications of these for both the environment and the community need to be considered. The potential bacterial biocontrol agent Pseudomonas fluorescens, carrying genes producing anti-Tipula proteins, was studied as a GMM model organism in this project. To achieve optimal effectiveness and to minimise risks of the application, the fate of the GMM in the target ecosystem, i.e. the soil, needs to be understood and, if possible, managed. The research required in this area was performed using enclosed environments, such as soil microcosms, and preceded any large-scale field trials.

The objectives of this project were firstly to develop strategies for the optimal tracking of GMMs introduced into the soil. The second aim was to study the ecology of introduced genetically modified organisms (GMOs) in the soil. Thirdly, the project aimed to design strategies for active or passive containment. The final objective was to develop a predictive model for the fate of GMOs on the basis of realistic soil parameters.

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Approach and methodology

Pseudomonas fluorescens was used as a model organism for the insertion of insect biocontrol (anti-Tipula) genes. It then served as a gene carrier vehicle for the delivery of the beneficial genes into the rhizosphere (the environment surrounding the plant root) of gramineous plants. The organism was also modified with promoter probe insertions in order to pinpoint genes responsive to the rhizosphere and soil environments, with the aim to use these signals for biological containment. In addition, information was gathered with other non-differentiating bacteria in order to pinpoint genes or triggers in their starvation stress response programmes, which could potentially be useful for biological containment. Finally, a mathematical model predicting inoculant fate in soil, based upon realistic soil parameters, was developed.
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Main findings and outcome

Tracking methods, including selective plating, immunofluorescence and polymerase chain reaction (PCR)-assisted detection using specific primers and probes for the GMMs under study, were developed and successfully applied, to monitor the Pseudomonas fluorescens gene delivery vehicles, and the 'foreign' DNA, in soil microcosms. Persistence of the gene in the absence of detectable surviving cells was found, which indicated the importance of further monitoring of the inserted gene in addition to that of the cells. Promoter probe insertions identified gene expression which is regulated by environmental triggers, i.e. by the rhizosphere (from root exudation) versus the bulk soil (carbon starvation). The rhizosphere responsive operons were found to react to the presence of proline which was abundantly present in the exudates of three gramineous plants. Several starvation-induced genes were further identified and their use in passive containment systems was indicated, whereas active containment using two selected host killing genes was not successful. The starvation response in non-differentiating bacteria was unravelled using two-dimensional gel electrophoresis as well as stress survival testing.

In vitro and in situ studies revealed that Pseudomonas fluorescens carrier bacteria quickly developed generalised cellular stress resistance upon starvation. This occurred via the programmed induction of a myriad of stress responsive genes. Testing in soil showed that introduced Pseudomonas species variants quickly responded to soil oligotrophy (nutrient scarcity, mainly organic carbon) by producing stress resistant cells. A mathematical model was developed that adequately described the dynamics of introduced Pseudomonas fluorescens populations in the soil.
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Conclusions

This project uncovered important results concerning potential biosafety hazards to both the community and the environment relating to the safety of the use of GMOs in crops. The study showed that the Pseudomonas-based potential biocontrol agents persisted for a limited time in the soil and wheat rhizospheres, however associated heterologous DNA persisted for a more extended period of time.
Environmentally-responsive genes were detected in the Pseudomonas-based biocontrol organisms. These are useful for environmentally-regulated expression of beneficial genes as well as biological containment. The model biocontrol agents responded to carbon starvation in soil by forming cells with enhanced stress resistance. This study also showed the potential for developing a mathematical model which can adequately describe the fate of inoculant bacteria in soil.

 

Major publications

Givskov M., Eberl L., Møller S., Poulsen L.K. and Molin S., “Responses to nutrient starvation in Pseudomonas putida strain KT2442: Analysis of general cross-protection, cell morphology, and macromolecular content”.
J. Bacteriol., 176, 1994, pp. 7-14.

Molin S., Kjelleberg S., “Release of engineered micro-organisms: biological containment and improved predictability for risk assessment”.
AMBIO, 22, 1993, pp. 242-245.

Van der Hoeven N., Van Elsas J.D., Heijnen C.E., “A model based on soil structural aspects describing the fate of genetically modified bacteria in soil”.
Ecol. Modelling, 89, 1996, pp. 161-173.

Van Overbeek L.S., Eberl L., Givskov M., Molin S, Van Elsas J.D., “Surival of, and induced stress resistance in, carbon-starved Pseudomonas fluorescens cells residing in soil”.
Appl Environ Microbiol, 61, 1995, pp. 4202-4208.

Van Overbeek L.S., Van Veen J.A., Van Elsas J.D., “Induced reporter gene activity, enhanced stress resistance, and competitive ability of a genetically modified Pseudomonas fluorescens strain released into a field plot planted with wheat”.
Appl Environ Microbiol, 63, 1997, pp. 1965-1973.
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imageResearch project
 

Contract number
BIOT-CT91-0288

Period
October 1991 - April 1994

Coordinator
J.D. van Elsas
Plant Research International (formerly IPO-DLO)
Wageningen (NL)

 
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Partners


S. Molin
Technical University of Denmark
Lyngby (DK)

S. Kjelleberg
University of New South Wales
Sydney (AU)

N. van der Hoeven
TNO Delft (NL)

 
 
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