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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Quantifying the impact of GMOs in the environment: development of non-disruptive biomarkers to monitor stability, expression and mobility of recombinant genes in polluted ecosystems

Background and objectives (1)

Surface reporters are outer membrane proteins bearing distinct antigens that become exposed on the bacterial surface and, therefore, can be detected non-disruptively with cognate monoclonal antibodies. Three reporters of this type have been developed. Each of the corresponding reporter genes have been placed in front of promoters of biodegradative pathways and their performance has been validated in activated sludge and rhizosphere microcosms. These antigenic markers have been used in combination with a suite of optical reporters. Two types of genes endowing optical properties to bacteria that harbour them have been thoroughly exploited as reporter systems. First, the luxAB genes of Vibrio harveyi that, when engineered into the chromosome of a target cell, makes detection of individual genetically modified cells possible using highly sensitive cameras connected to the microscope. The second development is the utilisation of the green fluorescent protein (GFP) from jellyfish as a general reporter system in bacteria. These genes have been used along with classical reporters (e.g. lacZ) in combination with fluorescent substrates.

This project aimed to develop genetic tools to follow the activity of bacteria destined for environmental bioremediation. The main focus was the design and validation of these and other novel markers that will allow quantitative monitoring of biodegradative activities and the presence of genetically modified organisms (GMOs) at the level of single cells.

(1) This project was a direct follow-on from EC project
BIOT-CT91-0293


image image Pollution.


Approach and methodology

Genetic markers, either optical or antigenic, were expressed through inducible promoters of catabolic pathways in Pseudomonas and their performance was examined in representative microcosms. The same markers were used, simultaneously, for the non-disruptive detection of gene transfer to or from GMOs engineered with various levels of biological containment.

A complete set of vectors to design circuits of conditional gene expression activated by DNA transfer were developed and their performance confirmed. Depending on the construction, these allow expression of a reporter gene (lacZ or GFP) as soon as the DNA sequence involved escapes the original host or enters a specific recipient.
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Main findings and outcome

The performance of the system has been examined in various configurations allowing non-disruptive detection of transfer frequencies as low as 10-5-10-6. Also, some regions of the TOL (toluene biodegradation) plasmid of P. putida involved in retrotransfer of chromosomal genes have been pinpointed through transposon mutagenesis, providing the basis to understand this ecologically important phenomenon of gene capture.

The results of this project address two major issues at the root of novel biodegradation and bioremediation approaches using GMOs as biocatalysts, namely, the development of tools to study community structure and the regulation of catabolic pathways. As a starting point, a reliable flow-chamber biofilm system was designed for establishing microbial communities under controllable conditions. The chambers may be mounted directly on a fluorescence microscope for on-line inspections. In situ rRNA hybridisation is then used for analysis of the species composition of the flow-chamber communities, as well as for detecting the presence and activity of genes involved, as a model, in biodegradation of toluene.

A simple but effective method has been developed to maintain the biofilm structure of these bacterial communities involving matrixing the consortia in polyacrylamide. This allows studies on community structure and three-dimensional organisation through confocal scanning laser microscopy that yields images of excellent quality and high resolution.

The mechanism of activation of the toluene-responsive transcriptional regulator XylR of the TOL pathway was determined. Genetic and biochemical data showed that effector binding to the N-terminal domain of the protein results in the release of the intramolecular repression exerted by this portion of the activator on the rest of the protein. These results lay the foundation for the rational design of transcriptional regulators responsive to predetermined signals. The regulation of the BPH system of Pseudomonas sp. LB400 for biodegradation of chlorinated biphenyls (PCBs) has been examined in the native host as well as in heterologous hosts able to colonize the rhizosphere of sugar beet plants. Although expression of the bph genes is not responsive to pathway substrates, the genes are expressed at sufficient levels to allow detection of biodegradative activities associated with the roots.
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Conclusions

Surface reporters have proved to be effective as monitors of specific promoter activity in untreated environmental samples. The use of jellyfish green fluorescent protein (GFP) as a reporter has been extended and generalised to microbial ecology studies. We have also developed genetic tools for non-disruptively detecting general or specific gene transfer and have described physical techniques for the assembly and structure analysis of bacterial communities. Various genes involved in responses to starvation and oxidative stress of non-enteric bacteria were found and employed as markers of the cell’s physiological status. On this basis, we conclude that there are no major technical limitations to the monitoring of GMOs released into the environment.

 

Major publications

Cebolla A., Guzmán C. and de Lorenzo V., “Non-disruptive detection of activity of catabolic promoters of Pseudomonas with an antigenic surface reporter system”.
Applied and Environmental Microbiology,
62, 1996, pp. 214-220.

Pérez-Martín J. and de Lorenzo V., “ATP binding to the s54-dependent activator XylR triggers a protein multimerization cycle catalysed by UAS DNA”.
Cell,
86, 1996, pp. 331-339.

Brazil D., Kenefick L., Callanan M., Haro A., de Lorenzo V., Dowling D.N. and O'Gara F., “Construction of a rhizosphere pseudomonad with potential to degrade polychorinated biphenyls and detection of bph gene expression in the rhizosphere”.
Applied and Environmental Microbiology,
61, 1995, pp. 1946-1952.

Munthali M., Timmis K.N. and Díaz E., “Restricting the dispersal of recombinant DNA: Design of a contained biological catalyst”.
Bio/Technology,
14, 1996, pp. 189-191.

Ramos J.L., Díaz E., Dowling D., de Lorenzo V., Molin S., O´Gara F., Ramos C. and Timmis K.N., “Behavior of bacteria designed for biodegradation”.
Bio/Technology, 12, 1994, pp. 1349-1356.
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imageResearch project
 

Contract number
BIO2-CT92-0084

Period
January 1993 – December 1995

Coordinator
V. de Lorenzo
CSIC
Centro Nacional de Biotecnología
Madrid (ES)

Follow-up of the project
Genetic tools for the construction of bacterial consortia in bio/mineral catalysts with activity on environmental pollutants (BIO4-CT97-2183).
Rational design of formatted catabolic segments for engineering superior bacterial biocatalysts for degradation of chloro and nitroaromatics (BIO4-CT97-2040).
Since these two projects focus on environmental remediation rather than GMO biosafety, they are not included in this review.

 
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Partners


K.N. Timmis
National Research Centre for Biotechnology (GBF)
Braunschweig (DE)

J.L. Ramos
CSIC
Estación Experimental del Zaidín
Granada (ES)

S. Molin
Technical University of Denmark
Lyngby (DK)

F. O'Gara
University College Cork (IE)

D. Dowling
Institute of Technology
Carlow (IE)

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

 
 
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