the impact of GMOs in the environment: development of non-disruptive biomarkers
to monitor stability, expression and mobility of recombinant genes in polluted
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.
project was a direct follow-on from EC project
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.
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.
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.
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 cells physiological status. On this basis, we conclude
that there are no major technical limitations to the monitoring of GMOs
released into the environment.
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.
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.
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.
January 1993 December 1995
V. de Lorenzo
Centro Nacional de Biotecnología
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
Since these two projects focus on environmental remediation rather
than GMO biosafety, they are not included in this review.
National Research Centre for Biotechnology (GBF)
Estación Experimental del Zaidín
Technical University of Denmark
University College Cork (IE)
Institute of Technology
University of New South Wales