tools for constructing genetically-modified micro-organisms (GEMs) with
high predictability in performance and behaviour in ecological microcosms,
soils, rhizospheres and river sediments
Many organic compounds including numerous pollutants are broken down by
microbes. However, polychlorinated biphenyls (PCBs) are recalcitrant to
microbial degradation. They persist in the environment, are toxic to micro-organisms,
may inhibit degradative pathways and can enter the food chain where they
present health hazards. Therefore, the removal of PCBs from the environment
is a high priority task. Since PCBs are found in large volumes in soils
and sediments, in situ degradation may be an effective approach.
The aim was to transfer the PCB degradation pathway from PCB-degrading
bacteria, which survive poorly in soil, into bacteria found associated
with plant roots and which can survive in soils. The result would be to
create recombinants with an improved ability to eliminate organic solvents.
Pseudomonas sp. LB400, the best known PCB degrader, was used as
a model system.
Approach and methodology
The catabolic bph genes were transferred into the chromosome of
indigenous bacteria, particularly those of the sugar beet rhizosphere.
The resultant recombinants were screened for their ability to degrade
of GEMs released into the environment has not been extensively documented,
therefore it is useful to restrict their survival to increase their predictability
in the environment. The Pseudomonas model was used to construct
contained GEMs. The gef killing gene was coupled to the PCB regulatory
system. The system was based on two elements. The control element consisted
of a fusion between the TOL meta-cleavage degradation pathway promoter
(Pm) and the LacI repressor protein, plus the xylS gene encoding
the XylS protein that respond to chlorobenzoate effectors. The killing
cassette element consisted of a fusion between the Plac promoter and gef.
Thus in the presence of PCBs the bacteria produce the LacI protein, which
prevents the expression of the killing gene. In the absence of PCBs the
expression of the killing cassette is no longer repressed and the bacteria
To limit the rate of lateral transfer of recombinant DNA (rDNA) a gene
containment system was developed. This consisted of a killing element
(the colE3 gene which encodes an RNase that cleaves all prokaryotic
16S rRNA) and a control element (the immE3 gene which encodes a
specific repressor of the lethal function). In the GEM the killing gene
is closely linked to the rDNA determining the new trait, whereas the control
element is not. Thus transfer of the rDNA would be accompanied by transfer
of the lethal gene but not the control gene and so if gene transfer occurred
the recipient organism would be killed.
Main findings and outcome
One major drawback of PCB-degraders is the responsiveness of the regulatory
circuit to the substrate. Therefore we examined the regulation of the
system in vivo, and particularly its potential to express the bph
gene under different conditions and its inducibility by PCBs. Bph
genes were constitutively expressed at high levels even in the absence
of PCBs. Exposure of the cells to PCBs resulted in a further increase
of bph expression. Thus bph genes can be expressed in diverse
The bph operon was transferred to Pseudomonas F113 and seventeen
Gram-negative bacteria isolated from river sediment. The functionality
of the operon in the modified organisms was confirmed by their ability
to grow on PCBs. The novel genetic trait was stably maintained for over
200 generations in the recombinant bacteria. The bph operon was
not transferred to related bacteria. The survival, colonisation and competitive
abilities of the recombinants were unaffected.
The TOL catalytic pathway was exploited to control gene expression through
artificial cascades to develop biologically contained strains.
A suicide containment system was incorporated randomly into the P.
putida chromosome. In both liquid cultures and sterile and non-sterile
soil microcosms the recombinant bearing the containment system behaved
as predicted. Mutants resistant to cell killing arose at a frequency of
around 10-5 to 10-6 per cell per generation. In
bacteria containing two copies of the killing cassette the frequency of
such mutants decreased to around 10-8 per cell per generation.
Mutations were therefore linked to the killing element and not to the
The introduction into the soil of the contained bacteria did not have
significant effects on natural PCB-degraders already present.
A system was designed using colicin E3 and immunity E3 as containment
genes and to decrease undesirable horizontal gene transfer. Colicin E3
kills all prokaryotes by inhibiting protein synthesis. This makes it a
powerful tool for decreasing dispersal of recombinant genes among indigenous
micro-organisms in ecosystems into which a GEM is deliberately or accidentally
We dissected the regulatory circuits involved in the control of a bacterial
pathway for degrading chemical pollutants. The genes for this pathway
were introduced into the host chromosome of indigenous bacteria isolated
from various ecological niches in which in situ bioremediation
treatment may be required. Ecologically, the recombinant bacteria behave
like the wild types. Thus, the first GEMs specifically designed to degrade
pollutants, equipped with either circuits for biological containment or
barriers to limit lateral transfer of rDNA, were developed. We showed
that the survival and behaviour of GEMs, and rDNA transfer, can be rendered
González-Pérez M.M., Ramos J.L., Gallegos M.T. and
Marqués S., Critical nucleotides in the upstream region
of the XylS-dependent TOL meta-cleavage pathway operon promoter
as deduced from analysis of mutants.
J. Biol. Chem., 274, 1999, pp. 2286-2290.
Espinosa-Urgel M., Salido A. and Ramos J.L., Genetic analysis
of functions involved in adhesion of Pseudomonas putida to
J. Bacteriol., 182,
2000, pp. 2363-2369.
Torres B., Jaenecke S., Timmis K.N., García J.L. and Díaz
E., A gene containment strategy based on a restriction-modification
Environ. Microbiol., 2,
2000 (in press).
Díaz E. and Prieto M.A., Bacterial promoters triggering
biodegradation of aromatic pollutants.
Curr. Opin. Biotechnol., 2000 (in press).
Ronchel M.C., Ramos C., Jensen L.B., Molin S., Ramos J.L., Construction
and behavior of biologically contained bacteria for environmental
applications in bioremediation.
Appl. Environ. Microbiol, 61,
1995, pp. 2990-2994.
Ronchel M.C., Molina L., Witte A., Lutbiz W., Molin S., Ramos J.L.,
Ramos C., Characterization of cell lysis in Pseudomonas
putida induced upon expression of heterologous killing genes.
Appl. Environ. Microbiol., 64,
1998, pp. 4904-4911.
Ronchel M.C., Ramos-Díaz M.A., Ramos J.L., Retrotransfer
of DNA in the rhizosphere.
Env. Microbiol., 2,
2000, pp. 319-323.
Molina L., Ramos C., Ronchel M.C, Molin S., Ramos J.L., Construction
of an efficient biologically contained Pseudomonas putida
strain and its survival in outdoor assays.
Appl. Environ. Microbiol., 64,
1998, pp. 2072-2078.
Molina L., Ramos C., Duque E., Ronchel M.C, García J.M.,
Wyke L., Ramos J.L., Survival of Pseudomonas putida
KT2440 in soil and in the rhizosphere of plants under greenhouse
and environmental conditions.
Soil Biol. Biochem., 32,
2000, pp. 315-321.
October 1991 September 1993
Estación Experimental del Zaidín
of the project
This project was continued in EC project: BIO2-CT92-0084.
Technical University of Denmark
K.N. Timmis, D. Dwyer
National Research Centre for Biotechnology (GBF)
V. de Lorenzo
Centro Nacional de Biotecnología
Institute of Technology