and effective pesticidal micro-organisms through customised environmental
Chemical pesticides are generally cost-effective in controlling pests
and diseases, and have in consequence become an integral part of modern
agriculture. However, these same chemical inputs are also implicated in
ecological, environmental and human health problems, so the need to find
effective alternative approaches with minimal deleterious effects is obvious.
Natural and genetically modified organisms (GMOs) provide an alternative
to chemical controls, however this alternative also has biological risks
associated. In particular, the implications of the biosafety risks of
using GMOs need to be carefully considered before they are implemented.
Ideally, a bacterium would persist for long enough in the environment
to achieve control of the pest (i.e. so it is effective), but no longer
(i.e. without ecological risk).
The specific aim of this project was two-fold. Firstly, to improve the
use of self-destruct systems to limit bacterial persistence,
and secondly, to construct a bacterial strain with enhanced desiccation
tolerance. The self-destruct system and the enhanced desiccation resistance
will be combined in a single strain, and its persistence will be tested
in greenhouse trials.
Approach and methodology
Two control systems were developed for killing genes in the
plant rhizosphere (the environment surrounding the plant root). This involved
construction of a prolonged persistence bacterial strain with a functional
self-destruct system. One system uses a cold-shock inducible
promoter isolated from Pseudomonas putida, which is being tested
for functionality in available suicide cassettes. The second system is
based on a range of lux genes inserted into the P. putida
chromosome, which can be screened on the basis of light emission from
the bacteria in response to corn root exudates. In addition, new broad
host range toxins for self-destructing bacteria were developed to circumvent
the possibility that low level read-through of the killing genes would
lead to the selection of mutants with non-functional lethal toxins. Two
strategies were employed. Firstly, mutations that rendered the host cells
resistant to the lethal action of the toxins were constructed and analysed.
An in vitro system was established for analysing the toxin activity.
Colonisation assays are being conducted in various crops including corn,
spinach and beans in pot assays in greenhouses to monitor the population
dynamics in the rhizosphere over a period of up to 12 weeks. P. putida
clones overproducing the osmoprotectant sugar trehalose, are being characterised
for desiccation, (using trehalose-accumulation, osmotic tolerance and
other growth characteristics of the genetically engineered bacteria).
Colonisation by the wild-type and the trehalose-overproducing clones will
be tested. The compatibility of the control systems for both desiccation
tolerance and self-destruct genes, as well as desiccation tolerance testing
in micro-organisms containing both genetic modifications, is under development.
Optimisation of drying protocols for Pseudomonas with inducible
trehalose synthase systems, formulation of desiccation-tolerant bacteria
for seed coating, and testing of the resistance of genetically engineered
bacteria to repeated cycles of desiccation and rehydration, are all being
Main findings and outcome
P. putida KT2442 is a root-colonising strain which can use proline,
one of the major components in root exudates, as its sole carbon and nitrogen
source. A P. putida mutant, unable to grow with proline as the
sole carbon and nitrogen source, was isolated after random mini-Tn5-Km
mutagenesis. The mini-Tn5 insertion was located at the putA
gene, which is adjacent to and divergent from the putP gene. The
putA gene codes for a protein of 1315 amino acid residues which
is homologous to the PutA protein of Escherichia coli, Salmonella
enterica serovar Typhimurium, Rhodobacter capsulatus, and several
Rhizobium strains. Regions of the P. putida PutA
protein showed homology to the proline dehydrogenase of Saccharomyces
cerevisiae and Drosophila melanogaster, as well as to the pyrroline-5-carboxylate
dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases.
This suggests that in P. putida, both enzymatic steps for proline
conversion to glutamic acid are catalysed by a single polypeptide. The
putP gene was homologous to the putP genes of several prokaryotic
micro-organisms, and its gene product is an integral inner-membrane protein
involved in proline uptake. The expression of both genes was induced by
the addition of proline to the culture medium and was regulated by PutA.
In a P. putida putA-deficient background, expression of both putA
and putP genes was maximal and proline independent.
P. putida KT2442 responds to root exudates, allowing gene expression
to be turned on or off depending on the niche being colonised. This strain
synthesises trehalose and an increase in the production of this carbohydrate
can increase persistence under desiccation conditions. Regulatory circuits
responding to plant exudates, and killing genes are being combined to
control, at will, the survival of the strain under environmental conditions.
Vílchez S., Molina L., Ramos C., Ramos J.L., Proline
catabolism by Pseudomonas putida: cloning, characterization,
and expression of the put genes in the presence of root exudates.
J. Bacteriol., 182, 2000, pp. 91-99.
Vilchez S., Manzanera M. and Ramos J.L., Control
of expression of divergent Pseudomonas putida put promoters
for proline catabolism.
Appl. Env. Microbiol., 66, 2000, pp. 5221-5225.
M.C. and Ramos J.L., Dual
system to reinforce biological containment of recombinant bacteria
designed for rhizoremediation.
Appl. Env. Microbiol., 67, 2001, pp. 2649-2656.
December 1998 - December 2000
Estación Experimental del Zaidín
University of Cambridge (UK)
Norwegian University of Science and Technology
Odense University (DK)