host-vector systems for the deliberate release of plant-beneficial Pseudomonas
Certain bacteria possess properties which make them beneficial to agriculture,
such as antagonism of phytopathogenic fungi and bacteria, antibiotic production,
plant growth hormone production, nitrogen fixation. Others, particularly
the Pseudomonads are particularly adept at the degradation of man-made
xenobiotic substances which pollute the environment, either accidentally
(e.g. polychlorinated aromatics or oil spills) or deliberately (pesticides
and herbicides). Genetic manipulation of such bacteria may render them
better able to perform these tasks or to catalyse new reactions not previously
possible. The purpose of this project was to stabilise the recombinant
genes present in such bacteria, to prevent horizontal transfer to other
bacteria, and to eliminate undesirable selective markers such as antibiotic
We aimed to construct safer recombinant DNA vectors for those bacteria
(e.g. Pseudomonas, Rhizobium) which might be considered desirable
to release into the environment for agricultural purposes, or for pollution
Approach and methodology
Recombinant DNA vectors are usually self-replicating plasmids. Unfortunately
these vectors can often be horizontally transferred to other soil bacteria
by conjugation, transformation or transduction. These properties make
them unsuitable for experiments involving the deliberate release of recombinant
bacteria into the environment. Furthermore, they are usually equipped
with antibiotic resistance genes, a feature which is also undesirable
for deliberate environmental release. For these reasons it was decided
to construct vectors which are based on transposons, which have reduced
horizontal transmission, being unable to self replicate. The horizontal
transmission could be reduced still further by removal of the transposase
gene. The antibiotic resistance genes were replaced by siderophore receptors,
which act as excellent selective markers, without being of medical importance.
Main findings and outcome
The project was successful and achieved the desired aim of constructing
improved vector systems for the deliberate release of recombinant micro-organisms
into the environment. Transposon vectors were modified to contain a maximum
number of useful restriction sites to facilitate cloning and gene manipulation.
These transposon vectors were carried on pBR322 based plasmids which had
also been modified to eliminate the corresponding useful restriction sites,
thus facilitating cloning into the transposon part of the DNA molecule.
This pBR322-based delivery vehicle was further equipped with the mobilization
genes of plasmid RP4 so that it could be transferred at high frequency
from E. coli to Pseudomonas, by conjugation in the presence
of a conjugative RP4 plasmid. Once inside the Pseudomonas cell,
the pBR322 plasmid replicon can no longer function and the plasmid acts
as a suicide vector and is rapidly lost from the cell. Thus, the transposon,
including the recombinant genes that it may carry, can only survive if
it transposes (jumps and integrates) to the host chromosome. In this location
it has greater stability and a much reduced rate of horizontal transfer
to other bacteria.
In a second stage, the undesirable antibiotic resistance genes of the
transposon vectors were replaced with the siderophore receptor pupA from
Pseudomonas putida WCS358. The presence of this receptor permits
the recipient bacteria to use the Pseudomonas WCS358 pseudobactin-358
siderophores as a source of iron. Thus, in the presence of both iron deprivation
and pseudobactin-358, bacteria carrying the transposon-borne siderophore
receptor are able to grow, while all others cannot. Thus, this siderophore
receptor marker provides a safer selection marker than the classical antibiotic
resistance and is able to permit the specific re-isolation of the original
bacteria introduced into the soil. Even in the case of horizontal transfer
to other bacteria, the transposon would confer little or no selective
advantage since selection depends on both iron deprivation and on the
simultaneous presence of the pseudobactin-358 siderophores.
This project showed the feasibility of constructing stable recombinant
DNA transposon vectors carrying selective markers other than antibiotic
resistance (which is undesirable for bacteria destined for large-scale
environmental release). These transposon based vectors were preferable
to the plasmid vectors in common use, since they show greatly reduced
horizontal transfer and thus reduce the possibility of escape of the recombinant
genes to other bacteria by horizontal transfer.
Davison J., Brunel F., Kaniga K. and Chevalier N., Recombinant
DNA vectors for Pseudomonas, in Pseudomonas: Biotransformations,
Pathogenesis and Evolving Biotechnology, S. Silver, A. Chakrabarty
and B. Iglewski (eds.).
American Society for Microbiology, 1990, pp. 242-251.
Davison J., Brunel F., and Phanopoulos A. and Kaniga K., Engineering
bacteria for environmental pollution control and agriculture,
in Biotechnology and Biodegradation, A. Chakrabarty and D.
Kamely (eds.), Portfolio Publishing Company, 1990, pp. 83-104.
Kaniga K. and Davison J., Transposon vectors for the expression
and stable integration of cloned genes in rhizosphere bacteria.
Gene, 100, 1991, pp. 201-205.
Leong J., Bitter W., Koster M., Venturi V. and Weisbeek P., Molecular
analysis of iron transport in plant growth promoting Pseudomonas
Biology of Metals, 4, 1991, pp. 36-40.
Raaijmakers J.M., Bitter W., Punte H.l., Bakker P.A., Weisbeek P.J.
and Schippers B., Siderophore receptor pupA as a marker to
monitor wild type Pseudomonas putida WCS358 in natural environments.
Applied and Environmental Microbiology, 60, 1994,
January 1989 December 1990
Utrecht University (NL)