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Appl Environ Microbiol, July 1998, p. 2634-2638, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Insertion Site and Metabolic Load on the Environmental
Fitness of a Genetically Modified Pseudomonas fluorescens
Isolate
Frans A. A. M.
De
Leij,1
Catherine E.
Thomas,1
Mark J.
Bailey,2
John M.
Whipps,3 and
James M.
Lynch1,*
School of Biological Sciences, University of
Surrey, Guildford, Surrey GU2 5XH,1
Natural Environment Research Council, Institute of Virology
and Environmental Microbiology, Oxford OX 3SR,2
and
Horticulture Research International, Wellesbourne,
Warwick CV35 9EF,3 United Kingdom
Received 20 May 1997/Accepted 5 May 1998
 |
ABSTRACT |
An isolate of Pseudomonas fluorescens (SBW25) was
modified with different marker genes (lacZY,
aph-1, and xylE). These marker genes were
inserted singly or in combination into two separate (1 Mbp apart)
and presumably nonessential sites (-6- and Ee) on the
chromosome of SBW25. This allowed the production of a range of
genetically modified SBW25 variants that differed with respect to
insertion site of the marker genes and metabolic burden. The environmental fitness of the different SBW25 variants was tested in
soil, in the rhizosphere of wheat and pea, and on the
phylloplane of wheat. Reduced environmental fitness of the different
variants was mainly attributed to the extra metabolic burden of novel
gene expression, whereas choice of insertion site was
of little significance. Changes in environmental fitness were dependent
on the environmental conditions; an environment, such as soil, with a
low microbial carrying capacity had a negative effect on the
environmental fitness of variants with a large metabolic load. In
environments with a larger carrying capacity, such as the rhizosphere
of pea, environmental fitness of variants with a large metabolic load
was not significantly different from that of variants with a
smaller metabolic burden.
| |
INTRODUCTION |
There is considerable interest in
the commercial development of microorganisms for use in bioremediation,
as biological control agents, as biofertilizers, or as a means to
protect plants against frost damage (13). The ability to
create a single organism with a variety of properties from different
organisms by using recombinant technology has allowed the possibility
of improving existing organisms for the benefit of human society and
the environment. However, before such genetically improved organisms
can be released into the environment, the wider environmental
consequences of such releases need to be determined (21,
22). This is especially important if there is reason to believe
that a genetically improved organism is environmentally more fit than
the wild-type organism from which it was derived. Environmental fitness
describes the interaction of an organism with its environment. The
environmental fitness of an organism will differ according to the
environment into which it is released. Because naturally occurring
permutations in the environment are almost limitless, it is impossible
to test environmental fitness empirically in contained environments. As a consequence, the lack of empirical data on the environmental fitness
of genetically modified microorganisms (GMMs) has led to legislative
measures that are designed to safeguard against the risks associated
with their release (22). Such risks include the unforeseen
environmental functions of heterologous genes combined with changes in
the persistence or impact of inocula.
To overcome the problem of unpredictable behavior of GMMs in the
environment, a fundamental understanding of the effects of gene
insertions on the GMMs' behavior is required. Experiments using
mixtures of GMMs and wild-type organisms often show that GMMs are less
fit than the wild-type organisms from which they are derived (6,
7, 19, 24). In some cases, however, no effects (2, 15,
17) and even enhanced survival of a GMM have been reported
(12). Several reasons have been suggested for decreased
environmental fitness of GMMs. Bromfield and Jones (7)
suggested that the decreased environmental fitness of triply marked
Rhizobium strains was due to a decrease of heterogeneity of
the recombinant strain, which would lead to a loss of genetic variability necessary to infect a heterogeneous population of clover.
Van Elsas et al. (24) suggested that either expression of
the Bacillus thuringiensis 
-endotoxin gene
(tox) in Pseudomonas fluorescens was a metabolic
burden for the organism or insertion of tox into the
chromosome led to disruption of essential gene functions, but no
evidence for either explanation was given. It generally is assumed that
the extra metabolic burden of expressing novel gene sequences is of
importance only if expression of the novel gene(s) is maintained at a
high level and not directly regulated or if the genes are on plasmids
with a high copy number (20, 25). It is thought that under
nutrient-deficient conditions, the extra energy demands due to the
presence and expression of novel genes in GMMs might lead to large
environmental fitness differences. In vitro, however,
nutrient stress does not seem to cause differences in
environmental fitness between parent and modified strains
(8). It also is assumed that any disruption of existing gene
functions that are necessary for bacterial growth and survival, due to
the introduction of the novel genetic elements into the genome of
an organism, can be minimized by appropriate selection
procedures during recombinant construction (4).
The aims of the experiments presented in this paper were to clarify
factors related to GMM fitness per se and to provide an insight into
mechanisms that influence the environmental fitness of GMMs in general.
For these purposes we used a range of genetically modified P. fluorescens variants that were derived from the same environmental
isolate (SBW25). Variants of SBW25 were altered with respect to genetic
and metabolic loads by changing the number of gene sequences used and
with respect to the positions of the gene sequences on the chromosome
(i.e., the same gene sequence was inserted at different positions). By
applying mixtures of these variants to contrasting environments which
differed in the ability to support the growth of P. fluorescens SBW25, the effects of the environments on the fitness
of different genetic SBW25 variants could be studied.
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MATERIALS AND METHODS |
Construction of genetically marked organisms.
SBW25, a
nonpathogenic, plasmid-free P. fluorescens strain, was
isolated from the phylloplane of sugar beet (Beta vulgaris cv. Amethyst). Two nonessential sites, -6- and Ee, approximately 1 Mbp
apart on the physical map of the 6.6-Mbp chromosome of SBW25 (18), were chosen for insertion of the different marker
genes by site-directed homologous recombination (2). Four
genetic variants derived from SBW25 were constructed for this study:
SBW25-6K was made resistant to 0.1% (wt/vol) kanamycin by insertion of the aph-1 gene into the -6- site of SBW25; SBW25EeK was made
resistant to 0.1% (wt/vol) kanamycin by insertion of the
aph-1 gene into the Ee site of SBW25; SBW25-6KX contained at
the -6- site of SBW25 the aph-1 gene for kanamycin
resistance coupled to the xylE gene, which encodes catechol
2,3-dioxygenase; and SBW25EeZY-6KX contained in addition the
lacZY genes (expressing lactose permease and
-galactosidase) at the Ee site of SBW25. The Tn903
kanamycin resistance gene, aph-1, was isolated on a 1.4-kbp
BamHI fragment from pUC4K (Pharmacia, St. Albans, United
Kingdom). The xylE gene originated from the TOL(pWWO)
plasmid (26) and was modified with the chloramphenicol acetyltransferase promoter (H. Joos, Plant Genetics Systems, Ghent, Belgium). The lacZY genes under the control of the
iucA promoter (5) were isolated from pMON117
(3). All genes were inserted by homologous recombination
into the two different sites and were expressed constitutively at high
levels (2). Cocultivation in nonselective L broth at 28°C
and 1,000-fold dilution daily for 20 days (approximately 200 generations) revealed no significant difference in competitive ability
between modified organisms and nonmodified wild-type organisms
(2).
Phenotypic characteristics conferred by the inserted genes.
The lacZY system (1, 10, 11) is one of the most
widely used metabolic markers (16). Bacteria of the genus
Pseudomonas cannot utilize lactose as a carbon source
(11), but the insertion of the Escherichia coli
genes lacZ (-galactosidase) and lacY (lactose
permease) enables modified organisms to do so. When bacterial colonies
expressing the xylE gene (catechol 2,3-dioxygenase) are sprayed with a 1% (wt/vol) catechol solution in water, they produce a
bright yellow product due to the formation of 2-hydroxymuconic semialdehyde. The kanamycin resistance gene, aph-1, provides
direct selection of recombinant cells in the presence of 0.1%
(wt/vol) kanamycin. When plated onto
Pseudomonas-selective medium (14) amended with 0.05% (wt/vol) X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) and
0.1% (wt/vol) kanamycin, the phenotypic characteristics conferred by
the different marker genes allowed differentiation of the SBW25 variants. SBW25-6K (aph-1 at the -6- site)
and SBW25EeK (aph-1 at the Ee site) formed white colonies,
SBW25-6KX (aph-1 and xylE at the -6- site) formed
white colonies that turned yellow after being sprayed with 1% (wt/vol)
catechol, and SBW25EeZY-6KX (lacZY at the Ee site and
aph-1 and xylE at the -6- site) formed blue colonies.
Culturing and inoculation.
SBW25 variants were stored at
70°C on beads treated with cryopreservatives (Technical Service
Consultants, Lancs, United Kingdom). Variants were cultured either on
tryptic soy agar (Oxoid) for 60 h at 25°C or in tryptic soy
broth with shaking (200 rpm) until late log phase. After separation of
the bacterial cells from the culture media by repeated washing in
0.25-strength Ringer's solution, cells were suspended in 0.25-strength
Ringer's solution to yield a concentration of approximately
1010 CFU/ml. Strain combinations were obtained by mixing
appropriate cell suspensions together. Wheat seeds (Triticum
aestivum cv. Axona) or pea seeds (Pisum sativum cv.
Montana) were soaked overnight in suspensions containing single SBW25
variants or variant mixtures. Before seeds were planted the number of
CFU of each strain was estimated by plating a 10-fold dilution range of
individually macerated seeds onto agar medium selective for
Pseudomonas (14) amended with 0.1% (wt/vol)
kanamycin and 0.05% (wt/vol) X-Gal (n = 4). Untreated
control seeds were soaked in sterile 0.25-strength Ringer's solution.
Bacteria were inoculated onto the leaves of spring wheat at tillering,
growth stage 22 (23), with a spray application of bacterial
cells (approximately 109 CFU/ml) suspended in 0.25-strength
Ringer's solution and 0.01% (vol/vol) Tween 80.
Environmental fitness of triply marked P. fluorescens
(SBW25EeZY-6KX) compared with the wild-type organism (SBW25).
Seventy-two pots (diameter, 23 cm) were filled with a mixture of 80%
silty loam (Hamble series) and 20% grit. Each pot was planted with 10 spring wheat seeds (T. aestivum cv. Axona). Plants were
grown in a greenhouse at temperatures between 20 and 30°C and a
photoperiod of 18 h. Pots were watered daily. At the seedling stage, growth stage 12 (23), plants in 36 pots were sprayed with a suspension of bacteria containing a 50-50 mixture of triply marked recombinant and wild-type SBW25 in 0.25-strength Ringer's solution and 0.01% (vol/vol) Tween 80 (treated). The remaining 36 pots
were sprayed with 0.25-strength Ringer's solution and 0.01% (vol/vol)
Tween 80 (untreated control). Subsequently, plants from 12 treated and
12 untreated pots were harvested immediately and after 7 and 43 days.
Recombinant and wild-type bacteria were quantified by plating a 10-fold
dilution range prepared from a weighted, macerated sample of leaves
onto a medium selective for Pseudomonas (14)
amended with 0.05% (wt/vol) X-Gal. After 7 days of incubation at
25°C, the number of CFU of each could be quantified and expressed as
a proportion of the total count. Control plants sprayed with
0.25-strength Ringer's solution and 0.01% (vol/vol) Tween 80 were
used to determine background populations of indigenous
Pseudomonas spp.
Effect of site of chromosomal insertion of marker genes on the
environmental fitness of recombinant organisms.
To investigate if
insertion of the novel genes had caused a disruption of essential gene
functions, genetic variants containing aph-1 at either the
-6- (SBW25-6K) or the Ee (SBW25EeK) site were compared in the
rhizosphere and phylloplane of wheat during competition experiments
with the triply marked variant, SBW25EeZY-6KX. Pairwise competition
against SBW25EeZY-6KX was necessary because the two kanamycin-resistant
variants (SBW25EeK and SBW25-6K) expressed the same phenotype.
Wheat seeds (T. aestivum cv. Axona) were soaked in a
suspension containing either a 50-50 mixture of SBW25EeZY-6KX and
SBW25EeK or a 50-50 mixture of SBW25EeZY-6KX and SBW25-6K. A 10-fold
dilution series of five replicate seeds from each treatment was plated onto Pseudomonas-selective medium (14) amended
with 0.05% (wt/vol) X-Gal and 0.1% (wt/vol) kanamycin. The proportion
and inoculum density per macerated seed of each variant were estimated.
Into each of 50 10-cm-diameter pots filled with a mixture of 90% silty loam (Hamble series) and 10% grit, three soaked spring wheat seeds (cv. Axona) were planted. Pots were placed in a greenhouse at air
temperatures between 20 and 32°C. The photoperiod was 18 h, and
plants were watered on a daily basis. Five pots from each treatment
were harvested weekly over a period of 35 days, and the population
densities of the different genetic SBW25 variants were estimated by
plating a 10-fold dilution series of macerated roots onto
Pseudomonas-selective medium (14) amended with
0.05% (wt/vol) X-Gal and 0.1% (wt/vol) kanamycin.
Effect of metabolic load on the environmental fitness of
recombinant organisms.
Variants SBW25-6K and SBW25-6KX were mixed
together with the triply marked recombinant SBW25EeZY-6KX in
0.25-strength Ringer's solution
(SBW25-6K/SBW25-6KX/SBW25EeZY-6KX ratio of 3:4.5:2.5) to
create a bacterial suspension containing approximately 1010
CFU/ml. Wheat (T. aestivum cv. Axona) and pea (P. sativum cv. Montana) seeds were allowed to soak overnight in this
bacterial suspension (treated) or in sterile 0.25-strength Ringer's
solution (control). Soaked individual seeds were planted in small pots (3 cm by 3 cm; 10 cm deep) filled with sieved sandy loam soil (Holiday
Hills series). Pots were placed in a growth chamber set at a day
temperature of 21°C, a night temperature of 15°C, and a photoperiod
of 18 h. Plants were watered daily. Four pots from each treatment
were harvested, and a dilution series of a macerated sample of seeds
(at 0 days) or roots (at 14 and 28 days) was plated onto
Pseudomonas-selective medium (14) amended with
0.1% (wt/vol) kanamycin and 0.05% (wt/vol) X-Gal. After 7 days of
incubation at 25°C, colonies were sprayed with 1% (wt/vol)
catechol in water and the SBW25 variants were quantified according to
their distinct phenotypes. Noninoculated control treatments were used
to determine the presence of naturally occurring pseudomonads with
similar phenotypes.
Comparison of the environmental fitness of recombinant organisms
in soil and the rhizosphere.
Pea seeds (P. sativum cv. Montana) and wheat seeds (T. aestivum cv. Axona) were soaked in suspensions with
SBW25-6K, SBW25-6KX, and SBW25EeZY-6KX (ratio of 3:4.5:2.5,
respectively). Growth conditions and experimental systems were the same
as described above. In addition, fallow soil was inoculated with the
genetic variants by pipetting 0.1 ml of bacterial suspension onto the
soil surface followed by watering. Immediately (at 0 days) and after 14 and 28 days, four pots from each treatment were harvested and a 10-fold dilution series of a macerated sample of seeds (at 0 days) or roots was
plated onto Pseudomonas-selective medium (14)
amended with 0.1% (wt/vol) kanamycin and 0.05% (wt/vol) X-Gal. The
different SBW25 variants in samples of seeds and roots were quantified
as described above. In addition, a 10-fold dilution series was prepared from each well-mixed sample of fallow soil. After 7 days of incubation at 25°C, colonies were sprayed with 1% (wt/vol) catechol in water and the SBW25 variants were quantified according to their distinct phenotypes. Noninoculated control treatments were used to determine the
presence of naturally occurring pseudomonads with similar phenotypes.
The experiments that were carried out to investigate differences in
environmental fitness among SBW25 variants are summarized in Table
1.
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TABLE 1.
Experiments carried out to investigate differences in
environmental fitness between different SBW25 variants in soil and
in the rhizospheres of pea and wheat
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Statistical analyses.
All treatments within each experiment
were completely randomized. After normalization of data sets by log or
logit transformation [formula for logit transformation, ln
x/(1x), with x being the proportion
of a given strain recovered), data were analyzed by analyses of
variance. Where more than two means were compared, significant
differences between treatments were analyzed by using least-significant-difference (LSD) values.
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RESULTS |
Environmental fitness of triply marked P. fluorescens (SBW25EeZY-6KX)
compared with the wild-type organism (SBW25).
Forty-three
days after spray application with a 50-50 mixture of the wild type
(SBW25) and the triply marked recombinant (SBW25EeZY-6KX), the wild
type became proportionally dominant (P < 0.001),
accounting for more than 70% of the total introduced
Pseudomonas population. Therefore, insertion of the
gene cassettes lacZY and aph-1-xylE into
the genome of P. fluorescens SBW25 resulted in
reduced competitive ability of the recombinant strain compared with
that of the nonmodified wild-type organism in the phylloplane of wheat.
Effect of site of chromosomal insertion of marker genes on
the environmental fitness of recombinant organisms.
In
competition with the triply marked strain, SBW25EeZY-6KX, no
significant differences in environmental fitness between
SBW25-6K and SBW25EeK were found in the rhizosphere of wheat,
indicating that insertion site choice had little effect on the
environmental fitness of the recombinant organisms (Table
2). Both SBW25-6K and SBW25EeK
outcompeted the triply marked strain during the 35-day test
period (P < 0.01). Differences in competitive ability
were most pronounced after 21 to 28 days (Table 2).
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TABLE 2.
Effect of insertion site on environmental fitness of
P. fluorescens SBW25 variants in the rhizosphere of
spring wheata
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Effect of metabolic load on the environmental fitness of
recombinant organisms.
Because no significant interactions between
the proportional survival of the SBW25 variants in the rhizosphere of
wheat and pea were observed, data obtained for the pea and wheat were
subsequently combined and analyzed. Even though proportionally more
(P < 0.01) SBW25-6KX variants were added to the
initial mixture, constitutive expression of the xylE gene
resulted in populations of this variant proportionally smaller
(P < 0.01) than those of the SBW25-6K variant after 14 and 28 days (Table 3). Similarly, the
triply marked organism, SBW25EeZY-6KX, was less fit (P < 0.01) than SBW25-6K. However, no significant difference in
environmental fitness was detected in the rhizosphere between
SBW25-6KX and the triply marked recombinant SBW25EeZY-6KX after
14 and 28 days, despite the greater metabolic burden of the latter
(Table 3).
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TABLE 3.
Effect of metabolic load on the environmental fitness of
P. fluorescens SBW25 variants in
the rhizospherea
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Effect of environment on the fitness of recombinant
organisms.
Environmental conditions (soil and rhizosphere
of wheat and pea) had a significant effect on the
relative survival of the recombinant SBW25 variants SBW25EeZY-6KX,
SBW25-6KX, and SBW25-6K. The proportion of the triply marked
SBW25EeZY-6KX within the three-membered introduced community
declined (P < 0.001) rapidly in soil (Table 4). Immediately after inoculation
SBW25EeZY-6KX comprised 25% of the total Pseudomonas SBW25
community, but this percentage declined to less than 1.6% of the total
SBW25 community 28 days after inoculation (Table 4). This did not
happen in the rhizosphere of either pea or wheat, where the proportion
of the triply marked strain stayed more or less constant (Table 4).
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TABLE 4.
Effect of different environments on the relative
survival of P. fluorescens SBW25EeZY-6KX in a mixture of
SBW25 variantsa
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| |
DISCUSSION |
The most likely explanation of the decreased environmental fitness
of SBW25EeZY-6KX compared with that of the nonmodified wild type was
probably the extra metabolic load of expressing the novel phenotypes
and possibly the accumulation of novel proteins in the cell. Because
marker gene expression is of no direct benefit to GMMs, the extra
energy demands imposed on the organisms are not offset by an increased
competitive capacity in the environment. The lacY,
lacZ, and xylE genes encode lactose
permease, -galactosidase, and catechol 2,3-dioxygenase,
respectively, and constitutive expression of these genes might be
metabolically expensive. Because kanamycin resistance is conferred by
protein production, and thus would put a potential metabolic burden
upon the GMM, all subsequent comparisons between the genetically
modified SBW25 variants were carried out with organisms that were
modified with the kanamycin resistance gene (aph-1).
Addition of the lacZY and xylE genes, therefore,
presented an extra metabolic burden on top of the metabolic burden of
expressing aph-1. Competition experiments with SBW25-6K (expressing kanamycin resistance) and SBW25-6KX (expressing kanamycin resistance and catechol 2,3-dioxygenase) confirmed that the extra metabolic load of expressing catechol 2,3-dioxygenase caused a decrease
in environmental fitness. Surprisingly, no significant difference
between the P. fluorescens variants SBW25-6KX (expressing kanamycin resistance and catechol 2,3-dioxygenase) and SBW25EeZY-6KX (expressing lactose permease and -galactosidase in addition to kanamycin resistance and catechol 2,3-dioxygenase) was found in the rhizosphere of pea and wheat.
However, when the organisms were applied to soil, additional expression
of the lactose permease and -galactosidase genes in SBW25EeZY-6KX
resulted in a significant reduction in environmental fitness compared
with that of SBW25-6KX (Table 4). This suggests that the extra
metabolic load of marker gene expression leads to a reduction
in environmental fitness only under certain environmental conditions.
Previous investigations showed that there was no significant difference
in environmental fitness between wild-type SBW25 and the triply marked
SBW25EeZY-6KX when the organisms were grown in nutrient broth
(9) or in the phytosphere of sugar beet (2). Both
these environments might contain enough nutrients for microbial growth
to offset the extra metabolic burden of marker gene expression.
Although the relation between metabolic burden and environmental
conditions might be difficult to predict, it is clear that an increase
in metabolic burden caused by constitutively expressed genes which are
of no benefit to the recipient organism is unlikely to lead to an
increase in environmental fitness of GMMs. From a risk assessment point
of view this is important: increasing the metabolic burden of an
organism carries a cost for that organism which might lead to reduced
environmental fitness under suboptimal growth conditions.
Unless this cost is offset by a clear environmental advantage
conferred by the novel genes, the extra metabolic cost will
almost certainly result in a reduced performance of the GMM in the
environment. Furthermore, taking into account the variability of the
natural environment, it is unlikely that an environmental fitness
advantage, if this were to occur, would apply to a wide variety of
microbial habitats. Extra metabolic activity of expressing novel gene
sequences and environmental variability are safeguards against
uncontrolled GMM multiplication in the environment. The ability to
monitor genetically marked organisms with great sensitivity, however,
is of great advantage in ecological and applied studies of
microorganisms in the environment. This advantage more than offsets the
fact that such genetic modifications might cause a GMM to be
slightly less fit than its nonmodified parental strain.
| |
ACKNOWLEDGMENTS |
This study was carried out under a contract from the Ministry of
Agriculture, Fisheries and Food (CSA 2739) following earlier contracts
from the Department of the Environment (PECD 7/8/161 and PECD 7/8/143).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH,
United Kingdom. Phone: 00 44 1483 259721. Fax: 00 44 1483 259728. E-mail: j.lynch{at}surrey.ac.uk.
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Abstract
Introduction
