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Applied and Environmental Microbiology, December 1998, p. 4904-4911, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of Cell Lysis in Pseudomonas
putida Induced upon Expression of Heterologous
Killing Genes
M. Carmen
Ronchel,1,2
Lázaro
Molina,2
Angela
Witte,3
Werner
Lutbiz,3
Søren
Molin,4
Juan L.
Ramos,1 and
Cayo
Ramos1,4
Department of Biochemistry and Molecular and
Cellular Biology of Plants, Estación Experimental del
Zaidín, Consejo Superior de Investigaciones
Científicas,1 and
GX-Biosystems
España S.L.,2 Granada, Spain;
Department of Microbiology, University of Vienna, Vienna,
Austria3; and
Department of
Microbiology, Technical University of Denmark, Lyngby,
Denmark4
Received 15 June 1998/Accepted 25 September 1998
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ABSTRACT |
Active biological containment systems are based on the controlled
expression of killing genes. These systems are of interest for the
Pseudomonadaceae because of the potential applications of
these microbes as bioremediation agents and biopesticides. The
physiological effects that lead to cell death upon the induction of
expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed.
P. putida CMC4 and CMC12 carry in their chromosomes a
fusion of the PA1-04/03 promoter to the Escherichia
coli gef gene and the 
X174 lysis gene E,
respectively. Expression of the killing genes is controlled by the LacI
protein, whose expression is initiated from the XylS-dependent Pm
promoter. Under induced conditions, killing of P. putida
CMC12 cells mediated by X174 lysis protein E was faster than that
observed for P. putida CMC4, for which the Gef protein was
the killing agent. In both cases, cell death occurred as a result of
impaired respiration, altered membrane permeability, and the release of some cytoplasmic contents to the extracellular medium.
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INTRODUCTION |
Active biological containment (ABC)
systems have been envisaged as a way to control the survival of
genetically modified microorganisms and the putative consequences of
their introduction into the environment (Fig.
1) (for reviews, see references
20 and 26). ABC systems are based
on the use of genes that encode killing proteins regulated by a control
element that activates (or derepresses) the killing function under
defined environmental conditions (1, 21).
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FIG. 1.
Detail of the biological containment system for
alkylbenzoates. This model consisted of the Pm promoter, which drives
the transcription of the meta-cleavage pathway of the TOL
plasmid, and the xylS gene, which encodes the sensor protein
that interacts with alkylbenzoates and stimulates transcription from
Pm. In the containment system, the lacI gene, coding for the
LacI repressor protein, was cloned downstream from Pm. The lethal
element consisted of the PA1-04/03 promoter fused to the
gef gene of E. coli or X174 gene E,
each of which codes for pore-forming proteins. The system was shown to
perform as follows. In the presence of 3-methylbenzoate, the XylS
protein became active and stimulated the synthesis of the LacI protein,
which in turn prevented the expression of the killing gene;
concomitantly, degradation of the alkylaromatic compound took place.
Once the compound was exhausted, the XylS protein became inactive, the
LacI protein was not made any longer, and expression from
PA1-04/03 led to the synthesis of Gef or E protein, which
in turn collapsed the cell membrane potential and led to the death of
the cell.
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The development of ABC systems for the Pseudomonadaceae is
of interest because of the potential applications of these microbes under field conditions. The so-called fluorescent
Pseudomonas group includes strains whose biochemical,
physiological, and genetic properties have been well characterized
(7, 27, 35). A number of genetic tools have made it possible
to design recombinant derivatives of this group of bacteria for the
biological control of pests (4, 24), the promotion of plant
growth (13, 17, 18), and the detoxification of polluted
sites (8, 27, 35).
Pseudomonas putida KT2440 is a DNA restriction-modification
system-negative strain derived from the soil bacterium P. putida mt-2, the natural host for the archetypal TOL plasmid pWW0
(39). Strain KT2440 has been shown to be a nonaggressive
soil rhizosphere colonizer (22, 25, 28). In addition, this
strain stably maintains and expresses heterologous genes, including
catabolic segments for the expansion of its metabolic versatility and
killing genes of interest for biological containment (5, 21,
26-28). The genes encoding killing functions successfully used
in P. putida were those that encode lysis proteins (1,
11, 14, 30), nucleases (3), and streptavidin
(34). In previous studies, we demonstrated that the
regulatory gene expression system of the TOL plasmid
meta-pathway for the metabolism of alkylbenzoates could be
combined with the gef gene of Escherichia coli,
which encodes a porin-like protein, in such a way that cell killing became a consequence of the absence of the substrate (and inducer) 3-methylbenzoate both under laboratory conditions (1, 11, 30,
31) and in soil microcosms (11, 30). We also showed that a P. putida strain carrying an ABC system on the host
chromosome functioned as expected under field conditions
(23). A modified version of this system based on lysis gene
E of bacteriophage X174 has also been constructed
(30). However, the above studies have not dealt in detail
with the physiological phenomena that lead to cell death in P. putida upon induction of the expression of killing genes in this
heterologous host. In E. coli, the natural host for X174
phage protein E-mediated cell lysis, killing occurs as the consequence
of the formation of a transmembrane tunnel made of E protein; the
tunnel fuses the outer and inner membranes and allows the escape of
cytoplasmic material (36-38). In E. coli, the
Gef protein forms dimers that are anchored in the cytoplasmic membrane
and lead to the collapse of the cell membrane potential (reviewed in
reference 20).
In this study, we show that once the Gef protein or the X174 E
protein is expressed in P. putida, cell death occurs as a consequence of the formation of membrane holes, which impair
respiration, alter membrane permeability, and release cell material.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The
P. putida strains used or constructed in this study are
derivatives of P. putida KT2440 (6). Their
relevant characteristics are given in Table
1. P. putida EEZ29
(31), CMC4 (23), and EEZ15K-3 (29)
were described before; these three strains bear the archetypal TOL
plasmid pWW0, which confers on them the ability to grow on
3-methylbenzoate. Strains that were constructed in the course of this
study are described below. P. putida strains were grown with
shaking at 30°C in modified M9 minimal medium (1) with 28 mM glucose or 5 to 15 mM 3-methylbenzoate as the sole carbon source.
In cloning experiments,
E. coli Mv1190
pir was
used to replicate the pMCC plasmids (Table
1). These plasmids are based
on
the R6K plasmid origin of replication, which is not recognized
in
P. putida strains, and behaves as a suicide replicon.
E. coli JM109 was used to maintain other plasmids or in
cloning experiments
with vectors that did not require the Pir protein
for replication.
E. coli strains were grown at 37°C in LB
medium (
19). The plasmids
used in this work are listed in
Table
1.
Antibiotics were used at the following final concentrations (micrograms
per milliliter): ampicillin, 100; chloramphenicol,
30; and kanamycin,
50. Potassium tellurite was used at 5 to 30
µg per
ml.
Construction of the killing cassette bearing the
PA1-04/03::gene E fusion.
Plasmid pUHE24-1 was described before (16). It carries
ampicillin and chloramphenicol resistance and exhibits two
NcoI sites. One of them lies 3' with respect to the
synthetic isopropyl-
-D-galactopyranoside (IPTG)-inducible promoter PA1-04/03, and the other lies at
the chloramphenicol resistance gene. To ensure that the plasmid
contained only the NcoI site 3' with respect to the
synthetic PA1-04/03 promoter, the plasmid was partially
digested with NcoI and treated with the Klenow enzyme and
the four deoxynucleoside triphosphates to fill in the sticky
NcoI ends. Apr clones were selected after
ligation and transformation. A Cms clone was selected, and
the plasmid that it bore was called pMCC26. We confirmed that the
single NcoI site remaining in this plasmid was located 3'
with respect to the synthetic PA1-04/03 promoter.
X174 gene
E was amplified by the PCR method with phage
DNA as a template. The oligonucleotides used for amplification
(5'-GTTTCTGGCCATGGTACGCTGGACTTTGTG-3'
and
5'-TCATTATCTTAAGCTTACGTTTTTTACCTTTAGA-3') were partly
complementary
to the ends of gene
E and were designed so
that
NcoI and
HindIII
sites would be
generated near the ends of the amplified DNA fragment.
The amplified
gene
E DNA was cleaved with
NcoI and
HindIII and
cloned into pMCC26 cut with
NcoI
and
HindIII, so that the amplified
promoterless
X174
gene
E was read from the synthetic P
A1-04/03 promoter. The resulting plasmid was called pMCC27 (Fig.
2). A
418-bp region from pMCC27
containing the P
A1-04/03::gene
E
fusion
was isolated after digestion with
XhoI and
HindIII and cloned
in pUC18Not digested with
SalI and
HindIII. The resulting plasmid,
pMCC30, was selected after transformation of the ligation mixture
into
E. coli JM109 (Fig.
2). Plasmid pMCC30 was digested with
NotI, and a 430-bp fragment containing the
P
A1-04/03::gene
E fusion
was cloned
into the unique
NotI site of pJMSB4 and transformed
into
E. coli Mv1190
pir. The resulting plasmid
carried a P
A1-04/03::gene
E fusion and
the tellurite-resistant determinant within mini-Tn5.
This
plasmid was called pMCC31 (Fig.
2).
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FIG. 2.
Construction of a Tn5-based insertion
delivery plasmid containing an inducible cell lysis system based on
lysis gene E from bacteriophage X174. Restriction sites
relevant for the constructions are shown. Plasmid pUC18Not and the
pUT-based plasmid pJMSB4 have been described elsewhere (9,
33). Abbreviations: geneE, lysis gene E of
bacteriophage X174; RBS, ribosome binding site from the E. coli expression plasmid pUHE24-1 (16);
PA1-04/03, synthetic lactose promoter from plasmid
pUHE24-1; ori, origin of replication; ori R6K, origin of
replication dependent on the Pir protein; ori ColE1, origin
of replication from plasmid ColE1; oriT RP4, origin of
transfer; tnp*, transposase; 'lacI, gene encoding the
repressor protein for the lac operon.
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Triparental matings.
Triparental matings were performed as
described by Herrero et al. (9). Equal numbers (about
108 cells) of the recipient strain P. putida
EEZ29, the donor strain E. coli
Mv1190pir(pMCC31) or E. coli
Mv1190pir(pJMSB4), and the helper strain E. coli HB101(pRK600) were mixed and deposited on a nitrocellulose
filter placed on the surface of an LB medium plate supplemented with 5 mM 3-methylbenzoate. Appropriate controls with unmixed cells, otherwise
treated identically to the mixture, were always included. P. putida transconjugants were selected on M9 minimal medium plates
containing kanamycin and tellurite and supplemented with
3-methylbenzoate as the sole carbon source. One random transconjugant
of P. putida EEZ29 that had received the minitransposon
mini-Tn5-Tel from pJSB4 was selected and called CMC13;
another random transconjugant that had received mini-Tn5-Tel PA1-04/03::gene E from pCMC31 was
selected and called CMC12.
Tests of killing efficiency.
Killing efficiency was tested
with liquid medium. Bacteria were grown in M9 minimal medium containing
glucose and 3-methylbenzoate and supplemented with the appropriate
antibiotics. Cells in the early exponential phase were harvested by
centrifugation (12,000 × g, 15 min), washed twice in
M9 minimal medium without a C source, and resuspended in M9 minimal
medium with glucose. The sample was divided in half. To one half, 5 mM
3-methylbenzoate was added; 1 mM IPTG was added to the other half. All
samples were then incubated at 30°C with shaking. For determination
of viable counts, triplicate samples from a series of dilutions of the
cultures were plated on LB medium plates containing 5 mM
3-methylbenzoate and the appropriate antibiotics.
Transmission electron microscopy.
P. putida cells were
harvested by centrifugation, immediately fixed with 2% (vol/vol)
glutaraldehyde-1% (vol/vol) formaldehyde in cacodylate buffer,
postfixed with osmium tetroxide in the presence of 2% (wt/vol)
potassium ferrocyanide, and embedded in Eponate 12. Thin sections were
poststained with uranyl acetate and lead citrate and examined in a
Zeiss transmission electron microscope at an accelerating voltage of 75 kV.
High-resolution scanning electron microscopy.
Scanning
electron micrographs were taken with a Hitachi S-800 field-emission
scanning electron microscope. The cells were fixed and prepared for
electron microscopy essentially as described previously
(38).
Protein analysis.
Proteins in culture supernatants of
P. putida were analyzed as follows. Whole cells were removed
by centrifugation at 12,000 × g for 2 min, and the
supernatant was concentrated by precipitation with 10% trichloroacetic
acid. Cells were then resuspended in Laemmli buffer and analyzed by
electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels
with the discontinuous buffer system of Laemmli (15). After
electrophoresis, the gels were silver stained (32).
Rubidium efflux.
P. putida cells were grown in 30 ml
of M9 minimal medium containing 15 mM 3-methylbenzoate as the sole
carbon source and supplemented with the appropriate antibiotics and 1 mCi of 86RbCl (1 mCi/mmol). Cells were harvested by
centrifugation (12,000 × g, 10 min), washed in M9
minimal medium without a C source, and resuspended in the same minimal
medium with either 15 mM 3-methylbenzoate or 28 mM glucose plus 5 mM
IPTG. The amount of 86RbCl retained intracellularly by the
cells was measured by harvesting 200-µl aliquots of the culture by
filtration. The pellets were resuspended in 200 µl of M9 minimal
medium and mixed with 500 µl of scintillation liquid, and emission
was counted with a Packard scintillation counter.
Oxygen uptake assays.
Oxygen consumption rates of whole
cells of P. putida were determined with a polarographic
Clark oxygen electrode. A 0.1-ml aliquot of a P. putida
culture was transferred to 1 ml of fresh medium kept at 30°C in the
chamber of the oxygen electrode. The rate of oxygen consumption was
then recorded for 5 to 10 min.
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RESULTS AND DISCUSSION |
Loss of viability of P. putida strains that express
heterologous killing genes.
Two different killing genes were
incorporated separately into the chromosome of P. putida
KT2440 cells. P. putida CMC4 carries mini-Tn5-Km
with a PA1-04/03::gef fusion
integrated in the host chromosome (23). P. putida
CMC12 carries mini-Tn5-Tel with a PA1-04/03::gene E fusion on the
chromosome (this study). To control expression of the killing genes,
the lacI gene, encoding the LacI repressor, was expressed
from the Pm promoter for the meta-cleavage pathway of the
P. putida TOL plasmid pWW0, whose expression is in turn
controlled by the xylS gene (10, 30). In the
presence of XylS effectors, such as 3-methylbenzoate, expression of the killing proteins is prevented and the strains survive. However, in the
absence of effectors and in the presence of IPTG, cell growth is
rapidly arrested as a consequence of expression of the lethal proteins
from the PA1-04/03 promoter (1, 11, 30). Such is
not the case with the control strains P. putida EEZ15-K3 and
CMC13, which do not bear the killing genes. (Note that in this series
of assays, IPTG was added to rapidly titrate out the LacI protein in
the cells).
In order to analyze whether cell growth arrest in cells bearing the
containment system was the result of a loss of cell viability,
we
counted viable cells after induction of the system by transferring
cells to a culture medium without 3-methylbenzoate and with IPTG.
Cells
of
P. putida EEZ15-K3, CMC4, CMC13, and CMC12 growing
exponentially
(about 10
6 to 10
7 CFU/ml) in M9
minimal medium with glucose and 3-methylbenzoate
were harvested by
centrifugation, washed with 50 mM phosphate
buffer, and then
resuspended at the same cell density in M9 minimal
medium containing
glucose and either 3-methylbenzoate or IPTG.
The number of cells of the
two control strains increased with
time regardless of the growth medium
(data not shown). In contrast,
the number of CFU of CMC4 and CMC12 per
milliliter increased with
time in medium with 3-methylbenzoate (Fig.
3) but not in the presence
of IPTG. After
an initial lag, the number of viable cells decreased
in both strains.
One hour after induction, about 33 and 4% of
the initial cells were
viable in cultures of
P. putida CMC4 and
CMC12, respectively
(Fig.
3). The initial lag probably represents
the time required for
LacI turnover and synthesis and accumulation
of the killer proteins.
The fact that
X174 lysis protein E-mediated
killing of
P. putida CMC12 cells was faster than that of
P. putida CMC4 cells, which expressed the
gef gene, might indicate
that
the critical concentration of protein E needed to trigger killing
is lower than that of the Gef protein or that lysis protein E
is more
efficient than the Gef protein in provoking cell death
when the cells
are growing exponentially. We assumed that there
were equal levels of
expression of the killing genes in both strains,
because the two
killing genes used in this study were expressed
from the same promoter
and the respective fusions were located
on the host chromosomes.
Nonetheless, prolonged incubation of
CMC4 and CMC12 with IPTG led to a
steady reduction in cell viability.
The number of viable cells was on
the order of 0.01% the initial
number for strain CMC12 7 h after
induction of the system, and
the number was even lower for strain CMC4.
Prolonged incubation
led to a further decrease in cell viability in
both strains, although
in some cases killing-resistant mutants
appeared.
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FIG. 3.
Cell viability of P. putida strains after
induction of the expression of killing genes. Cells of P. putida CMC4 (PA1-04/03::gef) (A)
and CMC12 (PA1-04/03::gene E) (B)
growing exponentially in M9 minimal medium with glucose and
3-methylbenzoate were transferred at time zero to medium containing
glucose and either 3-methylbenzoate (closed symbols) or IPTG (open
symbols). At the indicated times, viable cells were counted on LB
medium plates containing 3-methylbenzoate and the appropriate
antibiotics.
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Physiological consequences of the expression of killing genes.
As stated in the introduction, the cytoplasmic membrane is the ultimate
target of both X174 lysis protein E and Gef in E. coli.
The insertion of these proteins in the cell membrane of this
microorganism leads to cell death, most likely via an alteration of
cell membrane permeability.
To test the physiological consequences of the expression of the
heterologous
E. coli proteins in
P. putida, we
first monitored
oxygen consumption in
P. putida cells
expressing each of these
two killing proteins. At 30 min after
induction of expression
of the killing proteins, the rate of oxygen
consumption by
P. putida CMC12 and CMC4 (about 0.2 ± 0.1 µmol of O
2/mg of cell protein
per min) was about 8%
that in noninduced, control cultures kept
in the presence of
3-methylbenzoate (3.0 ± 0.4 µmol of O
2/mg of
cell
protein per min). The rate of respiration in the control
strains was
not significantly affected by the removal of 3-methylbenzoate
or the
presence of IPTG and was about 3.5 ± 0.5 µmol of
O
2/mg
of protein per min. These results suggest that the
cytoplasmic
membrane of
P. putida is indeed the target of
Gef and
X174 lysis
protein E and that the expression of these
proteins leads to alterations
in cell
respiration.
This hypothesis is also supported by the observation that induction of
the synthesis of these killing proteins in
P. putida CMC4
and CMC12 led to a rapid loss of K
+ ions from the
cytoplasm. To model K
+ loss, cells were preloaded with
86Rb
+ as described in Materials and Methods and
then transferred to
culture medium with or without 3-methylbenzoate. We
found less
retention of
86Rb
+ ions in the
cytoplasm of cells incubated without 3-methylbenzoate
(less than 3%
the loaded
86Rb
+) than in that of cells kept in
culture medium containing 3-methylbenzoate
(about 20 to 30% the loaded
86Rb
+). Control cells in culture medium with or
without 3-methylbenzoate
retained similar amounts of loaded
86Rb
+, which were in the same range as those
retained by strains bearing
the containment system and kept in culture
medium with 3-methylbenzoate.
The release of cellular material due to Gef- and
X174 lysis protein
E-mediated membrane damage was investigated. We used
SDS-polyacrylamide
gel electrophoresis to analyze the release
of proteins to the culture
supernatants of
P. putida EEZ15-K3,
CMC4, CMC13, and CMC12
in cultures with and without IPTG. No proteins
could be detected in
culture supernatants of control strains EEZ15-K3
and CMC13 during the
6-h experiment regardless of the growth medium
or in culture
supernatants of CMC4 and CMC13 grown with 3-methylbenzoate
but without
IPTG (data not shown). However, in culture supernatants
of CMC4 and
CMC12 incubated in the absence of 3-methylbenzoate
but in the presence
of IPTG, proteins were detected 90 min after
the addition of IPTG (data
not shown). In both cases, the total
amounts of proteins detected
increased with time, as deduced by
the number and density of the bands
in the gels (Fig.
4). These
results
indicate that lysis of
P. putida cells occurs after the
expression of Gef and
X174 lysis protein E. The differences in
the
patterns of proteins released (Fig.
4) from each of the two
P. putida strains after the induction of killing suggest that
Gef-mediated lysis and
X174 protein E-mediated lysis may occur
through different mechanisms.
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FIG. 4.
Protein release from P. putida CMC4
(PA1-04/03::gef) and CMC12
(PA1-04/03::gene E) after induction of
the expression of killing genes. P. putida cells growing
exponentially in M9 minimal medium with glucose and 3-methylbenzoate
were harvested by centrifugation, washed twice in M9 minimal medium
without a C source, and resuspended in M9 minimal medium with glucose
and 1 mM IPTG. Samples were taken at 0 h (lanes 1 and 3) and
6 h after induction (lanes 2 and 4). The released proteins were
analyzed by SDS-polyacrylamide gel electrophoresis as described in
Materials and Methods. Numbers to the left of each panel indicate
molecular masses (M) in kilodaltons.
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Ultrastructure of P. putida bacteria that express
killing genes.
The ultrastructure of P. putida
EEZ15-K3, CMC4, CMC13, and CMC12 cells was analyzed before and after
transfer to 3-methylbenzoate-free medium. The ultrastructure of the
control strain was not significantly affected by the culture medium;
almost 100% of the cells appeared electron dense when examined by
transmission electron microscopy (data not shown). In contrast,
significant differences were observed in CMC4 and CMC12, depending on
the culture medium. Before the induction of the killing genes, more
than 92% of the cells (counted in six different field exposures) of
these two strains were electron dense. These cells exhibited
well-defined outer and inner membranes and showed the typical rod
morphology of Pseudomonas (Fig. 5A and
C). Induction of the expression of Gef or
X174 lysis protein E was typically followed by a change in the
appearance of the cells; in both cases, the cells became almost
completely transparent (Fig. 5B and D). The occurrence of ghost cells
is evidence that cytoplasmic material has been lost, as discussed
above. Three hours after transfer to medium lacking 3-methylbenzoate,
80% of the cells (counted in six different field exposures) were lysed and appeared as ghosts (Fig. 5B and D).
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FIG. 5.
Transmission electron micrographs of ultrathin sections
of P. putida CMC4 (A and B) and P. putida CMC12
(C and D) cells. A and C, noninduced cells growing in M9 minimal medium
with glucose and 3-methylbenzoate. B and D, Induced cells 3 h
after transfer to M9 minimal medium with glucose and IPTG.
Magnification, ×4,000.
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P. putida CMC4 cells expressing the
gef gene
showed numerous holes in the cell envelope (Fig.
6A). In
P. putida CMC12, which
expressed gene
E, the holes appeared to be grouped together
instead
of distributed along the cell envelope. Cytoplasmic material
was
observed leaking out through these holes (Fig.
6B). Lysed cells
of
P. putida CMC12 were also observed by high-resolution
scanning
electron microscopy. Bleb-like structures could be seen
emanating
from some of the cells (Fig.
7). It is well known that in
E. coli,
protein E forms a unique transmembrane hole through which
cytoplasmic
material is released to the external medium (
37,
38). The
protein may act in a similar way in
P. putida.
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FIG. 6.
Transmission electron micrographs of ultrathin sections
of lysed P. putida CMC4 (A) and P. putida CMC12
(B) cells after the expression of killing genes. Magnification,
×40,000.
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FIG. 7.
Scanning electron micrograph of lysed P. putida CMC12 cells after the expression of gene E from
bacteriophage X174. Magnification, ×3,500.
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The results presented in this study indicate that the cytoplasmic
membrane is the target of Gef and
X174 lysis protein E
in
P. putida. After the expression of the genes for these two
E. coli killing proteins in
P. putida cells, the cell
envelope
of the heterologous host loses its integrity, numerous holes
appear,
and the cytoplasmic content is released to the extracellular
medium;
as a consequence, cell death occurs. Comparative studies on the
efficiency of host killing by these two genes under environmental
conditions should provide new insights into the potential of these
genes for the biological containment of
P. putida strains.
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ACKNOWLEDGMENTS |
This study was supported by grants from the Commission of the
European Communities (BIO4-CT97-2270), GX-Biosystems España S.L.,
and Comisión Interministerial de Ciencia y Tecnología (BIO97-0657).
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FOOTNOTES |
*
Corresponding author. Mailing address: Estación
Experimental del Zaidín, CSIC, Apdo. de Correos 419, E-18008
Granada, Spain. Phone: 34-58-121011. Fax: 34-58-129600. E-mail:
carmen{at}eez.csic.es.
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Applied and Environmental Microbiology, December 1998, p. 4904-4911, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
