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Appl Environ Microbiol, February 1998, p. 626-632, Vol. 64, No. 2
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Enterococcus faecalis Gene Transfer
under Natural Conditions in Municipal Sewage Water Treatment
Plants
Herbert
Marcinek,
Reinhard
Wirth,*
Albrecht
Muscholl-Silberhorn, and
Matthias
Gauer
Microbiology-NWFIII, University of
Regensburg, D-93053 Regensburg, Germany
Received 2 October 1997/Accepted 12 November 1997
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ABSTRACT |
The ability of Enterococcus faecalis to transfer
various genetic elements under natural conditions was tested in two
municipal sewage water treatment plants. Experiments in activated
sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a
resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory
conditions); and Tn916 (a conjugative transposon which is
transferred under laboratory conditions at low rates to gram-positive
bacteria and at very low rates to gram-negative bacteria). The transfer
rate between different strains of E. faecalis under natural
conditions was, compared to that under laboratory conditions, at least
105-fold lower for the sex pheromone plasmids, at least
100-fold lower for pIP501, and at least 10-fold lower for
Tn916. In no case was transfer from E. faecalis
to another bacterial species detected. By determining the dependence of
transfer rates for pIP1017 on bacterial concentration and extrapolating
to actual concentrations in the sewage water treatment plant, we
calculated that the maximum number of transfer events for the sex
pheromone plasmids between different strains of E. faecalis
in the municipal sewage water treatment plant of the city of Regensburg
ranged from 105 to 108 events per 4 h,
indicating that gene transfer should take place under natural
conditions.
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INTRODUCTION |
The discovery that a bacterial
phenotype can be "transformed" into a new phenotype dates back to
the time when the nature of genetic material was not yet known
(17). It was assumed for a long time that gene transfer
between different species of microorganisms is a very rare event at
best; later, this view changed. From gene-protein comparisons it was
concluded that, "the available evidence suggests that interspecific
transfer of genes has occurred between the three major groups of
organisms: archaebacteria, eubacteria and eucaryotes"
(33). There is very strong evidence that gene transfer occurs even between distantly related bacteria (25, 26),
which supports the idea that effective mechanisms prevent uncontrolled gene transfer, which otherwise might even interfere with the species concept.
The most spectacular example of gene transfer in nature involves the
ability of Agrobacterium tumefaciens strains to genetically engineer plants to produce the nutritive compounds nopalines and opines
by transferring and integrating the so-called T-part of their
endogenous Ti plasmids into plant genomes (14).
Interestingly, the principal mechanisms used for Ti-mediated T-DNA
transfer to plant cells and for bacterial conjugation seem to involve
similar systems (23). Nevertheless, T-DNA transfer from
A. tumefaciens to plants is one of the few examples of gene
transfer in nature. In almost all reports dealing with gene transfer
the workers performed experiments under laboratory conditions; e.g.,
the proof that T-DNA can be transferred from A. tumefaciens
to the yeast Saccharomyces cerevisiae was obtained in this
way (4).
For bacterium-bacterium gene transfer it has been postulated that
strains of the gram-positive eubacterium Enterococcus
faecalis "(the `Escherichia coli' of the
gram-positive bacteria?) may serve as reservoirs of genetic information
available for passage to other streptococci and even other genera by
conjugal processes" (5). This hypothesis stems from the
fact that various genetic elements have been found to be present in
great numbers in this species; these elements include transposons,
so-called conjugative transposons, cryptic plasmids, resistance
plasmids, and so-called sex pheromone plasmids (for reviews see
references 5, 8, and 36). Some of
these elements, the sex pheromone plasmids, have a very restricted host
range; except for pIP964, which reportedly also replicates in
Enterococcus faecium and Listeria monocytogenes (30), the sex pheromone plasmids are restricted to E. faecalis. Resistance plasmids apparently have a broad host range
for gram-positive bacteria; conjugative transposons are found primarily
in gram-positive bacteria, but they also can occur in gram-negative
bacteria. Indeed, the data for conjugal transfer of genes and genetic
elements between different strains of E. faecalis and
between this bacterium and other bacterial species (in both directions
[i.e., to and from E. faecalis]) under laboratory
conditions are too numerous to be cited here (for an overview see
reference 6).
On the other hand, reports of transfer of genetic elements from or to
E. faecalis in nature are rare. Resistance plasmid pAM
1 has been shown to transfer among E. faecalis, E. faecium, and Lactobacillus reuteri in the digestive
tracts of mice (27). (This plasmid also transfers between
Lactobacillus curvatus strains in fermenting sausages
[35].) Inducible transfer of conjugative transposon
Tn1545 from E. faecalis to Listeria
monocytogenes in the digestive tracts of gnotobiotic mice has also
been reported (12).
Here, we define natural conditions as conditions under which bacteria
are kept and grown under conditions prevalent in the natural biotope
(e.g., the digestive tract of an animal or a municipal sewage water
treatment plant with rather low water temperatures, partially toxic
components, etc.). Conditions under which conjugation between two
strains occurs on a filter which is placed on a (rich medium) agar
plate incubated at the optimal growth temperature are referred to as
laboratory conditions.
Since no data on E. faecalis gene transfer in sewage water
treatment plants are available, we performed such studies in the sewage
water treatment plants of two Bavarian cities, Munich (Klärwerk Marienhof; Dietersheim) and Regensburg (Klärwerk Barbing),
Germany. Our data indicate that under the natural conditions of the
Regensburg plant ca. 106 to 109 gene transfer
events between different E. faecalis strains should take
place per day.
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MATERIALS AND METHODS |
Bacterial strains.
E. faecalis OG1X (Smr,
containing no mobile genetic element) (20) was used as the
source of donor strains. Various derivatives of OG1X, which contained
sex pheromone plasmids pAD1 (34) and pIP1017
(19), broad-host-range plasmid pIP501 (18), and
conjugative transposon Tn916 (15) alone or in all
possible combinations, were constructed by standard conjugation
techniques (13, 15). E. faecalis FA2-2
(Rifr Fusr, containing no mobile genetic
element) (7) was used as the recipient. The following
bacteria (species were identified by routine tests performed at
University Hospital at Regensburg) were used as potential recipients
for the genetic elements listed above: Escherichia coli,
Bacillus subtilis, Enterococcus durans, Staphylococcus aureus, Streptococcus mutans, and
Streptococcus sanguis. In some cases spontaneous
antibiotic-resistant derivatives of these organisms had to be selected
on Todd-Hewitt broth (THB) plates so that we would be able to test for
conjugative transfer of the genetic elements used. Since
transconjugants were obtained in none of these experiments, the details
of the experiments are not described here.
Experiments in sewage water treatment plants.
Experiments in
activated sludge basins of municipal sewage water treatment plants had
to be performed in a microcosm (shown schematically in Fig.
1) so that there would be a sterile
compartment (composed of dialysis tubing) which could be placed at
various positions in the plants and protected from leakage caused by
debris. Each Nadir dialysis bag (Roth, Karlsruhe, Germany) had a
diameter of 50 mm and a nominal pore size of 2.5 nm. It was protected
from damage by a stainless steel cage having a pore size of ca. 0.75 mm. Experiments performed with phages present in the sewage water at
the Regensburg plant showed that the dialysis bags were phage proof.
Linear DNA, introduced into a microcosm (which was preequilibrated in
the activated sludge basin for 2 h) via a T connector and a sterile syringe, was degraded to nondetectable levels after 20 h;
this did not occur if the microcosm was preequilibrated in sterile 10 mM Tris-Cl (pH 7.5)-0.1 mM EDTA buffer. We concluded, therefore, that
our setup allowed an equilibrium between the dialysis bag contents and
the soluble components of sewage water to occur (within 90 min), but
that phages were excluded by the dialysis bag.
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FIG. 1.
Schematic diagram of the microcosm used for experiments
in the activated sludge digestors. The whole assembly could be varied
in length, by using nuts and bolts, from 15 to 45 cm and was sterilized
with the dialysis tubing connected to the polypropylene end caps via
autoclavable O rings. The arrows indicate connections via silicone
tubing to the recirculation system.
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All parts of the microcosm were constructed of noncorrosive materials
(the nuts, bolts, and protective cage were stainless
steel; the
dialysis tubing, O rings, and end caps were autoclavable
plastic); the
whole assembly, filled with 10 mM Tris-Cl (pH 7.5)-0.1
mM EDTA, was
autoclaved prior to each experiment. Silicone tubes
attached to the
inlet and outlet were closed immediately after
sterilization; they were
connected in the plant to a recirculating
pump (Isamatec type MV-Z).
The recirculating system contained
pH, temperature, and O
2
electrodes (WTW, Weilheim, Germany) and
was rinsed for 1 h prior
to use with 70% ethanol. Then the recirculating
system and the
microcosm were connected, and the liquid was replaced
via two T
connectors by sterile 1% NaCl. This assembly was circulated
(at a rate
of 600 ml/min) in the activated sludge basin for at
least 2 h,
after which the system was inoculated via one T connector
and a sterile
syringe. The total volume of the recirculating system
plus microcosm
was ca. 600 ml, and the microcosm contained ca.
300 ml. An
equilibration time of ca. 90 min for the microcosm
contents and sewage
water was determined by performing experiments
in which Tris-Cl buffer
(pH 9.5) was used and the pH was measured.
Donor strains of
E. faecalis (OG1X with various combinations
of genetic elements) and recipient strain FA2-2 were grown at
37°C
separately in THB (Oxoid, Wesel, Germany) to an optical density
at 600 nm of ca. 0.6, collected by centrifugation, washed with
1% NaCl
containing 10 mM Tris-Cl (pH 7.5), and resuspended in
the same buffer.
The cells were kept on ice for a maximum of 45
min and were mixed
immediately before inoculation into the microcosm
to obtain a
donor/recipient ratio of 0.1. A maximum volume of
20 ml was used for
inoculation; the syringe and T connector were
rinsed with another 20 ml
of buffer to mix all of the cells into
the recirculating system. After
3 min of recirculation, the first
sample (the zero-time sample) was
removed with a syringe from
the second T connector by first withdrawing
20 ml of waste and
then collecting an appropriate amount. Samples were
collected
1, 4, and 20 h after inoculation.
For experiments in anoxic sludge digestors cells were prepared as
described above. Only very slight turbulence occurred in
the digestors,
and thus there was no potential danger of damage
to the dialysis
tubing. Therefore, donor and recipient cells were
preincubated in
separate parts of a sterile dialysis bag for 90
min; the separating
clamp was removed after 90 min, and the contents
were mixed and
incubated for various times. The contents of the
dialysis bag were
mixed every hour by 3- to 5-fold inversion.
Determinations of gene transfer efficiency.
The numbers of
donor cells and recipient cells were determined for each sample by
counting the number of colonies resistant to streptomycin (500 µg/ml)
and the number of colonies resistant to rifampin (25 µg/ml) and
fusidic acid (25 µg/ml), respectively, for various sample dilutions.
Transconjugants were identified on THB agar containing fusidic acid
onto which various sample dilutions were plated. These plates also
contained 5% defibrinated horse blood (Oxoid, Augsburg, Germany) for
detection of pAD1-encoded cytolysin (3), 1,000 µg of
kanamycin per ml for detection of pIP1017, 10 µg of erythromycin per
ml for detection of pIP501, and 10 µg of tetracycline per ml for
detection of Tn916.
Gene transfer efficiency was calculated by determining the number of
transconjugants per donor cell by using the conventional
definition. It
should be noted, however, that this calculation
was to a certain extent
problematic for pAD1, since the pAD1-encoded
cytolysin kills recipients
over extended periods of time and therefore
the donor/recipient ratio
is not constant over time. Cell titers
were determined for donor
strains, recipient strains, and transconjugants
for each experiment in
parallel, and the duplicate values varied
by a maximum factor of 3. The
numbers shown in the tables below
are mean values from these duplicate
determinations of transfer
efficiencies. Also, under natural conditions
variables like sewage
water temperature, oxygen saturation, and
chemical composition
of the wastewater, etc., influence the results; in
our experiments
even greater variances occurred. Therefore, no
extensive statistical
analyses were done; we note that the experiments
performed in
the Regensburg plant (see Fig.
3) were performed in
triplicate
for the activated sludge basin and in duplicate for the
anoxic
sludge digestor. The values (determined in duplicate, as
described
above) which we obtained in these experiments varied by a
maximum
factor of 15. In the case of the Munich plant all of the
experiments
were performed only once; the identities of transconjugants
containing
pIP501 or Tn
916, both of which appeared at
unexpectedly high rates,
were determined by Southern hybridization (see
below).
A small inhibitory effect of the biotope sewage water treatment plant
on
E. faecalis was observed; reduction of the donor
and
recipient cell titers by a maximum factor of 2.7 was detected
in the
activated sludge basin, while the maximum factor for the
anoxic sludge
digestor was 16.3. This reduction occurred after
incubation for between
4 and 20 h, but was negligible between
zero time and 4 h
(data not shown). Since under laboratory and
natural conditions gene
transfer events increased with time up
to 5 h (data not shown),
most experiments were performed for 4
h. This also allowed us to
definitely conclude that experiments
in the sewage water treatment
plant were performed under sterile
conditions. In the few cases in
which sterility problems occurred
(due to dialysis membrane damage),
the problem very clearly was
detected as a reduction in donor and
recipient titers by at least
5 orders of magnitude, due to the massive
occurrence of phages
lytic for
E. faecalis in the sewage
water (data not shown).
In experiments in which less than 10 transconjugants were observed,
these were examined not only by determining the antibiotic
resistance
profile, but also by isolating total genomic DNA as
described by
Muscholl et al. (
28) and probing by Southern hybridization
by using the enhanced chemiluminescence protocol (Amersham,
Braunschweig,
Germany) and the following probes: two 1,600- and 700-bp
HindIII
fragments of pWM401 (
37) specific for
pIP501 and one 1,700-bp
HindIII-
Asp718
fragment of plasmid pAM120 (
16) specific for
Tn
916. In the case of pIP1017, transconjugants could be
detected
by pulsed-field gel electrophoresis (PFGE) of
BamHI-restricted
total genomic DNA (see Fig.
2) and the sex
pheromone-induced clumping
reaction. In the case of pAD1-containing
transconjugants, we used
not only the hemolytic phenotype, but also the
sex pheromone-induced
clumping reaction for identification.
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RESULTS AND DISCUSSION |
Gene transfer efficiencies under laboratory conditions.
Gene
transfer efficiencies under laboratory conditions were measured for the
following two reasons: (i) reference data were needed and (ii)
potential deviations of gene transfer efficiency values due to
different settings (laboratory or natural conditions) had to be
determined.
Transfer of single genetic elements under laboratory conditions was
within the expected size range (Table
1)
(
13). Cotransfer
of two genetic elements was clearly
observed, with the sex pheromone
plasmids showing a synergistic effect
at least for Tn
916 (the
conjugative transposon was
transferred at least 10- to 100-fold
better from a strain containing
not only Tn
916 but also pAD1 or
pIP1017). Since no
cotransfer of three genetic elements was observed,
such data are not
listed in Table
1.
We also determined if broad-host-range plasmid pIP501 and the
conjugative transposon Tn
916 were transferred under
laboratory
conditions into the gram-negative bacterium
Escherichia coli or
into the gram-positive bacteria
B. subtilis,
E. durans,
Staphylococcus aureus,
Streptococcus mutans, and
Streptococcus sanguis.
These
experiments were deliberately performed in liquid culture,
because
data for transfers on the surfaces of filters were available,
at least for some species (
2). In contrast to previously
reported
transfer efficiencies ranging from 10
6 to
10
8 for Tn
916 on solid surfaces, we did not
observe any transfer
in liquid medium (our detection limit was ca.
10
8); very probable mating pair formation is not
disturbed on solid
surfaces, in contrast to preparations incubated in
liquid. Since
gene transfer into the potential recipients was also not
observed
in a few orienting studies when we used the microcosm in the
activated
sludge basin of the Regensburg plant, no further studies of
gene
transfer into other species in sewage water treatment plants were
done.
Gene transfer efficiency in the sewage water treatment plants.
As Table 2 shows, the results obtained
under laboratory conditions were comparable for Munich and Regensburg;
therefore, the differences obtained in experiments performed in the
activated sludge basins of the Munich and Regensburg municipal sewage
water treatment plants, especially the differences observed with pIP501 and Tn916, were interpreted to be significant. Potential
factors which might have been responsible for the lower gene transfer efficiencies in the Regensburg plant than in the Munich plant are
discussed below.
For sex pheromone plasmid pAD1 no definite transfer efficiencies were
measured under natural conditions; they clearly were
less than
10
5. This stems from the fact that in the case of pAD1 no
direct
selection for transconjugants was possible; rather, the
phenotype
hemolysis of erythrocytes had to be determined. Not a single
hemolytic
colony was detected in these experiments, even if samples
were
plated on agar plates (22.5 by 22.5 cm). Our data clearly
indicated
that the activity of the sex pheromone system was reduced in
the
activated sludge basins of both plants by 4 to 6 orders of
magnitude
or that this system was not active at all. This could have
been
due to various reasons. (i) Active sex pheromone was rapidly
diluted
from the microcosm during the experiment. (Experiments in which
synthetic sex pheromone cAD1 was added to the microcosm at a titer
of
at least 100,000 together with
E. faecalis
OG1X::pAD1 did not
result in any clumping reaction. From
experiments in which we
used the microcosm in sterile buffer we
concluded that active
sex pheromone diffuses too fast into the
surrounding liquid [sewage
water in the activated sludge basin] to
induce a clumping reaction
[data not shown]). (ii) In addition,
degradation of the inducing
peptide in activated sludge seems to play a
role: 25 ml of filter-sterilized
activated sludge inactivated synthetic
sex pheromone at a titer
of 10,000 within 30 min at 37°C, but not if
the preparation was
pretreated for 5 min at 100°C. Such inactivation
did not occur
if sex pheromone and filter-sterilized activated sludge
were separated
by a dialysis bag (data not shown). (iii) Furthermore,
induction
of an aggregation substance (leading to the bacterial
clumping
reaction, resulting in very effective gene transfer specific
for
sex pheromone plasmids under laboratory conditions) by the sex
pheromone is not observed at temperatures below ca. 12°C. To our
knowledge, the latter effect has not been observed before; it
represents a true temperature regulatory phenomenon. Under laboratory
conditions transfer efficiencies for pAD1 and pIP1017 in liquid
culture
dropped from ca. 10
1 at 37°C to 10
7 at
temperatures below ca. 12 to 15°C. Since donor strains in
which an
aggregation substance was preinduced at 37°C showed normal
transfer
efficiencies of sex pheromone plasmids at 10°C, the regulatory
phenomenon seems not to involve the actual conjugation process.
In the case of broad-host-range plasmid pIP501 transfer efficiencies
under laboratory conditions were in the expected range;
they dropped
below the detection limit in the Regensburg plant,
but were
extraordinarily high in the Munich plant. The same phenomenon
was
observed for Tn
916, which had measured transfer efficiencies
ca. 10-fold lower than those for pIP501. Because these data were
rather
surprising, we checked transconjugants obtained in the
Munich plant for
the presence of pIP501 or Tn
916 by Southern hybridization.
The identity of the transconjugants as FA2-2 transconjugants was
tested
by performing PFGE with conventionally purified genomic
DNA. The fact
that OG1X and FA2-2 could be differentiated clearly
in such experiments
is shown in Fig.
2. In this case not only
was differentiation between OG1X and FA2-2 possible, but
differentiation
between OG1X::pIP1017 and
FA2-2::pIP1017 was also possible.
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FIG. 2.
Identification of FA2-2 transconjugants containing
pIP1017 by PFGE. Conventionally purified and
BamHI-restricted total genomic DNA was analyzed by PFGE.
Lanes 1 to 3, three independent clones, identified as
FA2-2::pIP1017 transconjugants; lane 4, FA2-2; lane 5, OG1X;
lane 6, OG1X::pIP1017. Lane 7 contained  DNA restricted
with Asp718 (30, 17, and 1.5 kb). The two arrows on the left
indicate the positions of bands specific for FA2-2 genomic DNA; the
arrow on the right indicates the position of a pIP1017-specific
fragment.
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There were several potential reasons for the extraordinarily high
transfer efficiencies observed for pIP501 and Tn
916 in the
activated sludge basin of the Munich plant compared to the Regensburg
plant. (i) Most of the experiments in the Munich plant were performed
during a very unusual weather situation in the fall of 1993 (heavy
rain
showers and even snowfall in October), which resulted in
very low
temperatures in the activated sludge basin and also unusually
dilute
sewage water. The Tn
916 experiments had to be performed
at
ca. 8°C, and all other experiments at the Munich plant were
performed
at temperatures between 10 and 13°C. In the Regensburg
plant,
experiments in the activated sludge basins were performed
at water
temperatures between 20 and 25°C (comparative data for
different
temperatures at the Regensburg plant are shown in Fig.
3). (ii) The efficiencies of aeration at
the Munich and Regensburg
plants differed extensively. An
O
2 saturation level of 1.4 to
2.2 mg/ml was measured at the
Regensburg plant, while the values
at the Munich plant were
consistently more than 10 mg/ml. (iii)
Although it is not possible to
directly compare the compositions
of the two sewage waters, it should
be noted that the Munich plant
receives relatively more industrial
wastewater than household
wastewater compared to the Regensburg plant
(indicated by a ca.
fivefold-higher load of heavy metals). Taken
together, our data
seem to indicate that under severe stress situations
very unusual
(high) gene transfer efficiencies can occur.
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FIG. 3.
Comparative data for gene transfer efficiency in the
Regensburg sewage water treatment plant. Efficiencies of conjugation
for sex pheromone plasmid pIP1017, broad-host-range plasmid pIP501, and
conjugative transposon Tn916 are shown. For the columns
labelled pIP501 (+ pIP1017) and Tn916 (+ pIP1017) the values
are values for transfer of the first genetic element from a donor
strain also harboring pIP1017. Values obtained under laboratory
conditions, obtained in the anoxic sludge digestor (35°C), obtained
during the summer in the activated sludge digestor (20 to 25°C), and
obtained during the winter in the activated sludge digestor (14 to
16°C) are indicated. Open bars indicate that no transconjugant was
obtained and indicate the detection limit.
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Comparative data for the Regensburg sewage water treatment
plant.
Figure 3 summarizes data for gene transfer efficiencies at
different temperatures in the activated sludge basin and the anoxic sludge digestor of the Regensburg sewage water treatment plant (for
comparison results obtained in the Regensburg laboratory are shown).
Since no actual numbers could be determined for pAD1 under natural
conditions (the values were <105 [see above]), data
for pAD1 under natural conditions are not included. While the
experiments performed in the activated sludge basin were performed with
the microcosm system (to simulate the extensive mixing in the basin),
incubations in the sludge digestor were in dialysis bags, the contents
of which were mixed every hour (to mimic the minor turbulence in the
digestor). Experiments under laboratory conditions were performed at
37°C; data for the activated sludge basin were collected during the
summer at temperatures between 20 and 25°C and during the winter at
temperatures between 14 and 16°C. For the anoxic sludge digestor only
one data set was collected, since the temperature was constant (35°C)
throughout the year.
It is evident that (i) in all cases gene transfer efficiencies were
highest under laboratory conditions; (ii) gene transfer
in the
activated sludge basin was more efficient at higher temperatures
(i.e.,
during the summer) than at lower temperatures; (iii) the
gene transfer
efficiencies in the anoxic sludge digestor were
higher than those in
the activated sludge basin (this effect can
be attributed to two facts,
the higher temperatures in the anoxic
sludge digestor and the far less
intense mixing in the digestor,
which did not disrupt potential mating
pairs); and (iv) the presence
of sex pheromone plasmids enhances
transfer of other genetic elements,
especially under laboratory
conditions (under natural conditions
this effect is less pronounced).
Our data showing that transfer efficiencies were highest under
laboratory conditions were mainly due to the fact that conjugation
efficiencies in THB are temperature dependent. The actual values
for
pIP1017 were 1 × 10
0, 2 × 10
3,
and 1.5 × 10
7 for experiments performed at 37, 25, and 10°C, respectively.
Nevertheless, other factors, such as growth
medium, also play
a role, since for experiments that were performed at
37°C under
laboratory conditions but in which filter-sterilized
activated
sludge was used instead of THB the efficiency of pIP1017
conjugation
was 1.2 × 10
4.
Calculation of endogenous transfer efficiencies for E. faecalis in the Regensburg plant.
In all of the experiments
described above we used comparable bacterial concentrations under
laboratory and natural conditions, which resulted in comparable data.
Therefore, the concentrations of E. faecalis introduced into
the microcosm (experiments performed in the activated sludge basin) or
the dialysis bags (experiments performed in the anoxic sludge
digestor), up to 107 donor cells/ml and 108
recipient cells/ml, were 3 to 4 orders of magnitude higher than the
actual numbers in the sewage water treatment plants. For different locations in the Regensburg plant we determined the following E. faecalis titers: 2.4 × 103 CFU/ml at the point
of entry into the plant; 7.5 × 102 CFU/ml in the
activated sludge basin; 4.8 × 104 CFU/ml at the
entrance of the anoxic sludge digestor; 4.7 × 102
CFU/ml at the exit of the anoxic sludge digestor; and 2.8 × 100 CFU/ml at the plant exit.
Since transfer efficiencies under natural conditions were highest for
pIP1017, experiments were performed with this genetic
element in the
activated sludge basin and the anoxic sludge digestor
by using reduced
titers of donor and recipient cells. We still
had to use titers of
donor cells (OG1X::pIP1017) of 10
4 to
10
5 to detect single transconjugants (our assumption that
10% of
all
E. faecalis cells could represent potential
donors is supported
by data from other workers [
9]).
The results of these experiments
are shown in Fig.
4. Since the limit of detection for gene
transfer
was ca. 100-fold higher than the actual concentrations of
potential
E. faecalis donor cells, extrapolations had to be
done to calculate
potential gene transfer rates. The extrapolated,
maximal gene
transfer efficiencies were ca. 10
8 for the
conditions in the activated sludge basin (the actual
E. faecalis titer was ca. 10
3 for donor plus recipient
cells) and the anoxic sludge digestor
(the actual
E. faecalis titer was ca. 10
4 for donor plus recipient
cells). By using these values the maximal
numbers of potential
transconjugants originating in 4 h under
natural conditions could
also be calculated (Table
3).
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FIG. 4.
Calculation of maximal transfer frequencies for pIP1017
at E. faecalis titers present in the Regensburg plant.
Transconjugation rates (numbers of transconjugants per donor cell) were
determined independent of the donor titer (note that the recipient cell
titer was in each case 10-fold higher). Since the limit of detection of
transconjugants was higher than the actual E. faecalis cell
number in either the anoxic sludge digestor or the activated sludge
basin, extrapolations had to be made to obtain calculated
transconjugation rates for total E. faecalis titers of
103 (activated sludge basin) and 104 (anoxic
sludge digestor).
|
|
The maximal numbers of calculated transconjugation events under natural
conditions, which range from 10
5 to 10
8, might
appear to be high at a first glance; it should be noted,
however, that
under these conditions only ca. 10
8 and
10
11 of all
E. faecalis cells in the anoxic
sludge digestor and the
activated sludge basin would be
transconjugants.
We want to emphasize that the above-mentioned calculations rely on
several numbers which could not be measured directly and
therefore
represent only rough estimates. In addition, conventional
statistical
analyses of data obtained under natural conditions
seem not to make
sense to us, since too many variables, which
cannot be controlled,
influence the actual numbers (see above).
A final comment relates the results of our study to other results,
including the results described below. (i) Our finding
that under very
special conditions (e.g., 30°C below the optimal
growth temperature
in the Munich plant), unexpectedly high efficiencies
of gene transfer
were observed is at least to some extent supported
by other findings.
Heat treatment resulted in enhanced gene transfer
from
Escherichia coli to various coryneform bacteria
(
31), as
did other stress situations, such as exposure to
organic solvents
or detergents and pH shifts (
32). (ii) Our
finding that gene
transfer efficiencies were highest under laboratory
conditions
is corroborated by the results of comparisons of
efficiencies
of gene transfer from
Alcaligenes eutrophus to
Variovorax paradoxus under laboratory conditions and in soil
(
29) and by the fact
that gene transfer rates between marine
bacteria were higher if
the strains were encapsulated in microbeads
than if the genes
were transferred in marine water (
1).
(iii) Gene transfer occurs
under natural conditions not only in soil,
marine water, and sewage
water treatment plants, but also in other
biotopes. For example,
transduction via phages occurs between
Pseudomonas aeruginosa strains on the surfaces of leaves
(
21); gene transfer occurs
between the fish-pathogenic
bacterium
Aeromonas salmonicida and
a human
Escherichia coli isolate in raw salmon on a cutting board
(see reference
22 for various other natural
settings); and gene
transfer between physically isolated bacteria is
enhanced by the
presence of burrowing earthworms as a biological factor
which
facilitates cell-to-cell contact (
10).
Finally, workers also have to be aware of the fact that not all genes
are transferred with the same efficiency (
11) and
the fact
that gene transfer mechanisms other than conjugation
also can be
extremely efficient in various biotopes (
24).
Conclusions.
Gene transfer efficiencies between different
strains of E. faecalis were highest under laboratory
conditions; under natural conditions in municipal sewage water
treatment plants the efficiencies dropped up to 6 orders of magnitude,
but still were measurable. Gene transfer from E. faecalis to
other bacterial species could not be detected in liquid media.
Calculations to determine potential gene transfer rates between
different strains of E. faecalis in the municipal sewage
water treatment plant of the city of Regensburg resulted in maximal
values of ca. 108 events per day. Even with a 100-fold
reduction (killing under natural conditions) 106
transconjugants per day would still be released into the River Danube.
These transconjugation events cannot be avoided since every person
excretes E. faecalis.
The argument that the possibility of gene transfer has to be totally
excluded in genetically engineered bacterial strains
does not make
sense if gene transfer occurs in nature. A case-by-case
discussion of
safety demands still seems to be justified; this
discussion should take
into account the enormous wealth of evidence
that gene transfer occurs
under natural conditions and the few
cases in which actual numbers have
been measured.
| |
ACKNOWLEDGMENTS |
This work was supported by grant D11 (program FORBIOSICH) from
the Bayerische Forschungsstiftung.
We thank H. Körner and B. Beckmann (of the Munich and Regensburg
municipal sewage water treatment plants) for their constant interest in
and enthusiastic support of this work. We especially thank workers in
the mechanical workshop of the Institute for Genetics and Microbiology
(Ludwig-Maximilians-Universität, Munich, Germany) for
construction of the microcosm.
H.M. and M.G. contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Mikrobiologie-NWFIII, University of Regensburg,
Universitätsstrasse 31, D-93053 Regensburg, Germany. Phone: (49)
941 943 1825. Fax: (49) 941 943 1824. E-mail: Reinhard.Wirth{at}biologie.uni-regensburg.de.
This paper is dedicated to Herbert Marcinek, who died in a tragic
accident.
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Abstract
Introduction
