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Applied and Environmental Microbiology, January 2001, p. 293-299, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.293-299.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of Biological and Physical Protection against Nuclease
Degradation of Clay-Bound Plasmid DNA
Sandrine
Demanèche,1
Lucile
Jocteur-Monrozier,1
Hervé
Quiquampoix,2 and
Pascal
Simonet1,*
Laboratoire d'Ecologie Microbienne, UMR
5557, Université Claude Bernard Lyon I, 69622 Villeurbanne
Cedex,1 and Laboratoire de Science
du Sol, INRA-ENSAM, 34060 Montpellier Cedex 01,2
France
Received 24 May 2000/Accepted 8 October 2000
 |
ABSTRACT |
In order to determine the mechanisms involved in the persistence of
extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of
nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the
clay mineral. The biological potential of the resulting DNA was
monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate
that adsorbed DNA was only partially available for transformation. This
part of the clay-bound DNA which was available for bacteria, was also
accessible to nucleases, while the remaining fraction escaped both
transformation and degradation. Finally, transformation efficiency was
related to the perpetuation mechanism, with homologous recombination
being less sensitive to nucleases than autonomous replication,
which requires intact molecules.
| |
INTRODUCTION |
In the environment, three
mechanisms are thought to be involved in gene uptake by bacteria
(31), namely, conjugation, transformation, and
transduction. Natural bacterial genetic transformation is the mechanism
by which a bacterium acquires naked DNA. Such a mechanism is thought to
have been involved in gene transfers during evolution and particularly
in transfers among unrelated organisms such as plants and bacteria
(1, 8, 21). However, numerous reports indicate that gene
transfer events may be very rare in the environment (14, 18,
33). This could be due to the numerous steps that are required
to achieve transformation. DNA released by organisms must persist under
adverse conditions such as those encountered in soils. Naked DNA must
then encounter competent recipient bacteria. Moreover, the incorporated
DNA will only be perpetuated if its nucleotide sequences exhibit
sufficient similarity to the recipient genome to allow recombination,
unless the sequences possess a replicon which is operational in the new
host (14, 16, 33).
Nevertheless, there is a general agreement that natural transformation
may occur in complex media such as soils. Indeed, large amounts of
naked DNA, which is the preliminary key factor for transformation, can
be detected in soils (7, 35, 40). Moreover, there is much
evidence that extracellular DNA can persist for periods of time up to
several months or years (8, 18, 25, 27, 28, 38).
Adsorption of DNA on soil components, particularly on clay minerals
such as montmorillonite, illite, and kaolinite, is thought to be
involved in protection of nucleic acids against nucleases, and could
explain the high content of DNA in soils (2, 10, 32).
However, soil or microcosm-based experiments have indicated that the
adsorption-related protection process has only limited impact
(3, 17, 25, 27). In fact, very little is known about the
protection mechanism itself, and the influence of parameters such as
clay type, the size of DNA or its conformation. For instance, does
adsorption physically prevent DNA from being attacked by nucleases? Or
is protection related to adsorption of enzymes on clay, which induces
conformation alterations leading to a decrease in activity (11,
17, 24, 33, 34)? Moreover, how is the remaining clay-adsorbed
DNA available for bacterial transformation, and what are the
consequences of a partial protection on the transformation efficiency?
Finally, will partial degradation have the same effect on
transformation frequencies when the processing of the transforming DNA
in the bacterial cell occurs via homologous recombination or autonomous replication?
In this study, we developed simple models to investigate the protection
mechanism related to DNA adsorption onto clay particles and the balance
existing between degradation and persistence of DNA. Agarose gel
electrophoresis was used to determine the role that adsorption of DNA
and nuclease could play in preventing DNA degradation. Proteins such as
DNase I and bovine serum albumin (BSA) were used in combination with
plasmid DNA in the presence of clay minerals. Particular emphasis was
focused on illite, which is a common clay mineral in soils of temperate
regions (6). The biological consequences of adsorption and
protection were monitored by transforming the highly efficient
naturally competent proteobacterium Acinetobacter sp. strain
BD413 with the DNA resulting from the various tested conditions
(4, 20). Insight into the different fates which could
characterize a similar gene originating from different hosts or even
replicons can be deduced from the fact that of the two plasmids we
used, one was able to replicate autonomously in the bacterium while the
genes of the second plasmid could only be integrated into the host
genome via homologous recombination.
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MATERIALS AND METHODS |
Bacteria, media, and DNA.
Acinetobacter sp.
strain BD413 was grown in Luria Bertani (LB) medium (containing [per
liter] 10 g of Bacto Tryptone, 5 g of yeast extract, and
5 g of NaCl). Natural transformation was processed in parallel in
LB medium and in LBm medium (LB medium plus [per liter] 5 g of Bacto
Tryptone and 2.5 g of yeast extract) according to the protocol
described below. Plasmid pGV1 (3.95 kb) (37), containing
an nptII gene conferring resistance to kanamycin, replicates
autonomously in Acinetobacter sp. strain BD413 after
transformation. Plasmid pAVA213-8 (10.5 kb) (19), a
pUC18-based plasmid containing an nptII gene is unable to
replicate in Acinetobacter sp. strain BD413. Presence of a
6.4-kb chromosomal DNA fragment from Acinetobacter sp.
strain BD413 permitted integration of the marker gene into the genome
by homologous recombination. Both plasmids were extracted from
Escherichia coli strains and purified with a kit from Qiagen
Inc., Chatsworth, Calif., according to manufacturer's recommendations
and stored in sterile purified water (milliQ) at 
20°C.
DNA sorption on clay minerals.
Properties of the clay
minerals used in this study are presented in Table
1. Adsorption was effected by mixing DNA
with clay in 1.5 ml of polyallomer centrifuge tubes, providing a final
concentration of DNA at 12.5 µg ml
1 and of clay at 15 mg ml1 (22). The adsorption process was run
by shaking the tubes at 23°C in a thermomixer (Roucaire, Courtabouef,
France) at 1,200 rpm for 2 h, followed by a centrifugation at
15,000 × g and 4°C for 20 min to settle all clay
particles. The pellet was washed twice by 2 volumes of sterile water.
Final concentrations were 30 mg of clay ml1 and 25 µg
of DNA ml1, corresponding to a complete adsorption of
DNA, which was verified on an agarose gel.
Desorption of bound DNA was carried out on pellets resulting from
centrifugation of 10 µl of adsorbed DNA (15,000 ×
g,
4°C,
20 min), by adding 10 µl of a saline solution containing 17 mM
lactic acid, 3 mM KH
2PO
4, 27 mM
Na
2HPO
4, 0.23 mM MgSO
4, 11 mM
NH
4Cl, 19 µM CaCl
2, 0.5 µM
FeSO
4, 86 mM sodium pyrophosphate,
and 57 mM EDTA. The
desorption was run out for 1 h at 23°C with
vigorous shaking
(thermomixer at 1,200 rpm), and the resulting
mixture was
electrophoresed on an agarose gel (0.8%).
The effect of the composition of culture medium on DNA sorption was
investigated as follows. Ten microliters of adsorbed DNA,
in the
presence of illite, with and without BSA, and 10 µl of
sterile water
were mixed to achieve the same concentrations as
in the transformation
assays. Sterile water or culture media (LB
and LBm) were added to
provide a final volume of 200 µl, and these
mixtures were incubated
for 90 min at 29°C. Tubes were then centrifuged
20 min at 15,000 ×
g and 4°C, and molecules in the supernatant
were
precipitated by ethanol. Precipitated pellets were resuspended
in 10 µl of sterile water and loaded on 0.8% agarose gel to detect
desorbed
DNA.
BSA adsorption.
BSA (Sigma Chemical Co., St. Louis, Mo.) was
diluted in water and dialyzed against sterile purified water. A
solution containing BSA (20 mg ml1) was added volume to
volume to clay-DNA complexes. Adsorption was carried out
extemporaneously by shaking the tubes at 23°C in a thermomixer
(Roucaire) at 1,200 rpm for 1 h, followed by centrifugation for 20 min at 4 °C and 15,000 × g. The nonadsorbed proteins
were removed by rinsing the pellet twice with 2 volumes of pure sterile
water. The final concentration of adsorbed DNA was maintained at 25 µg ml1.
DNase-mediated degradation tests.
Bovine pancreas DNase I
grade II (Boehringer, Mannheim, Germany) was diluted in water to avoid
bias resulting from interaction of the enzyme with divalent cations.
Sterile DNase I solutions were prepared by filtration through cellulose
acetate filters (pore size, 0.2 µm). Ten microliters of DNA at 25 µg ml1, free or adsorbed on clay particles, with or
without BSA, and 10 µl of DNase I at concentrations ranging from 0 to
20 µg ml1 were mixed together. Resulting mixtures were
kept on ice before incubation for 15 min at 37°C on a horizontal
shaker with a 110-rpm agitation.
Transformation of Acinetobacter sp. strain
BD413.
An overnight culture of Acinetobacter sp. strain
BD413 was diluted 25-fold into fresh medium and cultured for an
additional 2 h at 29°C. A bacterial suspension volume of 180 µl was added to 20 µl of DNA, free or adsorbed and with or without
BSA, previously submitted to the DNase I. Resulting mixtures were
thoroughly mixed and incubated for 90 min at 29°C. After incubation,
reaction tubes were chilled on ice. In a parallel control experiment,
DNA was replaced by water.
Antibiotic resistant transformants were selected on LB medium
containing kanamycin (50 µg ml
1). Colonies were counted
after 2 or 3 days of incubation at 29°C.
Results were expressed in
number of transformants per ml of culture
because the total number of
cells was 3.4 × 10
8 ± 4.9 × 10
7 cells ml
1 in all experiments. Three to
five repetitions were carried out
for each
sample.
In parallel, clay particles, including kaolinite, illite, and
montmorillonite, on which plasmid DNA had been adsorbed were
incubated
in culture media according to the previous conditions
(200 µl as
final volume, 90 min at 29°C). After centrifugation,
precipitated
molecules present in the supernatant of samples were
electrophoresed on
an agarose
gel.
| |
RESULTS |
Physical protection of DNA by clay minerals.
Electrophoresis
of isolated plasmids indicated that molecules were mainly under a
covalently closed circular (CCC) form, also called supercoiled, with
open circular and linear forms also being detected (Fig. 1A, lane
2). The first step of the experiment was to incubate this free DNA with different concentrations of DNase I
(Fig. 1A). The smallest concentration used (0.1 µg ml1)
did not produce any change in the plasmid pattern (lane 3). An increase
of the concentration of the DNase I to 0.5 µg ml1 led
to a shift among the relative amounts of the various plasmid conformations (lane 4). A drop of the CCC form corresponded to a strong
increase of the linear form. When the enzyme reached a concentration of
1 µg ml1, DNA was totally degraded (lane 5).
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FIG. 1.
Detection of action of DNase I on plasmid pAVA213-8. (A)
Free DNA; (B) DNA adsorbed on illite; (C) DNA adsorbed on illite
saturated with BSA. Lane 1: smart ladder (Eurogentec); lanes 2 to 7, 0, 0.1, 0.5, 1, 5, and 10 µg of DNase I ml1, respectively.
Abbreviations: CCC, covalently closed circular form; OC, open circular
form; L, linear form.
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In parallel, plasmid DNA at the same concentration as previously used
was adsorbed on illite before being submitted to the
various nuclease
treatments. Electrophoresis of clay-bound DNA
did not permit its direct
detection on the gel (data not shown);
thus, a desorption step before
electrophoresis was processed on
all samples. Plasmid profiles (Fig.
1B) appeared fuzzy due to
a migration of persisting clay particles
still bound to DNA. However
the three conformations of the plasmid were
still distinguishable.
The pattern corresponding to DNase I untreated
DNA (lane 2) was
still detected when illite-adsorbed plasmid molecules
were submitted
to DNase I concentrations ranging from 0.1 to 1 µg
ml
1 (lanes 3 to 5). Finally, even for the 5 and 10 µg
ml
1 DNase I concentrations, DNA was still detected,
although plasmid
conformation was shifted from CCC to linear forms
(lanes 6 and
7).
The same experimental procedures were used with illite-bound DNA
submitted to a BSA adsorption process before any DNase treatment.
We
found that the plasmid patterns were less fuzzy than previously
(Fig.
1C) and exhibited a greater similarity to the profiles obtained
with
naked DNA (Fig.
1A) than those exhibited in Fig.
1B (illite-bound
DNA
without BSA). Patterns corresponding to untreated (Fig.
1C,
lane 2) and
0.1 µg ml
1 DNase I-treated (Fig.
1C, lane 3) DNA were
similar, while the
two following DNase concentrations produced the
shift to the linear
form (lanes 4 and 5). Increasing nuclease
concentration to 5 µg
ml
1 led to a complete degradation
of DNA (lane
6).
In a second step, plasmid DNA was adsorbed onto kaolinite, illite, and
montmorillonite to be submitted to the same nuclease
concentrations as
previously (Fig.
2). DNA was detected for
all
DNase concentrations (lanes a to f) and patterns were quite
similar.
The only exceptions were detected for the 5 and 10 µg
ml
1 DNase concentrations which provided the shift to
linear forms
when plasmid DNA was adsorbed on illite (lanes e and f) as
previously
demonstrated on Fig.
1B.
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FIG. 2.
Comparison of the protective role of three clay
minerals kaolinite (K), illite (I), and montmorillonite (M)against
nuclease degradation by 0, 0.1, 0.5, 1, 5, and 10 µg of DNase I
ml1 (lanes a to f, respectively).
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Transformation of Acinetobacter sp. strain BD413.
Clay particles, including kaolinite, illite, and montmorillonite, on
which plasmid DNA had been adsorbed were incubated in culture media
according to the conditions used to transform Acinetobacter sp. strain BD413. Figure 3 clearly
demonstrates that large amounts of DNA were desorbed by LB medium
regardless of the clay tested. On the other hand, such an effect was
not noticed with the LBm medium, which exhibited a lower salt content
than the LB medium. The same result was obtained when incubation
occurred in sterile purified water (Fig. 3).
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FIG. 3.
Effect of the type of culture medium on sorption of DNA
to the three clay minerals kaolinite (K), illite (I), and
montmorillonite (M). Clay-bound DNA (20 µl) was incubated 1 h 30 min
at 29°C with 180 µl of each of the three media tested before
centrifugation and electrophoresis of the precipitated molecules
present in the supernatant of samples. Lane A contains smart ladder.
LBm is a low-salt concentrated medium, and LB is a high-salt
concentrated medium. H2O, ultra pure sterile water.
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Transformation of
Acinetobacter sp. strain BD413 was found
to decrease from 1 to 2 orders of magnitude in the LBm medium compared
to the LB medium, as evidenced by the use of free DNA with frequencies
dropping from 9.1 × 10
7 to 5.8 × 10
6 T ml
1 for pAVA213-8 and from 1.7 × 10
6 to 8.5 × 10
3 T ml
1 for
pGV1 (Table
2) (where T stands, for
transformants).
When transforming DNA was previously adsorbed on illite, the number of
transformants was nearly identical to that obtained
with free DNA for
the plasmid pAVA213-8, as only a 1% reduction
was observed (Table
2),
while a decrease of 54% was detected
for transformations conducted in
LB medium with plasmid pGV1.
In the LBm medium, a marked decrease was
observed between free
and adsorbed conditions, reaching a reduction of
74% for pAVA213-8
and 86% for
pGV1.
When submitted to an increasing range of DNase I concentrations, the
transformant numbers were found to change in similar
ways whatever the
conditions tested (Fig.
4). This included
a
first range of concentrations for which the number of transformants
remained identical to untreated DNase conditions. The various
curves
indicated that in a second step an increase of the nuclease
concentration led to a more or less marked decrease of transformation
efficiencies until the detection limit was reached. Evolution
of the
number of transformants when experiments were conducted
in LBm were
found to be very similar between free DNA and illite
adsorbed DNA with
or without BSA (Fig.
4C and D). On the other
hand, the three curves
resulting from experiments conducted in
LB medium differed for the
highest nuclease concentrations (Fig.
4A and B). For instance, when
Acinetobacter sp. strain BD413 was
transformed with
illite-bound DNA, plasmid pAVA213-8 submitted
to 10 µg of DNase I
ml
1 provided 1.8 × 10
2 recombinant
clones, while this number dropped below the detection
limit under the
two other conditions (Fig.
4A). For plasmid pGV1,
a DNase I (1 µg
ml
1) treatment produced a decrease from 8.4 × 10
4 T ml
1 with an illite-adsorbed DNA
inoculum to 5.8 × 10
3 T ml
1 with free
DNA and 100 T ml
1 under conditions including BSA (Fig.
4B). Transformations processed
in LB medium clearly indicated that
plasmid pGV1 exhibited a greater
sensitivity to the highest nuclease
concentrations than plasmid
pAVA213-8, as evidenced by the DNase I
concentration of 5 µg ml
1 which did not allow any
transformant to develop. Moreover, in
the LBm medium, a gradual
decrease of the number of transformants
was noticed for pGV1, while the
slope of the curves characterizing
pAVA213-8 was initially gentle and
became steeper for the highest
nuclease concentrations.
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FIG. 4.
Transformation of Acinetobacter sp. strain
BD413 by two types of plasmid DNA in two different media. (A)
Transformation with pAVA213-8 in LB medium; (B) transformation with
pGV1 in LB medium; (C) transformation with pAVA213-8 in LBm medium; (D)
transformation with pGV1 in LBm medium. Results were given as the mean
of transformants ± standard error (error bars), as the total
number of cells was 3.4 × 108 ± 4.9 × 107 ml1 in all experiments; the axes
represented data on a logarithmic scale.
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DISCUSSION |
Physical protection of DNA adsorbed on clay minerals.
The
first objective of our experiments was to confirm that soil components
provide some physical protection to extracellular DNA, which is
naturally released in soils. We focused our study on the effect of clay
minerals because of their essential implication in soils (5, 14,
32, 36). Comparison of Fig. 1A and B clearly demonstrates the
protective effect of illite for DNA against nucleases; the effect was
confirmed for the two other clays (Fig. 2). However, illite exhibited
less efficiency for the highest nuclease concentrations (Fig. 2, lanes
e and f). Intact CCC molecules or linearized forms resulting from
nuclease nicks were still detected when DNA was bound on the clays in
presence of DNase concentrations which would have totally degraded free
DNA. These results confirm that adsorption provides a physical
protection to DNA which relies on the type of the adsorbent (3,
10, 11, 26, 36).
Protection mechanism.
Two hypotheses can be proposed to
explain this protection mechanism. First, the clay mineral could be
considered as a niche providing DNA physical protection from nuclease.
An alternative hypothesis would involve simultaneous adsorption of the
enzyme on the clay, physically separating the enzyme from its
substrate. To examine this, we decided to prevent DNase I from
adsorbing onto illite by saturating potential sites with another
nonenzymatic protein (BSA) at a concentration guaranteeing complete
saturation (9, 23, 34). We also verified by agarose gel
electrophoresis that the BSA treatment did not desorb any DNA (data not shown).
The results (Fig.
1C) indicate that BSA treatment of illite improved
DNase activity by saturating the protein sites. Under
these conditions,
BSA blocked sorption of DNase I on the clay
surface so it remained
available to degrade DNA to nearly the
same extent as in the absence of
illite (Fig.
1A). These results
indicate that it is the adsorption of
the nuclease on the clay
itself that produces the protective effect.
Adsorption would physically
separate DNA and the nuclease on the clay
surface. This statement
is in agreement with the fact that bound
enzymes usually exhibit
reduced activity (
5,
11,
13,
24,
29,
32). Furthermore,
the lower level of protection found with
illite compared to kaolinite
and montmorillonite could be due to the
higher affinity of DNase
I for the two other clay minerals as we have
observed recently
(unpublished
results).
The hypothesis of a protective niche effect cannot be excluded since
persisting DNA could still be detected (Fig.
1C, lane
5) after
treatment with DNase I at 1 µg ml
1, a concentration
which totally degraded free DNA (Fig.
1A, lane
5). This discrepancy
could also be due to the persistence of some
sites permitting nuclease
adsorption in spite of the saturating
concentration of BSA we used.
However, the conformation and strength
of adsorption of both DNA and
nuclease depend on the mechanism
of adsorption and may affect
enzyme-substrate interaction and
degree of availability of DNA to
uptake for transformation and
attack by nucleases in
soils.
Biological availability of clay-bound DNA.
When considering
extracellular DNA in soils, a fundamental question is related to the
ability of these molecules to transform bacteria and thus to be
involved in evolution and adaptation mechanisms. The experiments
reported by Romanowski et al. (28) indicated a difference
between the transformation potential of plasmids inoculated and
reextracted from soils and their physical persistence detected by PCR
or Southern hybridization. This illustrates the importance of
considering both the physical integrity and the biological potential of
DNA. We used the natural transformation capacity of
Acinetobacter sp. strain BD413 as a tool to monitor the
availability of plasmid DNA adsorbed on illite and the consequences of
a DNase treatment.
The first parameter we considered was the chemical effect of the
environment on sorption of transforming plasmids to mimic
what could
occur in soils. We conducted these experiments in two
media in which
bacterial transformation occurred, although with
various efficiencies,
but which differed in their desorption potential.
Agarose gel
electrophoresis clearly demonstrated that most of
the bound DNA was
desorbed in LB medium, while this effect was
not noticed in LBm medium,
which exhibited a reduced salt concentration
(Fig.
3). When considering
transformation in the presence of clay
minerals, a question remained
about the availability of adsorbed
DNA. Are bacteria able to pick up
this clay-bound DNA or is a
desorption process necessary to release the
transforming DNA?
Transformation in the nondesorbing medium (LBm)
demonstrated that
a fraction of adsorbed DNA remained accessible to
bacteria, indicating
that it could still play a biological role in
soil. However, a
comparison of the transformation rates in the two
media with free
and previously adsorbed DNA demonstrated that another
part of
adsorbed DNA was not available for transformation. Finally, we
demonstrated that the LB medium increased DNA availability by
desorbing
it, as indicated by the number of transformants, similar
to that
obtained with free DNA (Table
2).
Chamier et al. (
3) suggested that DNA adsorbed on mineral
surfaces in a sedimentary or soil habitat may be available for
transformation of
Acinetobacter sp. strain BD413. Their
protocol
involved bacteria, which were applied to DNA-loaded microcosms
in a culture medium containing salts. It is probable that DNA
was
desorbed from the adsorbent surface by salts when cells were
added.
Thus, media containing salts should be avoided in experiments
monitoring DNA availability. Recently, Sikorski et al.
(
30)
showed that competent cells were able to take up DNA
bound on
soil particles, even in presence of microorganisms and DNases
indigenous to the soil. Introduced cells were washed before
introduction
in soil extract, and no bias occurred due to culture
medium. In
agreement with our results, the study by Sikorski et al.
(
30)
showed that bacteria could indeed take up adsorbed
DNA. Moreover,
our work demonstrates that only a fraction of adsorbed
DNA was
accessible to bacteria, while for another fraction, adsorption
prevented its availability for natural genetic
transformation.
Biological protection resulting from DNA adsorption.
When
exposed to DNase I, regardless of the conditions (free or adsorbed DNA,
with or without BSA), the transformant counts were found to change in
similar ways (Fig. 4). In contrast to previous data on the efficiency
of illite to physically protect DNA (Fig. 1), a lower protective effect
was detected when assessing the bacterial transforming availability of
the DNA treated with various nuclease concentrations.
Transformations conducted in the nondesorbing medium (LBm) could lead
to the conclusion that illite had no effect on transformation
efficiency, as demonstrated by the similarity of data obtained
for free
and adsorbed DNA (Fig.
4C and D). However, a comparison
with the
experiments in which DNA was desorbed (Fig.
4A and B)
did not confirm
this assessment. For the highest nuclease concentrations,
the number of
transformants was significantly higher in the presence
of illite
compared to free DNA. This suggests that a part of the
adsorbed DNA,
which was protected by illite, was not available
to transform bacteria.
Prevention of the specific nuclease adsorption
(presence of BSA)
suppressed the protection effect of illite on
DNA, confirming that DNA
was prevented from being degraded by
the adsorption of the nuclease
itself on illite, as previously
demonstrated on an agarose gel. When
environmental conditions
led to desorption, this DNA became accessible
to both bacteria
and
nucleases.
Influence of the processing of internalized DNA.
In nonsterile
soils, a successful transformation could be processed by integration of
the transforming DNA in the host genome by homologous or more or less
illegitimate recombination, or by autonomous replication of plasmids.
In order to monitor the involvement of such endogenous processes in the
perpetuation of genetic information in soils, we used two plasmids
differing by their processing in the host cell after DNA uptake.
Current knowledge on plasmid transformation in Acinetobacter
sp. strain BD413 indicates that this mechanism is not dependent on the
presence of multimers, as in Bacillus subtilis; that plasmid
recircularization is a RecA-independent mechanism; and that chromosomal
DNA and plasmid DNA compete for the same uptake system (12,
20). Our study completes these data, as our results indicate
that the perpetuation mechanisms affected the biological potential of
DNA submitted to nuclease degradation (Fig. 4). A higher sensitivity to
nucleases of the replicative plasmid was demonstrated compared to the
integrative one. This could be due to the integrity of the molecule
which was required to maintain a transforming activity, while partial degradation did not prevent integration of DNA as long as homologous sequences were preserved. This interpretation is supported by the
current state of knowledge on integration of chromosomal donor DNA into
the recipient chromosome and reconstitution of plasmid DNA molecules
after internalization of restricted single-stranded DNA (14, 31,
39).
Our results confirm previous data on the protection effect that the
presence of clay minerals in soils provides to extracellular
DNA,
protection which is mainly related to an efficient adsorption
of the
nucleases. While the occurrence of natural bacterial transformation
in
situ cannot be excluded, confirming that gene transfer could
occur
naturally, numerous factors contribute to maintaining a
low frequency
of such processes. Adsorption does not provide DNA
with a complete
protection against nucleases but does significantly
decrease its
availability for bacteria (
15,
27). These macromolecules
also have to cope with the changing environmental conditions which
certainly lead to rapid switches in the ratio between adsorbed
and
desorbed DNA. The combination of these various factors could
prevent
bacteria from acquiring a great amount of genetic information,
in
particular when the sequences have to be replicated
autonomously.
| |
ACKNOWLEDGMENTS |
We are very grateful to K. J. Hellingwerf, University of
Amsterdam, Amsterdam, The Netherlands, who provided
Acinetobacter sp. strain BD413 and plasmids pAVA213-8 and
pGV1. Clay minerals were kindly provided by C. Chenu, INRA Versailles,
Versailles, France. We are grateful to Stephane Peyrard for technical assistance.
This work was supported by a grant from the Ministère de la
Recherche et de l'Education and as part of the Biotechnology program
of the Ministère Français de l'Enseignement
Supérieur et de la Recherche.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire
d'Ecologie Microbienne, UMR 5557, Université Lyon I, 43 bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France. Phone: 33 4 72 44 82 89. Fax: 33 4 72 43 12 23. E-mail:
simonet{at}biomserv.univ-lyon1.fr.
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Applied and Environmental Microbiology, January 2001, p. 293-299, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.293-299.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
