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Applied and Environmental Microbiology, June 2001, p. 2617-2621, Vol. 67, No. 6
Laboratoire d'Écologie Microbienne,
UMR 5557, Université Lyon I, 69622 Villeurbanne Cedex, France
Received 12 October 2000/Accepted 7 March 2001
Little information is available concerning the occurrence
of natural transformation of bacteria in soil, the frequency of such
events, and the actual role of this process on bacterial evolution.
This is because few bacteria are known to possess the genes required to
develop competence and because the tested bacteria are unable to reach
this physiological state in situ. In this study we found that two soil
bacteria, Agrobacterium tumefaciens and Pseudomonas
fluorescens, can undergo transformation in soil microcosms
without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile
soil microcosms but failed to do so in the various in vitro conditions
we tested. A. tumefaciens could be transformed in vitro and
in sterile soil samples. These results indicate that the number of
transformable bacteria could be higher than previously thought and that
these bacteria could find the conditions necessary for uptake of
extracellular DNA in soil.
According to gene sequence analyses
and experimental data, gene transfer by natural transformation among
microorganisms is involved in bacterial evolution and is likely to
occur at present, providing bacteria the flexibility to rapidly adapt
to changing environmental conditions (22, 26). However,
such transfers remain very difficult to detect under natural
conditions, particularly in soil, indicating they occur at very
low frequencies (25, 27, 29). According to PCR data,
large amounts of extracellular DNA are readily found in most soils and
persist for months, indicating that the turnover of naturally released
extracellular DNA would be quite slow (10, 19, 23, 24,
31). Extracellular DNA is thought to persist, because
soil particles, particularly clay minerals, absorb macromolecules,
a process which would not totally inhibit the transforming
activity of DNA (7).
The main limitations to natural transformation in soil would be related
to the recipient bacteria. Relatively few bacteria have been shown to
carry the genes required to develop a natural state of competence;
those described belong to phylogenetically distant taxa, including
gram-positive and proteobacteria species (16). However,
their number could be significantly higher considering that isolates
are not systematically tested for this property and that more than 99%
of all soil bacteria remain uncultured in vitro. Another limitation to
transformation is related to the development under natural conditions
of the competence state permitting active uptake of DNA. In soil,
bacteria tend to live in a state of dormancy due to prevailing
oligotrophic conditions, which would not be particularly favorable for
the development of competence (16, 17). For instance,
Acinetobacter sp. strain BD413, which exhibits high
frequencies of transformation in vitro, is unable to develop a
competence state when grown in soil. Moreover, this physiological state
is lost very rapidly in situ when in vitro-prepared competent cells are
inoculated into soil (18).
In this study, we found that Agrobacterium tumefaciens and
Pseudomonas fluorescens, two bacteria present in most soils
and used as models for numerous molecular, physiological, and
ecological studies, can be transformed without any physical or chemical
treatment. Interestingly, the various conditions we tested did not
permit P. fluorescens to reach the competence state in
vitro, while transformants were detected in situ.
Bacterial strains, culture media, growth conditions, and cell
enumeration.
Bacterial strains and plasmids used in this study are
listed in Table 1. Escherichia
coli, A. tumefaciens, and P. fluorescens strains were
grown overnight at 37°C for E. coli and 28°C for the two
other bacteria in liquid Luria-Bertani (LB) medium (containing, per
liter, 10 g of Bacto Tryptone, 5 g of yeast extract, and
5 g of NaCl) supplemented with the appropriate antibiotics
according to the concentrations given in Table 1. Bacterium counts were determined as the number of CFU on agar-solidified LB medium incubated overnight or for 48 h when targeting E. coli or the
other soil bacteria, respectively.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2617-2621.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Natural Transformation of Pseudomonas
fluorescens and Agrobacterium tumefaciens in
Soil
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Inoculum preparation. Bacterium inocula were prepared by diluting an overnight culture 20-fold into fresh medium and incubating it for an additional 2-h period (to an optical density of 0.6). These cultures were then centrifuged for 10 min at 6,500 × g before resuspension in the appropriate volume of sterile water to provide a 100-fold bacterial concentration. Using pure sterile water rather than a minimal salt medium avoided modification of competence development and natural transformation.
Soil microcosms.
Microcosms consisted of 50-ml Falcon tubes
in which were placed 30 g of air-dried and sieved (2-mm pore size)
samples of a sandy loam soil (sand, 50%; silt, 41%; clay, 9%;
organic matter, 40.6 g kg of dry soil
1; pH 5.6)
collected at La Côte Saint-André (Isère, France). Sterile soil conditions were obtained by gamma-irradiating the microcosms at a dose of 25 kGy from a 60Co source
(Conservatome, Dagneux, France). Moisture was adjusted to 3 g of
water for each microcosm according to the following protocol: 2.5 g of pure sterile water was added initially to the 30 g of dried
soil, which was left to drain naturally for 2 days at room temperature.
During the next 3 days, soils were mixed manually from time to time to
homogenize moisture. Soils were then seeded with 15 µg of DNA in 0.25 ml of water and 0.25 ml of the concentrated recipient strain in water
to provide a soil microcosm with 0.5 µg of DNA and 5 × 109 bacteria g of dry soil1.
1.
Control experiments were systematically conducted with pure water to
replace each of the two biological inocula.
Enumeration. Following a 3-day incubation at 28°C to stabilize the population level (11), soil microcosms were treated with DNase I to degrade any persistent extracellular DNA (incubation for 3 h at 28°C with 1,000 U of DNase I in 90 ml of LB medium). Appropriate dilutions of the soil suspensions were spread on selective (Table 1) and LB media to enumerate transformants and recipient cells, respectively. Colonies were counted following a 2-day incubation at the appropriate temperature. Repetitions were performed as follows. For every experimental combination, six independent microcosms were treated separately, and each one was plated on five petri dishes. Thus, a total of 30 replicates were counted to detect transformants. Moreover, this entire procedure was repeated at least once subsequently.
Recipient cells were selected through their specific resistance to antibiotics (Table 1). The presence of plasmids in the growing colonies was confirmed by using a plasmid extraction kit (Qiagen Inc., Chatsworth, California), according to manufacturer instructions.Identification of strains. The PCR amplification method included the use of 1 µl of bacterial cultures as template and a hot start procedure (3 min at 95°C) to release DNA, to avoid initial mispriming and to enhance primer specificity. Primers used, pA and pH (9), were complementary to part of the 16S ribosomal gene amplifying 1.6-kb-long PCR products. Restriction enzymes were from Boehringer Mannheim (Meylan, France) and used according to manufacturer instructions before fragments were separated on agarose gels.
Recovering of transformants from soil. We put 1, 10, 100, or 1,000 CFU of the plasmid-containing Agrobacterium or Pseudomonas into the sterile soil seeded with the stabilized plasmidless bacteria in the same way as described previously, followed by a 3-h incubation at 28°C in 90 ml of LB medium. Appropriate dilutions of the soil suspensions were spread on selective (Table 1) and LB media to enumerate plasmid-containing strains and plasmidless cells, respectively. Colonies were counted following a 2-day incubation at 28°C. Three independent microcosms were treated separately, and each was repeated subsequently. Each one was plated on five petri dishes.
In vitro transformation protocols.
Plasmids were introduced
into E. coli and P. fluorescens strains according
to standard electroporation techniques (8). The
development of a natural competence state in P. fluorescens was tested by mixing 20 µl of 100-fold-concentrated recipient cells
from overnight cultures with 0.5 µg of DNA (20 µl of a 25-µg ml1 concentrated plasmid solution) or 20 µl of
100-fold-concentrated donor cells and deposited onto a GTTP filter
(Millipore, Bedford, Massachusetts) on each of the solid media
described below. After drying, the Petri dishes were incubated at
28°C for 24 h before bacteria were resuspended in 2 ml of pure
sterile water and plated out on the same media under the appropriate
selective conditions. Liquid conditions were also tested by mixing 40 µl of recipient cells from overnight cultures with 950 µl of each
of the liquid culture media and 10 µl of a 100-µg ml1
concentrated plasmid solution. The tubes were incubated for 24 h
at 28°C with shaking before the bacteria were plated out on media
with appropriate antibiotics. Additional tests included a second
10-µl inoculation of a 100-µg ml1 concentrated
plasmid solution 12 h after the first one and incubation was
continued for another 12-h period. The various media we tested included
(i) the standard P medium (60 mM lactic acid, 11 mM
KH2PO4, 95 mM Na2HPO4,
0.81 mM MgSO4, 37 mM NH4Cl, 68 µM
CaCl2, 1.8 µM FeSO4, and 1 ml of trace
element liter1) (21) and derived media, each
with a specific deficiency obtained by decreasing the concentration of
the corresponding nutrient as follows. To obtain a carbon-limited
culture, only 20 mM lactic acid was added. For the nitrogen limitation,
only 3 mM NH4Cl was added together with 60 mM lactic acid.
The potassium limitation was obtained by adding only 50 µM
KH2PO4 and 30 mM
Na2HPO4 together with 60 mM lactic acid
(20); (ii) the MM medium [containing, per liter,
KH2PO4, 3.4 g;
(NH4)2SO4, 0.5 g;
MgSO4 · 7H2O, 0.05 g;
FeSO4 · 7H2O, 0.125 mg; thiamine, 0.25 mg; glucose, 2 g; glycerol, 4 ml]; (iii) two soil-based media: a
rough soil medium consisting of 50% (wt vol1) ground
soil in sterile distilled water used as a liquid medium or solidified
by adding 15 g of agar liter1 and a mineral soil
medium in which the previous soil suspension in water was centrifuged
twice for 15 min at 4,500 × g before the supernatant
was filtered (pore size, 0.2 µm; Millipore). The filtered solution
was used directly as a liquid medium or supplemented with 15 g of
agar liter1 to be used as a solid medium. Five
repetitions were carried out for each sample, repeated at least twice
subsequently. Controls consisted of filters with donor or recipient
strains alone.
Statistical analysis of data.
Assuming that each petri dish
received roughly the same number of bacteria, each petri dish was
considered to be a sampling unit, and the presence or absence of any
transformant was considered to be the variable. In other words,
the result was considered negative only if no transformants appeared.
We calculated the probability that the in vitro results were due to a
sampling effect and compared this to results of soil experiments. We
assumed as a null hypothesis that the frequency of transformation and
the probability of observing negative plates were the same in soil and in vitro experiments. We estimated the probability of
obtaining negative plates in soil experiments and calculated the
probability of observing no positive plates in the in vitro experiments
under this hypothesis. We used the zero term of binomial distribution as in the most-probable-number method of Cochran (6), but
using only one dilution:
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(1) |
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Transformation of A. tumefaciens and P. fluorescens in soil.
Transformation experiments were
conducted directly in soil microcosms by inoculating recipient bacteria
and DNA. Detection of clones exhibiting the appropriate antibiotic
resistance at frequencies significantly higher than the detection limit
(number of recipient cells1) and possessing the plasmid
would indicate the ability of the tested bacteria to be naturally
transformed. Because the plasmids we used were lacking the
tra function and could not transfer autonomously, they could
not be mobilized and could not form cointegrates with additional
conjugative plasmids; the only way to transfer the marker genes to the
recipient strain was via transformation. We did not detect mutants in
the various DNA-less and bacterium-less controls. This would indicate
that the spontaneous mutation rate remained below the detection limit.
We tested the quantity of transformants (1 to 1,000 CFU) that we could
recover in sterile soils containing 5 × 109
plasmidless bacteria under the same conditions as those in
transformation experiments. The method we used allowed the detection of
18 ± 15 CFU g of dry soil1 when 10 CFU was seeded
in the 30-g soil microcosms thanks to the enrichment step.
1. When the soils were
inoculated with a plasmid solution at 0.5 µg of DNA g of dry
soil1, transformants exhibiting resistance to specific
antibiotics and carrying plasmid pPZP111 (Fig.
1) were detected at frequencies reaching
1.3 × 108 (Table 2),
corresponding to 12 ± 15 transformants g of dry
soil1. We also carried out experiments in which the
recipient bacteria were coinoculated with donor cells of P. fluorescens AK15 containing the plasmid pPZP111. In such cases,
transformants were also detected (Fig. 1) at frequencies reaching
2.7 × 109 (Table 2), corresponding to 6.7 ± 13.3 transformants g of dry soil1.
|
|
1 after 4 days. When the soils were inoculated with
plasmid pKT230, transformants exhibiting resistance to kanamycin and
streptomycin and carrying the plasmid were detected (Fig.
2) at frequencies reaching 8.3 × 108, corresponding to 20 ± 16.3 transformants g of
dry soil1 (Table 2). We also carried out experiments in
which the recipient bacteria were coinoculated with donor cells
(E. coli containing the plasmid pKT230). In such cases,
transformants were also detected (Fig. 2) at frequencies reaching
5.8 × 108 (Table 2), corresponding to 33.3 ± 36.5 transformants g of dry soil1. E. coli and
P. fluorescens strains were identified by comparison of
restriction patterns from PCR-restriction fragment length polymorphism analysis conducted on 16S DNA with restriction enzyme AluI
(data not shown).
|
1 after 4 days in
soil, while the E. coli donor strain dropped to 3.2 × 105 ± 1.2 × 105 cells g of dry
soil1. Among colonies growing on the selective medium
(867 ± 76 cells g of dry soil1), some were identified as
P. fluorescens. This was assessed by the similarity of the
16S ribosomal restriction patterns of PCR products obtained from these
clones compared to those of the recipient strain (data not shown).
Moreover, these clones harbor an 11.9 kb plasmid, which exhibited
the restriction patterns corresponding to the donor plasmid pKT230
(Fig. 2). The presence of indigenous soil bacteria resistant to
the four selective antibiotics prevented us from determining the
precise transformation rate. However, the transformation rate in
nonsterile soil seems to be higher than that in sterile soils. We
cannot justify this observation, but we suggest that it may be due to
the presence of an organic compound in soil that is in part destroyed
by soil sterilization.
Detection of transformants in vitro. The two bacteria which exhibited evidence of transformation in soil, A. tumefaciens GMI9023 and P. fluorescens LP59JG, as well as E. coli 1504 as the negative control, were tested in vitro for their ability to take up, internalize, and express extracellular DNA.
When transformed with the plasmid pPZP111 harboring the resistance genes to kanamycin and chloramphenicol (Table 1), A. tumefaciens provided recombinant clones at frequencies reaching 6 × 109 (14 ± 10 transformants
ml1), while the detection limit remained at 1.6 × 1010 (Table 3). Moreover,
detection of plasmids of the expected size (11.9 kb) (Fig. 1) helped
confirm that a transformation event occurred, allowing the recipient
strain to take up and replicate the donor plasmid DNA.
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9 (Table 3). However, development of
competence in naturally transformable bacteria is known to depend upon
the chemical composition of the medium, with requirements varying
greatly. For some bacteria, specific nutrients are required to induce
this physiological state, while in other cases the presence of
particular substances inhibits the development of competence. For
instance, media with limited carbon and energy sources or deficient in
nitrogen and phosphorus have been found to favor the development of
competence in Pseudomonas stutzeri (15). On the
other hand, bacteria such as Acinetobacter sp. strain BD413
(14, 21) and Ralstonia solanacearum
(5) were transformable in media with totally different
requirements. In order to cover a wide range of conditions, we tested
various media described as promoting competence development in most of the naturally transformable bacteria. This included the use of the P
and the MM media favoring competence development in
Acinetobacter sp. strain BD413 and R. solanacearum, respectively, but also media characterized by a
specific deficiency to test whether inhibition of competence
development would be related to the presence of a specific substance.
Each medium was tested according to two protocols based on incubation
of bacteria on agar-solidified media and in liquid. Finally, we also
tested two soil-based media, including a rough medium containing the
whole soil sample in water and a medium containing only the soil
mineral fraction. None of the conditions tested enabled us to detect
transformants, which would have indicated that P. fluorescens can develop competence in vitro. In spite of our
efforts and the use of rich, soil-based or deficient media, we were
unable to determine whether an essential component for transformation
present in the soil would be missing in vitro or whether the media we
used contained an inhibiting product. Another hypothesis could involve
a rapid degradation of the transforming DNA by excretion of an active
nuclease before the cells became competent, as evidenced in
Bacillus subtilis (2). However, in spite of a
modification of the protocol to include two DNA inoculations separated
by a 12-h lag time (see Materials and Methods), transformants remained
undetectable. These data confirm that bacterial physiology remains
largely unknown with numerous specific limitations. In the case of
P. fluorescens, these limitations prevent the development of
a competence state in vitro, while for more than 99% of the indigenous
microflora it is the growth of colonies on petri dishes which is inhibited.
Transformation mechanism. A major question regarding these results is related to the mechanism by which bacteria can take up DNA. A first hypothesis deals with the presence of the molecular machinery permitting bacteria to develop a competence state. Tests should be conducted on bacteria belonging to the 99% nonculturable fraction of the soil microflora (28). Another hypothesis to explain the transformants we obtained with A. tumefaciens and P. fluorescens deals with a mechanism that would not be related to the well-defined natural transformation. E. coli transformation has been observed in various environments such as freshwater (4) and even foodstuffs (3). The naturally existing chemical (Ca2+ concentration) or physical (heat shock) parameters could provide E. coli with optimal conditions for a natural process such as transformation. Moreover, it has been hypothesized that induction of competence in E. coli would involve a physiological rather than a physicochemical mechanism (4). From our results, it can be supposed that soils, unlike other media, do not provide the required conditions for physiological or physicochemical transformation of E. coli. This could be due to the fact that E. coli is not a usual inhabitant of soils. When inoculated into nonsterile soil samples, the number of cells decreases rapidly, and so the competitive potential of E. coli becomes much lower than that of indigenous soil bacteria (23). However, the actual status leading to a physical, chemical, physiological, or genetic induction of competence in A. tumefaciens and P. fluorescens, which are natural inhabitants of soils, remains unknown. Additional experiments are necessary to determine whether the currently living strains of these bacteria are fitted with the genes involved in the development of competence, as observed in typical naturally competent bacteria, or whether they can take up DNA by other mechanisms. We can thus assume that the list of bacteria naturally capable of transformation is certainly longer than previously expected. Moreover, the fact that our findings revealed this property in well-known bacteria such as A. tumefaciens and P. fluorescens points out the interest of a more exhaustive study among available isolates, keeping in mind that development of competence was found to be species and even strain specific in bacteria (16).
Whatever the induction mechanisms operating in current bacterial strains, our results, based on experiments simulating natural conditions more closely than previous studies, demonstrate that transformation-mediated gene transfers can occur in soils. Whether such transformation-mediated gene exchange occurs at sufficiently high frequencies to contribute significantly to bacterial genome evolution remains to be investigated. However, factors such as the number of bacteria in the biosphere (5 × 1030 bacterial cells) (30), but also the time scale (3 billion years) will have to be considered carefully when tracking bacterial genome evolution and the related mechanisms.| |
ACKNOWLEDGMENTS |
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We are grateful to Stephane Peyrard for technical assistance.
This work was done as part of the Biotechnologies program supported by the Ministère Français de l'Enseignement Supérieur et de la Recherche (MENRT). S.D. and E.K. were funded by a grant from the MENRT.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire d'Écologie 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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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Applied and Environmental Microbiology, June 2001, p. 2617-2621, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2617-2621.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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