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Applied and Environmental Microbiology, December 1999, p. 5303-5306, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transformation of Escherichia coli with
DNA from Saccharomyces cerevisiae Cell Lysates
Ana Cristina
Adam,
Gracia
González-Blasco,
Marta
Rubio-Texeira,
and
Julio
Polaina*
Instituto de Agroquímica y
Tecnología de Alimentos, Consejo Superior de
Investigaciones Científicas, Apartado de Correos 73, E-46100 Burjassot, Valencia, Spain
Received 18 March 1999/Accepted 16 September 1999
 |
ABSTRACT |
We developed a system to monitor the transfer of heterologous DNA
from a genetically manipulated strain of Saccharomyces
cerevisiae to Escherichia coli. This system is based
on a yeast strain that carries multiple integrated copies of a
pUC-derived plasmid. The bacterial sequences are maintained in the
yeast genome by selectable markers for lactose
utilization. Lysates of the yeast strain were used to transform
E. coli. Transfer of DNA was measured by determining the
number of ampicillin-resistant E. coli clones. Our results show that transmission of the Ampr gene to E. coli by genetic transformation, caused by DNA released from the
yeast, occurs at a very low frequency (about 50 transformants per µg of DNA) under optimal conditions (a highly
competent host strain and a highly efficient transformation procedure).
These results suggest that under natural conditions, spontaneous
transmission of chromosomal genes from genetically modified organisms
is likely to be rare.
| |
INTRODUCTION |
The increasing use of
genetically modified organisms (GMOs) in biotechnology has
generated controversy concerning the safety of these organisms.
Specifically, whether recombinant sequences are transferred from hosts
to other organisms is of interest. Also, if such transfers occur, it is
necessary to determine by what mechanism and at what frequency, since
safety regulations require estimates of potential risk (10).
The flow of genes between species, which is termed horizontal transfer,
is well-documented (19). Therefore, transfer of genetic
material from GMOs to other organisms, which may lead to undesirable
effects, is possible. For example, in vivo transfer of an antibiotic
resistance gene from lactic acid bacteria to mouse intestinal bacteria
has been reported (9).
The mechanisms by which horizontal transfer can occur are conjugation,
transformation, and virus-mediated transduction (2, 11).
Conjugation, which can mediate gene exchange in virtually all
eubacteria, also can transfer genetic material from bacteria to yeasts
and plants (8, 17, 22). Transformation and transduction are
known to occur spontaneously only in bacteria. Although yeasts and
other eukaryotic organisms can be transformed, the conditions required
for this to occur are quite different from conditions found in the
natural environment.
The aim of this study was to determine the conditions under which and
the frequency at which heterologous DNA present in a recombinant
Saccharomyces cerevisiae strain could transform
Escherichia coli. The yeast S. cerevisiae is a
convenient experimental organism since it is widely used in the food
industry and has been well-characterized genetically. Transformation is
the most likely mechanism by which DNA can be transmitted from a
eukaryotic organism to bacteria. While transformation of E. coli by plasmids extracted from yeast cells is a routine
laboratory procedure, transfer of genes integrated into a chromosome
has not been extensively studied. We studied this process by
using a S. cerevisiae strain that carries multiple copies of a recombinant plasmid integrated into its genome.
| |
MATERIALS AND METHODS |
Strains, plasmids, and culture conditions.
The E. coli strain used in this work was strain DH5
(7).
Cultures of this strain were grown in Luria-Bertani (LB) medium at
37°C.
The S. cerevisiae strain used was strain MRY247. This strain
carries at the ribosomal DNA (rDNA) locus (RDN1 in
chromosome XII) multiple copies of two plasmids, pAA11 and pMR4
(1, 13) (Fig. 1). Plasmid
pAA11 (length, 9.4 kb) contains a complete copy of pUC18
(21), the defective leu2d gene of S. cerevisiae, the bglA gene encoding a 
-glucosidase
from Paenibacillus (Bacillus) polymyxa
under control of the S. cerevisiae CYC-GAL promoter, and a
fragment of rDNA from S. cerevisiae that targets the plasmid in the yeast genome (1).
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FIG. 1.
Physical structure of the RDN1 locus of
strain MRY247. The bars represent the tandemly repeated units of the
locus. The solid bars represent units of rDNA. The open bars represent
insertions of plasmid pAA11, whose genetic map is shown at the top. The
shaded bars represent insertions of plasmid pMR4, whose genetic map is
shown at the bottom.
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|
The
-glucosidase of
P. polymyxa has some
-galactosidase activity, which allows it to cleave lactose
(
15).
S. cerevisiae strains expressing the
bglA gene can assimilate lactose if they
have permease
activity for sugar uptake. Plasmid pMR4 (length,
9.6 kb) contains the
leu2d gene, pUC18, and the
Saccharomyces rDNA
fragment (as in pAA11). It contains the
LAC12 gene encoding
the lactose permease from
Kluyveromyces lactis under control
of
the
CYC-GAL promoter (
13). Growth in a medium
containing lactose
as the carbon source selects for lactase and lactose
permease
functions supplied by the integrated plasmids. The lactase
activity
of the
blgA -glucosidase is quite low. The gene
must be overexpressed
for the host yeast to grow on lactose. The
permease activity provided
by pMR4, however, is not required at such
high levels. Thus, many
copies of pAA11 are present (ca. 200 copies per
genome), whereas
there are fewer copies of pMR4 (ca. 20 copies per
genome).
Yeast cultures were grown at 30°C in standard media prepared as
described by Sherman et al. (
16) with either glucose or
lactose as the carbon source. Minimal media were supplemented
with the
necessary auxotrophic
compounds.
Plasmid pUC18 DNA (
21) was used as a control for
E. coli transformation.
Preparation of transforming DNA.
We used cell lysates of
S. cerevisiae MRY247 as the source of transforming DNA.
Cultures of this strain were grown overnight in liquid YPD medium. The
cells were collected by centrifugation (4,000 × g, 15 min), washed with TE buffer (10 mM Tris, 1 mM EDTA; pH 7.5), and
resuspended in 4% of the original volume of the same buffer. The cells
were broken either by mechanical disruption or by chemical lysis.
Mechanical disruption was accomplished by vortexing a cell suspension
in the presence of 0.25 g of glass beads (diameter, 425 to 600 µm; Sigma Chemical Co., St. Louis, Mo.). The cells were vortexed for
1 min and then placed on ice for another 1 min to avoid excessive
heating. This process was repeated several times. Cell disruption was
monitored by visual inspection with a microscope. Disruption was
considered sufficient when a majority of the cells (ca. 70 to 80%)
were broken. Cell debris was removed by centrifugation
(13,000 × g, 15 min), and the supernatant was used in
transformation experiments. Alternatively, cell extracts were prepared
by chemical lysis by using the fast procedure for yeast DNA
purification of Polaina and Adam (12), as modified by
Rubio-Texeira et al. (13). The DNA concentrations in the
extracts prepared by the two procedures were 10 to 50 µg/ml. These
extracts were used in transformation experiments either directly or
after precipitation of the DNA with ethanol and resuspension in 10% of
the original volume.
DNA obtained from the yeast lysates was cut with excess
HindIII (0.5 µg of MRY247 DNA and 20 U of enzyme in a
20-µl [final
volume] mixture). The effectiveness of the digestion
procedure
was monitored by gel electrophoresis. Ligation of
HindIII-digested
DNA was carried out with 0.5 µg of
DNA and 1 U of T4 DNA ligase
(Amersham Iberica, Madrid, Spain) in 30 µl of 1× ligation buffer.
After overnight incubation at 16°C, the
DNA was ethanol precipitated
and resuspended in TE buffer at a final
concentration of 50 µg/ml.
Transformation of E. coli.
Transformation by DNA
from cell lysates of S. cerevisiae was assayed by using
competent cells of E. coli DH5 prepared by the following
two procedures: treatment with rubidium chloride (RbCl) (6)
and electroporation (5, 20) with a Gene Pulser electroporation system (Bio-Rad Laboratories, Hercules, Calif.). Ten-microliter aliquots of the yeast lysate were mixed with 100 µl of
a suspension of RbCl-treated competent E. coli cells
containing ca. 109 cells/ml. Alternatively, 1 µl of cell
extract, or the same volume of concentrated DNA was mixed with 40 µl
of electrocompetent cells. After the DNA was mixed with the E. coli cells, transformation was completed by using standard
procedures (5, 7, 20), and the cells were plated onto LB
medium supplemented with ampicillin in order to score the formation of
transformant (Ampr) colonies.
HindIII-digested DNA (25 µg/ml) and DNA treated with
ligase (50 µg/ml) were used to transform
E. coli; 10- and
1-µl aliquots
of this material were used for the RbCl and
electroporation procedures,
respectively.
Other molecular biology procedures were carried out as described by
Sambrook et al. (
14).
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RESULTS |
Gene transfer from disrupted yeast cells to E. coli.
MRY274 lysates prepared by either mechanical or
chemical cell disruption and concentrated DNA from the lysates
(fragments more than 20 kb long) were mixed with untreated cells from
bacterial cultures. No transformation was observed regardless of the
amount of DNA used (up to 50 µg/ml) or the physiological state
of the culture (exponential- or stationary-phase cells grown in LB
medium). Therefore, we assayed transformation of highly competent
E. coli cells. Several experiments were carried out
with cell extracts containing different concentrations of DNA (10 to
500 µg of DNA/ml) prepared by mechanical cell disruption and
different transformation procedures (RbCl or electroporation). No
transformants were obtained in these experiments. A similar set of
experiments was carried out with extracts prepared by chemical
cell disruption. In this case, transformation was observed when high
concentrations of DNA (100 to 500 µg/ml) were used and the DNA was
introduced by electroporation (Table 1).
Preparation of cell extracts by the chemical procedure involved partial
deproteinization and yielded cleaner DNA. This probably resulted in
more efficient transformation.
The disrupted yeast cells released high-molecular-weight DNA. We
digested the cell extracts with
HindIII, an endonuclease
that cuts pUC18 once. The digested DNA (5 to 10 kb; 10 to 50 mg/ml)
was
used to transform competent
E. coli cells, but no
transformants
were
recovered.
Circular plasmids are much more efficient for transformation than
linear forms are (
4). Therefore, we circularized the
linear
fragments generated with
HindIII by treating them with
DNA ligase, and the transformation frequencies increased (Table
1).
This result shows that functional bacterial DNA that could
be
rearranged to transform
E. coli was present in the
recombinant
yeast.
Analysis of plasmid DNA recovered from E. coli
transformants.
We analyzed plasmids recovered from the
Ampr transformants (Table 1 and Fig.
2). The physical maps of plasmids pAA11
and pMR4 are similar, but these two plasmids can be distinguished by
two characteristic restriction sites (an XbaI site in pMR4
and an SmaI site in pAA11). All of the plasmids recovered
could have arisen from pAA11, which may be attributable to the
differences in the number of plasmid copies in MRY247.
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FIG. 2.
Physical maps of pMR4, pAA11, and plasmids recovered
from E. coli clones transformed by DNA of S. cerevisiae MRY247. (A and B) Plasmids recovered from clones
transformed by untreated DNA. (C through G) Plasmids recovered from
clones transformed by DNA treated with HindIII and
ligase. Only restriction sites relevant for characterization of the
plasmids are shown. Open and solid arrowheads indicate restriction
sites that are critical for identification (XbaI in pMR4 and
SmaI in pAA11, respectively). The solid bars in maps B, E,
F, and G represent DNA regions that were not characterized.
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The majority of the plasmids isolated from transformants obtained with
untreated DNA (8 of 10 plasmids) exhibited the same
restriction pattern
as pAA11 (Fig.
2A). The physical maps of the
remaining two plasmids
could have originated by a deletion event
(Fig.
2B).
When the maps of plasmids isolated from transformants obtained with DNA
digested with
HindIII and treated with ligase were
analyzed, a wider range of patterns was observed. Of the 34 transformants
analyzed, the members of the largest group (26 transformants)
had plasmids whose maps corresponded totally (7 transformants)
or partially (19 transformants) to the map of
plasmid pAA11 (Fig.
2C and D). A second group, consisting of six
clones, contained
plasmids whose maps corresponded to the map of
pAA11 plus a large
insertion (Fig.
2E). Finally, two transformants
carried plasmids
whose maps bore no resemblance to any of the other
maps (Fig.
2F and
G).
| |
DISCUSSION |
Our results suggest that spontaneous transfer of
chromosome-integrated sequences is a rare event. Although E. coli can develop natural competence (3), the conditions
under which we detected transformation (high concentration of DNA, high
degree of cell competence, and use of electroporation) are very
different from the conditions found in natural habitats. In addition,
our results agree with the general observation that transformation is a
rather inefficient mechanism of exchange for nonplasmidic genes
(18). The increase in transformation frequency observed
after DNA circularization supports safety regulations that prohibit
episomal plasmids in commercial GMOs.
When E. coli is transformed with a linear plasmid, the
majority of the transformants contain perfectly recircularized
molecules of the plasmid (4). The genome of MRY247 has
multiple copies of pAA11 in a tandem array. Therefore, linear fragments
of DNA from MRY247 extracts able to transform E. coli give rise to circular plasmids with the physical pattern of
pAA11. An interesting possibility is the existence in the yeast nucleus
of circular molecules of pAA11 generated as loop-outs by homologous
recombination. Digestion of MRY247 DNA with
HindIII followed by ligation leads to the formation of
plasmid molecules whose maps are shown in Fig. 2C and D. As expected,
these patterns are the ones most frequently recovered. Ligation of
heterogeneous DNA fragments and in vivo recombination events explain
the generation of the other forms isolated.
Even though horizontal transfer of chromosome-integrated sequences is
rare, our results indicate that under certain circumstances it is
possible. The nature of the plasmids recovered from E. coli, which have a bacterial origin of replication and a gene that confers a
selective advantage under certain circumstances, means that a single
transfer event could change an entire population and lead to
unpredictable consequences. Therefore, it is advisable to prevent the
presence in GMOs of genetic elements that could help spread recombinant
DNA sequences.
| |
ACKNOWLEDGMENT |
This work was supported by grant ALI-0362-97 from CICYT.
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FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Agroquímica y Tecnología de Alimentos, Consejo Superior
de Investigaciones Científicas, Apartado de Correos 73, E-46100 Burjassot, Valencia, Spain. Phone: 34-963 90 00 22. Fax: 34-963 63 63 01. E-mail: jpolaina{at}iata.csic.es.
Present address: Kimmel Cancer Institute, Jefferson Medical
College, Philadelphia, PA 19107.
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Applied and Environmental Microbiology, December 1999, p. 5303-5306, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
