Note: Descriptions are shown in the official language in which they were submitted.
13~ S
Case 130-4004
I~PROVEHENTS IN OR RELATING TO ORGANIC SYST~S
The present invention relates to a method of transforming
Bacillus thuringiensis cells.
The terms "transforming" and "transformation", as used herein are
intended to relate to a mechanism of genetic transfer whereby
exogenous DNA is introduced in a recipient bacterium, thereby inducing
genetic changes in said recipient bacterium.
Bacillus thuringiensis (BT) are gram-positive bacteria containing
a crystal protein, the delta-endotoxin (DET) which is toxic to the
larvae of a number of insects. Depending on the sub-species, BT is
used as a selective biological pesticide against different pests. The
sub-species thuringiensis, alesti and dendrolimus are for example
pathogenic against Lepidoptera: the sub-species israelensis,
darmstadiensis 73-E-10-2, kyushuensis and morrisoni PG14 against
Diptera; the sub-species tenebrionis against Coleoptera; the
sub-species kurstaki HD-l, kenyae, aizawai and colmeri against
Lepidoptera and Diptera, whereas the sub-species dakota, indiana,
tokokuensis and kumamotoensis are not known to be toxic to any pests.
From the industrial and ecological point of vlew lt ls deslrable
to have additlonal biological pesticides with different e.g. higher or
broader spectrwn of activity.
This aim can, for example, be achieved by the development of new
isolates from nature, by conjugatlon of bacteria or by transformation
of bacteria.
Thus new BT strains with interestlng actlvity have been isolated
recently (e.g. var. tenebrionis with activity against beetles) and
recent successes with regard to the conjugation of BT strains have
been reported as well.
Transformation of bacteria has the advantage that, if successful,
it allows the introduction of specific genetic information into
bacteria.
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Thus a gene coding for a DET has been cloned in various micro-
organisms such as Escherichia coli, Bacillus subtilis and Pseudomonas
fluorescens and even in higher plants (tobacco) by recombinant DNA
techniques, more specifically by transformation.
Such genetically manipulated organisms produce however low
amounts of DET compared to the amounts produced by natural BT strains.
The commercial value of such organisms is accordingly questionable, at
least as long as no way has been found to improve the expression of
the exogenous DNA encoding for DET.
It would accordingly appear indicated to try and obtain a better
expression of exogenous genes (DNA) by using a BT bacterium as
recipient bacterium in transformation techniques.
Known transformation techniques are essentially effected
employing either cells or protoplasts.
The transformation of cells implies the presence of competent
cells, i.e. cells in a precise physiological stage allowing binding
and uptake of exogenous DNA. There is however no evidence for the
existence of competent BT cells.
The transformation of BT protoplasts by DNA has been reported to
succeed only in very low yields, i.e. substantially lower than those
obtained with the transformation of B. subtilis. The low yields may be
partly due to the poor regeneration of the protoplasts, including the
transformed protoplast. Although Shall et al. (Fundamental and applied
aspects of invertebrate Pathology, edited by R.A. Samson, J.M. Vlak
and D. Peters, 1986, page 402) report that they optimized the
protoplasting procedure and developed improved regeneration media to
transform BT or B. cereus with plasmid DNA, they do not specify the
nature of the optimization or improvement.
The transformation frequencies indicated by Shall et al. are
accordingly difficult to interprete.
The present invention now provides an improved method of
transforming BT. It is based on the finding that BT microorganisms
develop a so-called competence status when they are introduced in a
hypertonic aqueous medium.
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The term hypertonic as used herein refers to a medium which is
hypertonic vis a vis the conventional BT (cell culture or growth)
media.
The method of the invention involves the steps of
a) growing BT cells in a hypertonic aqueous medium
b) introducing in the cell culture obtained by step a)
and in the presence of polyethylene glycol, exogenous DNA while
maintaining the hypertonic status, and
c) isolating and resuspending the thus treated BT cells in hypertonic
aqueous medium to allow expression.
In principle any compound which does not pass the semi-permeable
cell membrane and is not metabolized by or toxic to BT cell may be
employed to obtain the desired hypertonic status. In general, the
desired hypertonic status will conveniently be achieved with the aid
of saccharides, particularly mono- or disaccharides, which are not
metabolized by BT. Suitable examples of such saccharides are sucrose
and lactose.
The concentration of saccharides to be employed to achieved the
desired hypertonic status is conveniently of the order of 0.4 M
saccharide per litre of aqueous medium or higher. In general, good
results will be obtained with concentrations which are essentially
isotonic with respect to the BT cytoplasm. Such osmotic status is in
general obtained with a concentration of from 0.4 M to 0.5 M of
saccharides per litre of aqueous medium. Hlgher saccharides
concentrations may however be employed, but offer in general no
advantages.
The term "hypertonic" employed hereinafter refers to a status or
medium as specifled hereinbefore.
It is important that the hypertoni~ conditions are essentially
maintained throughout the various steps a) to c) of the process.
The hypertonic aqueous media should be essentially neutral, i.e.
they should conveniently have a pH of 7 + 2, more preferably of
7 + 1.
In addition to the saccharides (to maintain the hypertonic
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status) and eventually buffers (to maintain an essentially neutral
status of the medium) other ingredients may and will be added e.g. to
allow growth and development of the BT culture when required, etc.
Such additional ingredients are conventional and known by those
skilled in the art, they comprise e.g. nutrients and salts.
Examples of suitable nutrients are e.g. beef extract, yeast
extract, peptones, tryptones, amino acids (e.g. tryptophan),
nucleosides such as thymidine and the like.
Examples of suitable salts are NaCl and MgC12.6H20. A suitable
hypertonic medium may contain from 0.05 to 0.1 M of salts per litre.
The salts wil comprise preferably magnesium salts, such as MgC12.6H20.
The BT cell culture (starting material) will conveniently be
prepared and grown under conventional conditions, i.e. with aeration
and at ambient temperature, in an appropriate nutrient medium, e.g. in
the minimal medium disclosed by J. Spizizen in Proc. natl. Acad. Sci.
(Wash) 44, 171-175 (1958), eventually supplemented with amino acids,
salts, e.g. catalytic amounts of a manganese salt such as MnS04, etc.
It is advantageous to employ in step a) a BT cell culture which is in
the exponential growth phase.
The freshly prepared BT cell culture is then diluted in a
hypertonic medium to a starting cell concentration of substantially
less then 109 cells per ml, e.g. of 104 to 106 cells per ml and the
cell culture is grown, in said hypertonic medium up until a cell
concentration of slightly less than 109 cells per ml, e.g. 108 to
5.108 cells per ml medium i9 obtained-
The hypertonic medium employed to dilute the freshly prepared BTcell culture is conveniently at 20 to 40C, e.g. at 37C. The culture
is then allowed to grow at this temperature. Thorough aeration should
of course be ascertained. A slight amount of silicon is conveniently
added to the cell culture medium to prevent foaming.
When the desired final cell concentration (o slightly less than
109 cells per ml) is reached, the thus prepared competent BT cells may
be treated with DNA in the presence of polyethylene glycol (PEG),
according to step b) of the process of the invention.
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It is however advantageous to treat the competent BT cells,
obtained according to step a) of the invention, with moderate
concentrations of lysozyme in hypertonic medium, and to isolate and
resuspend the lysozyme treated BT cells in hypertonic medium, before
subjecting them to process b). The amount of lysozyme to be employed
should be less than that normally used for the preparation of
protoplasts. Such amount (concentration) will of course depend on
various factors such as the osmotic pressure of the medium, its
temperature, the desired reaction time etc. In general a suitable
lysozyme concentration is of 20 to 300 microgram, e.g. of 200
microgram per ml of hypertonic aqueous medium (which is substantially
lower than the 2 to 15 mg per ml which would be normally required for
protoplasting purposes). Adequate distribution of lysozyme in the cell
culture medium should be ascertained. The reaction time will i.a.
depend on the concentration and the quality of the lysozyme
solution employed. The optimum reaction time may be determined by
standard assays.
The reaction temperature is conveniently between 20 to 40C,
preferably above room temperature, e.g. at about 37~C.
During the lysozyme treatment the hypertonic status, as specified
above, should be maintained.
The treatment with lysozyme is then terminated by centrifugation
of the cell suspension and resuspension of the pellet in hypertonic
medium, conveniently at room temperature.
The thus prepared 3T cell culture - obtalned according to step
a), optionally followed by treatment with lysozyme - is then treated
with DNA, e.g. plasmid DNA, in the presence of polyethylene glycol
(PEG). For that purpose, the DNA as well as the PEG are employed as
suspensions/solutions in a hypertonic solutions, such that the osmotic
pressure of the cell suspension remains essentially unchanged after
addition of DNA and PEG to said cell suspension.
The amount of PEG employed will be conveniently selected such
that its concentration in the BT cell culture lies within the range of
from lOOg to 400g per litre, e.g. at 300~ per litre cell culture medium.
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The transformation step b) can essentially be effected under the
conditions known to be appropriate for conventional protoplast
transformation processes.
Accordingly, the selection of the appropriate amount and type of
PEG and of the appropriate amount of DNA to be employed can
conveniently be made by those skilled in the art of protoplast
transformation.
Thus, an example of PEG suitable for use in this process is PEG
6000.
DNA amounts of from lO0 nanogram to 20 microgram per 108 to 109
BT cells will in general allow good results.
The incubation is convenien~ly effected with gentle mixing at
room temperature. Tlle required incubation time is short, in general of
the order of a few minutes (see the example).
The suspension comprising the transformed cells is then worked up
employing conventional methods but while securing the hypertonic
status of the solvent of the cells (when in solution/suspension). Thus
the suspension is for example diluted with hypertonic solution, the
suspension mixed, centrifuged and the pellet resuspended in hypertonic
medium.
The resulting suspension is then incubated at a temperature of 20
to 40C, e.g. at 37C, to allow expression. The suspension is
conveniently aerated, employing e.g. a shakin~ water bath. An
appropriate incubation time ls 30 nlit1utes to .~ hours, more preferably
between 2 to 4 hours, e.g. 3 hours.
Appropriate dilutions of the thus obtained cèll cultures may then
be placed on culture plates for determination of colony forming units
(CFU). The transformation frequency may be determined by known methods
employing standard techniques such as antibiotic containing culture
plates, visual observation etc.
The method of the present invention allows the transformation of
BT cells in high yields. Transformation allows gene cloning of genomic
libraries in BT cells, cloning and expression of DET genes in BT,
cloning and expression of in vitro and in vivo modified DET genes in
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BT, the synthesis of useful polypeptides, etc.
Where the transformed BT cells are intended for use as biological
pesticides they are conveniently employed in insecticidal composition
form, e.g. in suspension concentrate form or powder form. Such
compositions may be obtained in conventional manner.
In the following non-limitative example the starting materials
(BT cells and plasmid DNA) were selected such that the results are
unambigous and cannot be due to plasmid interaction; the BT cells used
as starting material did not contain plasmids, the plasmid DNA used as
transforming agent encodes for resistance against tetra-
cycline.
It will be appreciated that other BT cells and/or exogenous DNA,
particularly plasmid DNA may be used in the method of the invention
with similar results.
Temperatures are in centigrade and parts by weight unless
specified otherwise.
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EXAHPLE
Star_ing Materials
Strain : Bacillus thuringiensis subsp. kurstaki HDl cry B, (obtained
from M.-M. Lecadet, Institut Pasteur, Paris) having no
plasmids.
DNA : pBC16.1 (Kraft. J. et al. (1978) Molec. gen. Genet. 162 :
59-67) extracted from HDl cry B (pBC16.1), in which it was
introduced by conjugation via cell mating with B. subtilis
BD224 (pBC16.1), coding for tetracycline resistance.
Media
SA Trp : Spizizen minimal medium (Spizizen J. (1958) Proc. natl. Acad.
Scl (Wash.) 44 : 171-175) supplemented with 1% Casamino
acids (Difco), 5xlO 6 M MnS04 and 20 ~g/ml Tryptophan.
Hypetonic medium (HM) :
Beef Extract1.50 g/l
Peptone 5.00 g/l
NaCl 3.50 g/l
Sucrose171.15 g/l
Maleic Acid2.32 g/l
gC12 . 6H204.07 g/l
pH 6.7
Luria Medium ~A? :
Tryptone 10 g/l
Yeast Extract 5 g/l
NaCl 10 g/l
Agar (Difco Bacto) 15 g/l
Thymidine20 mg/l
Antibiotics : Tetracycline, 10-100 ~g/ml in LA plates
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Solutions
SMM : Sucrose 171.15 g/l
Maleic Acid 2.32 g/l
gC12 . 6H204.07 g/l
pH 6.5
PEG . PEG 6'000 40 g
SMM ad 100 ml
Lysozyme : 2 mg/ml in HM, freshly prepared.
Method
An overnight culture of HDl cry B is prepared in 15 ml of SA Trp
and grown with aeration at 20C. The following morning, the culture is
diluted 50-100 x in prewarmed HM medium to a starting cell concen-
tration of 7.5 x 105 / ml. Silicon (2 ~1) is added to prevent foaming.
The culture is grown at 37C with moderate aeration for 3h 30 min.,
i.e. to a cell concentration of 2.5 x 108 _ 3 x 108/ml. Lysozyme is
added to a final concentration of 200 ug/ml and 1 ml of cell
suspension is incubated for 30 min. at 37C in a shaking water bath
(150 rpm). The cell suspension is then centrifuged 1 min. at 10'000 g
and the pellet is resuspended in 1 ml fresh HM at room temperature.
0.5 ml cell suspension is added to 50 ~1 SMM to which 100 ng-10
~g of plasmid DNA have been added. The cells are transformed by
addition of 1.5 ml of PEG solution, gentle mixing and a 2 min.
incubation at room temperature. 5 ml of HM is added to the cell
suspension, which is gently but thoroughly mixed snd centrifuged for
20 min. at 3'000 g. The pellet i9 re~uspensed in 0.6 ml of HM and
incubated 3h. at 37C in a shaking water bath (150 rpm) to allow
expression. Appropriate dilutions are plated on LA plates for CFU
determination and on Tetracycline-containing LA plates for
transformant selection.
1-2 x 103 transformants per ~g of intact plasmid DNA, with a
frequency of 5 x 10 5 - 10 4 are obtained.
