Note: Descriptions are shown in the official language in which they were submitted.
20491l3!3
Pvs-l8l99lA
RESTRICTION-DE~ICIENT MUTANT
The present invention relates to partially restriction-deficient mutants of Bacfllus
thuringiensis and Bacillus cereus and to methods for the preparation of said mutants. The
invention relates also to methods of overcoming restriction barriers in Bacillusthuringiensis and/or Bacillus cereus using said restriction-deficient mutants.
Reslriction/modification systems are very common in microorganisms and have beenknown for a long time. More than 600 restriction enzymes and approximately 100 of the
associated methylases have now been described in the scientific literature [Kessler &
HBltke (1986)~.
By far the greatest number of restriction/modification systems described hitherto belong to
the so-called class II systems. These class II systems are characterised by their low degree
of complexity and accordingly use relatively simple proteins. A class II restriction
enzyme, for example, is capable of recognising and of cleaving a specific DNA sequence,
provided that the latter has not already been specifically methylated by the associated
methylase. Neither enzyme requires ATP. The genes coding for these enzymes
[methylases] frequently lie closely coupled adjacent to one another on the bacterial
genome.
The few known restriction/modification systems of the more complex classes I and III
originate from Escherichia coli, Salmonella and Haemophilus [Kessler cec Holtke (1986)].
In addidon to these "classic" systems there have been found in recent years, first in E. coli,
and then in other species, restricdon enzymes that, in a reversal of the situadnn with the
class II systems, recognise and cleave a DNA sequence only when that system has been
speci~lcally methylated [Raleigh & Wilson (1986); Sladek et al (1986) and MacNeil
(1988)].
20491~3
The classic restriction enzymes of class II are one of the most important tools in rDNA
technology and therefore constitute one of the basic essentials of modern molecular
genetics. Their primary application is in the construction of recombinant DNA molecules.
Their natural function in the cell, however, is to defend against undesired foreign DNA,
for example viral or bacterial DNA. Because of this natural function, which is based on
the degradation of foreign DNA that has penetrated into the cell, some drastically reduced
transformation frequencies have to be expected when working with recombinant DNA.
This is referred to in terms of the presence of so-called restricdon barriers [Matsushima et
al (1987); Miller et al (1988) and McDonald & Burke, (1984)]. Even slight restriction
barriers can be sufficient substantially to prevent the construction of a representative gene
bank, since the restriction effect increases as the size of the recombinant plasmid
increases. When there is restriction, therefore, fully intact genes can be isolated only with
great difficulty. In the case of B. sub~ilis, for example, it has been shown that the
restriction enzyme BsuM occurring in that strain [Uozumi et al (1977); Hoshino et al
(1980) and Bron et al (1988)] is one of the main causes of the structural instability of
recombinant plasmids in that organism [Haima et al (1987)].
Overcoming the restriction barriers brought about by the restriction enzymes of class II
has therefore been, and continues to be, one of the main problems to be solved in
molecular genetics. This can be achieved today in several ways.
One possible approach is to use non-restricting intermediate hosts, DNA of which can be
transferred into the actual host without restriction. This approach is suitable, for example,
for methylation-specific restriction systems. There may be used as the intermediate host,
for example, a non-methylating mutant of E. coli or a naturally methyladon-deficient
strain [MacNeil (1988); European Patent No. 0 341 776 A2]. This approach is, however,
rather complicated and has the additional disadvantage that these methylation-deficient
E. coli strains are generally difficult to transform and are in no way ideal host strains.
In a further approach, heat-labile restriction enzymes are temporarily inactivated in vivo
[Bailey & Winstanley (1986); Engel (1987)]. This approach is limited by the relative
heat-sensitivities of the host and the restriction enzymes.
A further possible means of overcoming existing restriction barriers is the speci~lc
methylation of the DNA used. If the specific methylase for a certain restriction system is
3 ;~:049~.~3, 3
known and has been purified, the DNA can be specifically methylated in vitro before
transformation and thus be protected against restriction (Vehmaanpera, 1988). In a
situation where the corresponding methylase gene has been cloned, it is possible in
principle for the DNA that is to be transformed to be methylated directly in vivo in a
suitable host.
The solution that is probably the most satisfactory, and therefore the preferred solution, for
the genetic engineer is, however, the isolation of mutants that do not synthesise the
restriction enzymes responsible for the restriction barriers. Most of the E. coli K strains
used as host strains carry, for example, a mutation in the gene for EcoK, an enzyme of
class I. Since DNA of higher eukaryotes is generally strongly methylated, recently E. coli
strains have been constructed that additionally carry mutations for the methylation-
specific restricdon systems mcrA, mcrB and mrr [Kretz et al (1989)]. This approach is
difficult in strains that have a relatively large number of restriction systems, for example
Streptomycesfradiae [Matsushima e~ al (1987)].
Restriction barriers are not, however, reslricted to E.coli, but are to be found also in a
large number of other microorganisms that are used for cloning experiments.
In B. thuringiensis and B. cereus, restriction/modification systems are very little known as
yet, since it is only relatively recently that it has been possible to transform these Bacillus
species efficiently (Schurter et al. 1989) and there has therefore been only a limited
amount of interest in studying restriction/modification systems in those organisms.
Accordingly, restriction barriers have hitherto been mentioned only in passing in
connection with B. thuringiensis and B. cereus. Azizbekjan et al (1983), for example,
describe an AvaII isoschizomer of B. thuringiensis var. israelensis.
With the newly created means for the efficient transforrnation of B. thuringiensis and/or
B. cereus and hence for the use of those organisms as cloning vehicles, however, these
restriction barriers have become a problem also in the case of B. thuringiensis and
B. cereus.
If, for example, a shuttle vector that is capable of replication both in B. thuringiensis and
B. subtilis and in E. coli is used for the electroporation of B. thuringiensis strain ~IDlcryB
[Stahly et al tl978)], a crystal-body-free derivative of B. thuringiensis var. kurstaki ~)1,
the transformation frequency achieved depends primarily on the strain from which the
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plasmid DNA was isolated. The absolute values obtained in each case may vary, but the
relative frequencies remain substantially constant.
A representative example of these differences in the transformation frequencies is shown
in Table 1. The results show that, for example, the transformation of the 'shuttle' vector
pHY300PLK is poorer by a factor of at least 103 when the plasmid is isolated from an
E. coli host rather than a B. fhuringiensis host. If the original strain is B. sub~ilis, the
transforrnation frequency is reduced by a factor of approximately 10. If, on the other hand,
the plasmid DNA is reisolated from the B. thuringiensis strain HDlcryB, it can be
transformed back into HDlcryB at a high frequency.
This restricdon effect undergoes a further increase if additional DNA is cloned into the
shuttle vector, for example in the form of a protoxin gene. In this case the restriction
barrier is found to have increased markedly again in the case of transformation into a
B. thuringiensis host [see Tab. 4, pXI204, pXI93]. The reason for this is presumably that
the DNA additionally integrated into the vector has further restriction cleavage sites,
which repreænt an additional site of attack for the postulated host-specific [HDlcryB]
enzymes. These restriction barriers can reach a value of approximately 104 - 105x, which
makes the probability of a successful transformation of HDlcryB using these shuttle
vectors very low.
In this case also the said plasmids can be transformed "normally" into HDlcryB only if
they have previously been isolated from HDlcryB. Very often, however, it is desirable to
clone DNA in a heterologous intermediate host, which, however, in many cases is
incompatible or only slightly compatible with the restriction system present in B. thurin-
giensis and/or B. cereus. Transformation of B. thuringiensis and/or B. cereus with vector
DNA that has previously been isolated from such a heterologous, non-compatible inter-
mediate host then often leads to resu1ts that are not very satisfactory.
The problem that was to be solved within the context of the present invention was
therefore primarily to identify and to characterise restriction barriers in B. thuringiensis
and/or B. cereus and to develop methods of overcoming those barriers. This problem has
now been solved within the context of this invention, surprisingly, by making available a
partially restriction-deficient mutant of B. thuringiensis, as can be seen in detail from the
following detailed description.
2049~
The present invention therefore relates especially to a mutant of B. ~huringiensis and/or of
B. cereus that has a partial deficiency of the restriction barriers inherent in the unmutated
starting strain and that may therefore be transformed significantly better than the said
non-mutated starting strain when vector DNA fiom a heterologous intermediate host that
is naturally subject to restriction in B. thuringiensis and/or B. cereus is used.
Within the context of this invention, a heterologous intermediate host is to be understood
as being a host organism that is suitable for the cloning of vector DNA and that is not
identical at least with the B. thuringiensis and/or B. cereus strain from which the DNA to
be cloned was originally isolated.
The invention relates especially to a partially restriction-deficient mutane of
B. thuringiensis and/or B. cereus which, when shuttle vectors from a non-compatible
intermediate host, such as E. coli and/or B. subtilis, are used, may be transformed
significant1y better than the non-mutated starting strain, preferably by a factor of > 102 and
especially preferably by a factor of 102-104.
Within the context of this invention there is to be understood by a non-compatible
intermediate host a host organism that has not developed any mechanisms for effectively
protecting its DNA against restriction after transformation into a B. thuringiensis or B.
cereus strain having a restriction system disclosed within the context of the present
invention.
An especially preferred embodiment of the present invention is a partially
restriction-deficient mutant of the B. thuringiensis strain HDlcryB which, when shuttle
vectors from E. coli and/or B. subtilis are used, exhibits a transformability that is better
than that of the non-mutated starting strain by a factor of 2102, and especially by a factor
of 102-104, depending on the particular transformation vector used.
Special preference is given wi~in the context of the present invention to the partially
restriction-deficient mutant B. thuringiensis var. kurstaki HDlcryB Res9, which is
obtainable by means of spontaneous mutation from the B.t. strain HDlcryB and can be
obtained by means of enrichment methods known per se, and to mutants and variants
thereof that are derived directly from that strain and that still have the distinguishing
restriction-reducing characteristics of the stalting strain.
20~9
- 6-
The present invention relates also to the methods of preparing the said restriction-deficient
mutants.
Preference is given to a method wherein, essentially, spontaneous mutants having a
reduced restriction barrier are enriched by means of a series of several transformation and
selection cycles, there preferably being used for the transformation shutde vectors that
code for different selection markers and that guarantee a sufficiently high rate of trans-
formation, preferably a transformation rate of from 106 to 108 transformants, and those
mutants are selected that, in addition to having a reduced restriction barrier, still exhibit a
high degree of transformation efficiency.
The invention relates further to methods of reducing restriction barriers in B. thuringiensis
and/or B. cereus using restriction-negative mutants, especially using the restriction-
negative mutant B. fhuringiensis HD lcryB Res9.
The invention relates also to a method of reducing restriction barriers in B. thuringiensis
and/or B. cereus, wherein a restTiction-negative mutant, especially the restricdon-negative
mutant B. thuringiensis HDlcryB Res9, is used in combination with a specific methylase
which, by methylating the inserted DNA, protects the latter from being digested by
restriction enzymes inherent in B.thuringiensis and/or B. cereus and thus further increases
the efficiency of the method.
As defined within the context of the present invention, a restriction-negative or
restriction-deficient mutant is one that may be better transformed, by a factor of 2 102, by
a shuttle vector that has been isolated from E. coli than the unmutated starting strain.
However, spontaneous mutations generally occur very rarely (10-6-10-8) in the case of
B. thuringiensis and B. cereus, so that these rare mutants first have to be enriched by one
of the known enriching methods. This can be achieved, preferably, by passing through
several, preferably from 1 to 8, cycles consisting of transformation and selection, the most
promising transforrnants being selected at the end of each cycle and used again in the next
transformation/selection cycle. Once one of these rare mutants has been transformed,
enrichment by a factor of 102 - 103 can be expected in each new cycle consisting of
transformation/selection, that is to say, after only a few cycles the cell population should
comprise a majority of mutated cells.
Since spontaneous mutants are rare (10-6 10-8), and therefore the probability that one of
S3
- 7 -
those mutants will be transformed is correspondingly low, the transformation conditions
must be so selected that high rates of transformation, preferably transformation rates of at
least 106-lOg, are achieved. This applies especially to the ~lrst two transformation cycles.
These positive transformants can then be enriched, identified and finally isolated in further
transformation/selecdon cycles.
The particular difficulty that the isolation of restriction-deficient mutants entails is
avoiding the occuIrence of undesired mutants.
Thus, for example, the enrichment of spontaneous, generally chromosomally coded
resistance mutants can be avoided or at least kept as low as possible by using in each new
transformation/selection cycle preferably plasmids having different selection markers.
In order to enrich restriction-deficient mutants, therefore, it is preferable to use efficiently
transformable vectors that allow a specific and selective choice of the positivetransformants. Suitable vectors contain preferably one or more marker genes that impart to
the host cell a characteristic by which the cells transformed with the vector can be easily
identified and subsequently selected. Preference is given to marker genes that code for
antibiotics resistance. Some examples of suitable resistance genes are especially those that
code for the antibiotics ampicillin, chloramphenicol, erythromycin, tetracycline,
hygromycin, G418, kanamycin, bleomycin, neomycin or thiostrepton.
Preference is given also to marker genes that code for enzymes for which a chromogenic
substrate is available. Examples of such marker genes are the lacZ gene [chromogenic
substrate: X-gal ~ 5-bromo-4-chloro-3-indolyl-B-D-galactoside]; the xylE gene [chromo-
genic substrate: catechol]; or the luxAluxB operon which, with long-chained aldehydes as
substrate [for exarnple n-decanal], generates the emission of light.
The transformed colonies can then be detected very easily by means of a specific colour
reaction.
Also suitable for use in the method according to the invention are genes that impart a
resistance to heavy metals, such as mercury.
Special preference is given within ~e context of this invention to a series of mutually
compatible shuttle vectors that code for different markers and preferably have a certain
- 8- 2049~
instability in B. thuringiensis and/or B. cereus. Owing to this instability, it is relatively
simple later to prepare plasmid-free derivatives from potentially restriction-negative
mutants.
Owing to their specific chaT~cterisdc, these vectors can also be used more than once for
transformation within the context of mutant enrichment.
A further precondition for finding suitable mutants is initially high transformation rates in
order to ensure a high degree of probability that one of those rarely occulring mutants will
be transforrned at that same time. Special preference is given to initial transformation rates
of at least 106-108 or more transformants.
The transformation of B. thuringiensis and/or B. cereus using shuttle vectors suitable for
the selection of transformants is preferably caTried out by means of electroporation, as
described, for example, in Schurter et al, 1989 or in EP-A 342 633.
In a specific embodiment preferred within the context of this invention, the
B. thuringiensis and/or B. cereus cells are first of all incubated in a suitable nutrient
medium with adequate ventilation and at a suitable temperature, preferably at from 20C
to 35C, undl an optical density ~ODsso) of from 0.1 to 1.0 is reached.
The age of the Bacillus cultures provided for the electroporation has a marked influence
on the transformation frequency. Special preference is therefore given to an optical density
of the Bacillus cultures of from 0.1 to 0.3, especially of 0.2. It should be pointed out,
however, that it is possible to achieve good transformation frequencies also with Bacillus
cultures from other growth phases, especially with overnight cultures.
Fresh cells or spores are generally used as staTting material, but deep-frozen cell material
can equally well be used. The deep-frozen cell material used is preferably in the form of
suspensions of B. thuringiensis and/or B. cereus cells in suitable liquid media, to which a
certain amount of antifreezing agent is advantageously added. Suitable antifreezing agents
are especially mixtures of osmotically active components and DMSO in water or a
suitable buffer solution. Further suitable components that may be considered for use in
solutions of antifreezing agents include sugars, polyhydric alcohols, such as glycerol,
sugar alcohols, amino acids and polymers, such as polyethylene glycol.
204918~3
g
If B. thuringiensis spores are used as starting material they are first inoculated in a suitable
medium and incubated overnight at a suitable temperature, preferably at from 25C to
28C and with adequate ventilation. This batch is then diluted and treated further in the
manner described above.
In order to induce sporulation in B. thuringiensis, any medium that induces suchsporulation can be used. ~reference is given within the context of this invention to a GYS
medium according to Yousten AA and Rogoff MH (1969).
The introduction of oxygen into the culture medium is generally effected by agitation of
the cultures, for example using a shaking device, speeds of from 50 rpm to 300 rpm being
prefer~
The culturing of B. thuringiensis spores and of vegetative microorganism cells within the
context of the present invention is effected in accordance with known, generallycustomary methods, the use of liquid nutrient media being preferred for practical reasons.
The composition of the nutrient media can vary slightly according to the strain of
B. thuringiensis or B. cereus used. In general, complex media having poorly defined,
readily assimilable carbon (C) and nitrogen (N) sources are preferred, such as are
customarily used for culturing aerobic Bacillus species.
Apart from the LB medium preferably used within the context of the present invendon, it
is possible to use any other culture medium suitable for culturing B. thuringiensis and/or
B. cereus, for example antibiotic medium 3, SCGY medium etc.. Sporulated
B. thuringiensis cultures are preferably stored on GYS media (slant agar) at a temperature
of 4C. [The precise composition of the media referred to is given in the section "Media
and buffer solutions".]
Once the cell culture has reached the desired cell density the cells are harvested by means
of centrifugation and suspended in a suitable buffer soludon that has preferably been
cooled beforehand with ice. Especially suitable buffer solutions within the context of this
invention are osmotically stabilised phosphate buffers that comprise as stabilising agent
sugars, such as glucose or sucrose, or sugar alcohols, for exarnple mannitol, and that have
been adjusted to pH values of from 5.0 to 8Ø Special preference is given to phosphate
buffers of the PBS type having a pH value of from 5.0 to 8.0, preferably from 5.5 to 6.5,
20~91~
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that contain sucrose as stabilising agent in a concentration of from O.lM to l.OM,
preferably from 0.3M to 0.5M.
The incubation period for the Bacillus cells before and after electroporation is preferably
from 0.1 to 30 minutes, especially 10 minutes. The temperature can be freely selected
within a wide range. A temperature range of from 0C to 35C, preferably from 2C to
15C, and most preferably a temperature of 4C is preferred.
Aliquots of the suspended bacillus cells are then transferred to cuvettes or any other
suitable vessels and incubated, together with the DNA to be transformed, for a suitable
period, preferably for a period of from 0.1 to 30 minutes, especially from 5 to lS minutes,
and at a suitable temperature, preferably at a temperature of from 0C to 35C, especially
at a temperature of from 2C to 15C and most preferably at a temperature of 4C.
When working at low temperatures, it is advantageous to use precooled cuvettes or any
other suitable precooled vessels.
The DNA concentration preferred for B. thuringiensis or B. cereus is in a range of from 1
ng to 20 ~g. Especially preferred is a DNA concentration of from 10 ng to 2 llg.
The whole batch, comprising B. thuringiensis andlor B. cereus cells and the plasmid DNA
to be transformed, is then introduced into an electroporation apparatus and subjected to
electroporation, that is to say, exposed briefly to an electrical pulse.
Electroporadon apparatuses that are suitable for use in the method according to the
invendon are now available from various manufacturers, such as Bio Rad (Richmond, CA,
USA; 'Gene Pulær Apparatus'), Biotechnologies and Experimental Research, Inc. (San
Diego, CA, USA; 'BTX Transfector 100'), Promega (Madison, WI, USA; 'X-Cell 2000
Electroporadon System'), etc..
It is of course possible to use any other suitable apparatus in the method according to the
invendon.
The capacitance setdng at the capacitor is advantageously from 1 ~,IF to 250 ,uF, especially
from 1 ~F to S0 ~F and most preferably 25 ~F. The choice of starting voltage can be made
freely within a wide range. Preference is given to a starting voltage V0 of from 0.2 kV to
,
,
.
204~
- 11 -
50 kV, especially from 0.2 kV to 2.5 kV and most preferably from 1.2 kV to 1.8 kV. The
spacing of the electrode plates depends inter alia on the dimensions of the electroporation
apparatus. It is advantageously from 0.1 cm to 1.0 cm, preferably from 0.2 cm to 1.0 cm.
Especially preferred is a plate spacing of 0.4 cm. The spacing of the electrode plates and
the starting voltage set at the capacitor produce the ffeld strength values that act on the cell
suspension. These values are advantageously in a range of from 100 V/cm to
50 000 V/cm. Especially preferred are field strengths of from 100 V/cm to 10 000 V/cm,
especially from 3 000 V/cm to 4 500 V/cm.
The exponential decay time preferred within the context of the method according to the
invention is from approximately 2 ms to approximately 50 ms, especially from
approximately 8 ms to approximately 30 ms. Special preference is given to an exponential
decay time of from approximately 14 ms to approximately 20 ms.
The fine adjustment of the freely selectable parameters, such as capacitance, starting
voltage, plate spacing, etc., depends to a certain extent on the architecture of the apparatus
used and can therefore vary from case to case, within certain limits. It is therefore possible
in certain cases also to exceed or to fall below the limit values given, insofar as this might
be necessary in order to achieve optimum field strength values.
The actual electroporation operation may be repeated once or several times, until an
optimum transformation frequency for the particular system has been reached.
The electroporation can advantageously be followed by further incubation of the treated
Bacillus cells, preferably for a period of from 0.1 to 30 minutes, at a temperature of from
0C to 35C, preferably from 2C to 15C. The electroporated cells are then diluted with a
suitable medium and incubated again for a suitable period of time, preferably for from 2 to
3 hours, with adequate ventilation and at a suitable temperature, preferably at from 20C
to 35C.
At the end of each new electroporation cycle with one of the vectors described in detail
hereinbefore, the treated Bacillus thuringiensis and/or B. cereus cells are transferred to a
selective medium and are incubated there at a temperature of from 10C to 40C,
preferably at a temperature of from 20C to 35C and most preferably at a temperature of
from 30C to 33C. The selective medium contains as selective substance preferably one
of the above-mentioned antibiotics, depending on the vector used, and optionally a
suitable solidifying agent, such as agar, agarose, gelatin, etc.
- 12- 2049~
For further assessment of potentially restriction-negative mutants, first of all plasmid-free
derivatives of the enriched mutants are prepared. For that purpose, individual colonies of
the enrichment cultures obtainable from the most recent transfoIInation/selection cycle in
each case are selected and incubated on a suitable medium without selection.
The cultures obtainable in this manner are then diluted and plated out on suitable solid
media, preferably on L agar, without selective markers. Suitable dilutions are then
replicated on a suitable selective medium, preferably an L medium, that contains different
selection markers in different concentrations. Special preference is given within the
context of this invention to tetracycline, chloramphenicol and erythromycin in aconcentration that inhibits the growth of sensitive cells but still allows the growth of the
cells having the corresponding resistance plasmids. In this manner, it is relatively simple
to find derivatives that exhibit no further growth in the presence of the specifically used
selection markers.
In order to analyse the restriction barriers, the remaining isolates are then transformed
using a suitable vector that may originate from E. coli on the one hand and from the
unmutated starting strain of B. thuringie~sis and/or B. cereus on the other. In this case also
the transformation is preferably carried out as described above, via electroporation.
In the manner described hereinbeforej therefore, it is possible to select restriction-deficient
mutants of B. thuringiensis and/or B. cereus that, when vector DNA from a heterologous
intermediate host that is naturally subject to restriction in B. thuringiensis and/or B. cereus
is used, may be transformed significantly better, preferably by a factor of 2 102, than the
said non-mutated starting strain.
Special preference is given to a pardally restriction-deficient mutant of the B.thuringiensis strain HDlcryB which, when using shuttle vectors from a non-compadble
intermediate host, such as E. coli and/or B. subtilis, has a transformability that is better by
a factor of 21~2 than that of the non-mutated starting strain.
Special preference is given within the context of the present invention to the pardally
restriction-deficient mutant B. thuringiensis var. kurstaki HDlcryB Res9, which is
obtainable by means of spontaneous mutation from the B.t. strain HDlcryB and can be
obtained by means of enrichment methods that are known per se, and to mutants and
21~491
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variants thereof that are derived directly from that strain and that still have the
distinguishing restriction-reducing characteristics of the starting strain.
The restriction-deficient mutants described hereinbefore are outstandingly suitable, owing
to their improved transformability, for use as host strains for the cloning and optionally the
expression of genes or other useful DNA sequences, especially of protoxin genes.
In detail, the procedure is preferably as follows: first of all
(a) the said genes or DNA sequences are isolated from a suitable source or are
synthesised;
(b) the isolated or synthesised genes or DNA sequences are operably linked to expression
signals that are capable of functioning in Bacillus thuringiensis and/or Bacillus cereus and
that may be of homologous or heterologous origin in relation to the genes or DNAsequences used;
(c) the chimaeric genetic construction according to section (b) is transforrned using
suitable vectors, including those that are naturally subject to restriction in B. thuringiensis
and/or B. cereus, into a restriction-deficient mutant of Bacillus thuringiensis and/or
Bacillus cereus; and
(d) a corresponding gene product is optionally expressed and, if desired, isolated.
In order further to increase the efficiency of the method according to the invention, it is
possible to include in the method an additional step wherein the vector DNA is incubated
in vitro in a suitable reaction mixture, together with a specific methylase that is capable of
methylating one or more bases within the recognition sequence of a host-specificrestriction-endonuclease, and the methylated vector DNA is then transformed into a
restriction-deficient mutant of B. ~huringiensis and/or B. cereus.
Special preference is given within the context of this invention to a method for the cloning
and optionally the expression of genes or other useful DNA sequences, in which method
vectors that originate from E. coli or B. subtilis are used.
In addition to structural genes, it is of course also possible for any other useful DNA
sequences to be used, such as non-coding DNA sequences that have a regulatory function.
There may be mentioned at this point by way of example an 'anti-sense' DNA that is
transcribed into RNA, but is not translated into protein.
-14- 20491~
Using the restriction-deficient mutants it is, moreover, possible for the first time routinely
to set up representative gene banks in B. thuringiensis and/or Bacillus cereus, the
procedure preferably being as follows: first of all
(a) the total DNA of Bacillus thuringiensis is disintegrated into fragments mechanically
or, preferably, using suitable restriction enzymes;
(b) fragments of suitable size are isolated;
(c) the said fragments are inserted into a suitable vector, including those that are naturally
subject to restriction in B. thuringiensis and/or B. cereus;
(d) restriction-deficient Bacillus thuringiensis and/or Bacillus cereus cells are transformed
with the said vector; and
(e) there are selected from the transformants, using suitable screening methods, those that
comprise the novel and desired DNA sequences.
In this case also the efficiency of the transformation can be increased further by means of
additional methylation of the DNA to be inserted.
Special preference is given within the context of the present invention to a method
wherein the said Bacillus thuringiensis is a strain that has a restriction/modification
system comparable to that of the Bacillus thuringiensis strain HDlcryB.
This invention relates further to methods of reducing restriction barriers in B. thuringiensis
and/or B. cereus using restriction-negative mutants, especially using the restricdon-
negative mutant B. thuringiensis HDlcryB Res9.
A further increase in the efficiency of this method by repeating the reduction of the
restriction barriers can be achieved by using a restriction-negative mutant, especially the
restriction-negative mutant B. thuringiensis HDlcryB Res9, in combination with aspecific methylase that, by methylating the inserted vector DNA, protects the latter from
being digested by restriction enzymes inherent in B. thuringiensis and/or B. cereus and
thus further increases the efficiency of the method.
In detail, it is possible to proceed as follows: the vector DNA, together with a specific
methylase that is capable of methylating one or more bases within the recognition
sequence of a host-specific restriction endonuclease, is incubated in vitro in a suitable
2049~
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reaction mixture and the methylated vector DNA is thcn transformed into a restriction-
deficient mutant of B. thuringiensis and/or B. cereus, especially into the restriction-
deficient mutant B. thuringiensis HDlCryB Res9.
An example of such a methylase, which is in no way to be regarded as limiting, is the
enzyme M-FnuDII, which methylates specifically the first cytosine within the recognition
sequence of the restriction enzyme FnuDII or of its isoschizomers, such as BthKI~*CGCG] and therefore protects the vector DNA treated with the said methylase from
being digested by that enzyme. In this manner it is possible significantly to reduce or
completely to eliminate residual acdvity remaining after the exclusion of the Dam-specific
restriction, which residual activity is nevertheless still responsible for reducing the
transformation frequency of unmodified DNA by a factor of from 10 to 50.
In order to illustrate the rather general description and to provide a better understanding of
the present invention, reference is made below to specific examples which are not,
however, of a limiting nature, unless there is a specific indication to the contrary. The
same applies to all lists given by way of example in the foregoing description.
- 16 - 2049~8~3
NON-LTMlTING EXAMPLES
Example 1: Isolation of a restriction-nePative mutant of the strain HDlcrYB
1.1 Shuttle vectors used for the selection of mutants
For the isolation of a restriction-deficient mutant of the Bacillus thuringiensis strain
HDlcryB [Stahly DP et al (1978)], the following E. coli/B. thuringiensis shuttle vectors,
all isolated &om E. coli, are used, in the following order:
Vector selection origin/reference
(in B.thuringiensis)
1. pAM401 Cm Wirth et al, 1986
2. pHY300PLK Tc Toyobo Co., Osaka, 530 Japan,
Order No. PHY-001
3. pAM401 Cm Wirth et al, 1986
4. pHP13 Em, Cm Bacillus Genetic Stock Center,
Ohio State Univ., Columbus, Ohio
43210, USA, Strain No. lP50
Since the plasmid pAM401 is extremely unstable and without selection is rapidly lost
again, it can be used for transformadon more than once. Since the other shuttle vectors
also exhibit a certain instability in B. thuringiensis, plasmid-free derivatives can later be
obtained relatively readily &om potentially restriction-negative mutants.
1.2 Transformation of Bacillus thurinRiensis HDl crYB
The transformation of Bacillus thuringiensis using the shutde vectors listed under 1.1 is
carried out by means of electroporation.
1.2.1 Standard protocol for the transformation of B. thurinRiensis HDlcrYB via electro-
poration
10 ml of an LB medium (tryptone 10 g/l, yeast extract 5 gll, NaCl S g/l) are inoculated
with spores of B. thuringiensis var. kurstaki HDlcryB [Stahly DP et al (1978)], a
20~
- 17-
plasmid-free vaIiant of B. thuringiensis var. kurstaki HDl.
This batch is incubated overnight at a temperature of 27C on a rotary shaker at 50 rpm.
The B. thuringiensis culture is then diluted 100-fold in from 100 ml to 400 rnl of LB
medium and cultured further at a temperature of 30C on a rotary shaker at 250 rpm until
an optical density (OD5so) of 0.2 has been achieved.
The cells are harvested by means of centrifugation and suspended in 1/40 volume of an
ice-cooled PBS buffer (400 mM sucrose, 1 mM MgCI2, 7 mM phosphate buffer pH 6.0).
The centrifugation and subsequent suspension of the harvested B. thuringiensis cells in
PBS buffer is repeated once.
The cells thus pretreated can then be either electroporated directly or stored, after the
addition of glycerol to the buffer solution [20% (w/v)], at from -20C to -70C and used at
a later date.
400 ~1 aliquots of the ice-cooled cells are then transferred into precooled cuvettes and
plasmid DNA is then added in a suitable concentration and the whole batch is incubated
for from 0.1 to 10 minutes at 4C.
When using deep-frozen cell material, first of all a suitable aliquot of frozen cells is
thawed in ice or at room temperature. The subsequent treatment is effected analogously to
the procedure for fresh cell material.
The cuvette is then introduced into an electroporation apparatus, where the
B. thuringiensis cells present in the suspension undergo electroporation by being
subjected, in a single discharge from a capacitor, to voltages of from 0.1 kV to 2.5 kV. A
voltage of 1.3 kV is preferred.
The capacitor used in this case has a capacitance of 25 ~F, the cuvettes have an electrode
spacing of 0.4 cm, which, in the case of a discharge, depending on the setdng, leads to an
exponentially decreasing field strength having initial peak values of from 0.25 kV/cm to
6.25 kV/cm, especially of 3.25 kV/cm. The exponential decay dme is in a range of from
lOmsto20ms.
For the electroporadon experiments described, for example an electroporadon apparatus
~:04918~3
- 18-
supplied by Bio Rad may be used ('Gene Pulser Apparatus', No. 165-2075, Bio Rad, 1414
Harbour Way South, Richmond, CA 94~04, USA).
Of course any other suitable apparatus can be used in the electroporation methoddescribed hereinbefore.
After a further period of incubation of from 0.1 - 10 minutes at 4C, the cell suspension is
diluted with 1.6 ml of LB medium and incubated for 2 hours at a temperature of 30C on a
rotary shaker at 250 rpm.
Suitable dilutions are then plated out on L agar (LB medium solidified with agar, 15 glt)
that contains as additive an antibiotic suitable for the selection of the newly obtained
plasmid. In the case of pHY300PLK, this is the antibiotic tetracycline, which is added to
the medium in a concentration of 20 mg/t.
Bacillus cereus cells can be transformed in the same way as B. thuringiensis cells in
accordance with the above protocol.
1.2.2 SDecific modifications within the individuat transformation/selection cvcles
1st electroPoration: The Bacillus thuringiensis strain HDlcryB is cultured in accordance
with the standard protocol [see Section 1.2.1] and concentrated 500-fold in order to
achieve high transformation frequencies. [Detailed information on the electroporation of
Bacillus thuringiensis cells can be found in European Patent Application EP-A 0 342 633].
After electroporation of the Bacillus thuringiensis HDlcryB cells with S llg of pAM401
plasmid DNA, the cells are diluted 5-fold in LB medium and incubated for 2 hours at
250 rpm and 30C. The transforrnants are then selected using 20 ,ug/ml of
chlorarnphenicol for 4 hours at 250 rpm and 30C and are diluted 200-fold in LB medium
for the next electroporation.
2nd electroporation: After 2 hours' incubation at 150 rpm and 30C in accordance with the
standard protocol lsee Section 1.2.1L the cells are washed and concentrated 400-fold.
After electroporation with 12 )lg of pHY300PLK plasmid DNA, the cells are diluted
5-fold in LB medium and incubated for 1.5 hours at 250 rpm and 30C. After a further
5-fold dilution, the transformants are selected overnight using 20 ~,lg/ml of tetracycline at
19 204918B
150 rpm and 30C and are diluted 200-fold in LB medium for the next electroporation.
3rd electroporation: After incubation for 1.5 hours at 250 rpm and 30C in accordance
with the protocol, the cells are washed and concentrated 100-fold. After electroporation
with 5 ~,lg of pAM401 plasrIud DNA? the cells, as described for the second electroporation,
are diluted in LB, incubated and diluted again with LB and the transformants are selected
using 10 ~lg of chloramphenicol for 2.5 hours at 250 rpm and 30C. The culture thus
selected is then stored at room temperature.
4th electroporation: The above culture is diluted S0-fold and in accordance with the
standard protocol incubated overnight and then cultured further as a sub-culture, washed
and concentrated. After electroporation with 5 ~g of pHP13, the cells are diluted 5-fold
and incubated for a period of 3.5 hours at 250 rpm and 30C. The cells are then diluted
again and plated out on L agar with 200 ~lg/ml of erythromycin.
The 3rd and 4th electroporations already exhibit markedly increased transformation
frequencies, which is a first indication of the enrichment of restriction-deficient mutants.
1.3 Preparation of plasmid-free derivatives
For further assessment of potentially restriction-deficient mutants first of all plasmid-free
derivatives are prepared. For ehat pulpose 10 individual colonies are picked from the
above selection plates [after ehe 4eh eleceroporation, see Section 1.2.2] and incubated in
10 ml of LB medium overnight at 50 rpm and 27C without selection.
The cultures are ehen diluted and plated out on L agar wiehout selective markers. Suieable
dilutions of these cultures are then replicated on L agar plates each containing one of the
following selection markers: tetracycline [20 ,~-gtml], chloramphenicol ~10 ~g/ml] or
erythromycin 1200 llg/ml].
In this manner, of the original 10 colonies, it is relatively easy to find derivatives of 6 ehae
exhibit no further growth in the presence of all three selection markers. In none of ehose
derivaeives can any of the previously insereed plasmids be detected.
1.4 Transformation with the shuttle vector pHY300PLK
In order to analyse the restriction barriers, the remaining 6 isolates are transformed wieh
pHY3û0PLK which originates on the one hand from E. coli and on the other from
2~ 8
- 20-
B. thuringiensis HDlcryB. The transformation is carried out by means of electroporation
[see Example 1.2.1].
The results of Table 3 show that 4 of the 6 isolates tested exhibit markedly reduced
restriction barriers as compared with the parent strain HDlcryB. Of those mutants,
designated ResS, 7, 8 and 9, ResS, 7 and 9 have a restriction barrier that is approximately
SO-lOOx smaller than that of HDlcryB. Res5 and 7, however, seen in absolute terms, are
10 times less transformable than HDlcryB. Although Res8 exhibits an extremely low
restriction barrier, seen in absolute terms its transformability is approximately 500x poorer
than that of HDlcryB, which cancels out the advantage of the reduced restriction barrier.
Only the restriction-negative mutant Res9 combines the advantages of a high degree of
transformation efficiency with those of reduced restriction barriers.
The advantages of the mutant Res9 become even clearer in the case of transformation with
recombinant plasmids. It is clear from Table 4 that shu~tle vectors isolated from E. coli
that contain a protoxin gene lpXI204, pXI93], can be transformed into B. thuringiensis
HDlcryB only at a very low frequency, whereas the mutant Res9 allows efficient trans-
formation using the sarne plasmids.
1.5 Taxonomic characterisation of the Bacillus thurin~iensis strains used
1.5.1 Strain~)lcrvB
Identification of strain DSM 4574
Characteristics of the strain
Bacillus
Width /llm 1.0-1.2
Length /llm 3.0-5.0
Mobility +
Spores +
204918~3
- 21 -
ellipsoid +
round
swollen sporangium
Gram reacdon +
Catalase +
Anaerobic growth +
VP reaction +
pH in VP medium 4.9
Maximum temperature
Growth positive at C 45
Growth negative at C 50
Growth in
Medium pH 5.7 +
NaC15% +
7 %
10%
Acid from
glucose +
L-arabinose
xylose
mannitol
Gas from glucose
Lecithinase
Hydrolysis of
starch
;:049~8~3
- 22 -
gelatin +
casein +
Utilisation of
citrate +
propionate
Degradation of tyrosine
NO2 from NO3 +
indole
phenylalanine desaminase
arginine dihydrolase +
unusual characteristic:
no lecithinase activity
1.5.2 Strain HDlcryB Res9
Identification of strain DSM 5854
Characteristics of the strain
Bacillus
Width /~m 1.0-1.2
Length /~,lm 3.0-5.0
Mobility +
Spores +
ellipsoid +
round
-` 2049~8.~3
- 23 -
swollen sporangium
Gram reac~ion +
Catalase +
Anaerobic growth +
VP reaction +
pH in VP medium 4.7
Maximum temperature
Growth positive at C 45
Growth negative at C 50
Growth in
Medium pH 5.7 +
NaCl S % +
7%
10%
Acid from
glucose +
L-arabinose
xylose
mannitol
Gas from g1ucose
Lecithinase
Hydrolysis of
starch +
gelatin +
casein +
:.
'
.
20491~
- 24 -
Utilisation of
citrate +
propionate
Degradation of tyrosine
NO2 from NO3
indole
phenylalanine desaminase
arginine dihydrolase +
unusual characteristic:
no lecithinase activity
Exam~le 2: Characterisation of the restriction barriers of the strains HDlcrYB and Res9
2.1 Restricdon enzYme BthKl
In crude extracts of the strain HDlcryB it is impossible to detect sequence-specific
restriction enzymes, since the test DNA is totally degraded by non-specific nucleases.
These interfering nucleases can to a large extent be separated off in a simple manner by
means of a dextran/polyethylene glycol phase distribution [Schleif (1980)]. Depending on
the characteristics of the nucleases and restriction endonucleases that are present, ionic
strengths can be found at which the different enzymes prefer different phases. ln the case
of HDlcryB the non-specific nucleases can be removed at all ionic stren~ths. At NaCI
concentrations lower than 10 mM or higher than 250 mM, a sequence-specific nuclease
can be detected in the aqueous phase.
Further purification and separation of the restriction enzymes present in the strain
HDlcryB have been carried out as follows by means of affinity chromatography on a
heparin column [Bickle (1977)]. The cells are harvested from S litres of culture by means
~Oa~9
- 25 -
of centrifugation and disintegrated in a cell-disintegration apparatus [for example a French
Pressure CelU and the cell detritus is separated off by centrifugation. Nucleic acids are
separated off by precipitation with polyethyleneimine (Pirrotta, 1980) and the proteins are
precipitated with (NH4)2S04 at 70 % saturation and taken up in a small volume of buffer.
The proteins are then loaded onto a heparin column and eluted with an NaCl gradient
[OM-lM]. Aliquots of the eluted fractions are then tested for the presence of restriction
enzymes [Bickle (1977)]. The substrate used is pHY300PLK, a plasmid that is known to
be subject to restriction and that is isolated from E. coli. In spite of incomplete separation
of the non-specific nucleases, a sequence-specific restriction activity that generates a
defined pattern of DNA fragments has been demonstrated. The restriction activity elutes at
approximately 0.6M - 0.8M.
The enriched enzyme cleaves the plasmid pHY300PLK if it has previously been isolated
from E. coli or B. subnlis, but not if it has been isolated from HDlcryB. Characteristically,
therefore, in this case also the producer of the restriction enzyme is protected against
digestion of its own DNA, probably as a result of sequence-specific methylation.
The characteristics documented above thus all indicate a lestriction enzyme of class lI. In
accordance with the accepted nomenclature [Szybalski et al (1988)], the enzyme isolated
from B. thuringiensis HDlcryB receives the designation BthKl.
The present invention relates also to this restriction enzyme BthKl.
2.1.1 SPecific reco nition sequence of BthKl
The specific recognition and cleavage sequence of the restriction enzyme isolated from
HDlcryB can be deterrnined as follows.
The DNA used as substrate is generally cut into many small fragments, which leads to the J
assumption that it is an enzyme the recognition sequence of which consists of only four
nucleotides. Furthermore, DNA having an average to high GC content is cleaved
frequently, DNA having a low GC content is cleaved rather less frequently, which in turn
leads to the conclusion that the recognition sequence is rich in GC. In an en~yme that
recognises a four sequence, this indicates that the sequence consists exclusively of GC.
Using as substrate DNA of plasmids pC194 [Horinuchi and Weisblum (1982)~ and pBC16
[Bernhard et al (1978)1 isolated from B. subtilis and by means of a comparison with
commercially available restriction enzymes, it has been shown unequivocally that the
restriction enzyme BthKl is an isoschizomer of the commercially available enzymes Thal
,
.
2~)4918~
- 26 -
[GIBCO BRL, Gaithersburg, Maryland 20877, USA], Acc2 [Stratagene, La Jolla, CA
92037, USA], BstUl [New England Biolabs, Beverly, MA 01915-5510, USA] and Mvnl
[Boehringer Mannheim, D-6800 Mannheim, FRG], that is to say, it recognises and cleaves
the sequence CGCG if the latter has not been specifically methylated. The specific
methylase M.BthKl postulated for the restriction enzyme BthKl provides protecdon not
only against digestion by BthKl, but also against digestion by Thal or Acc2. Since the
methyladon of those two enzymes is known, it can be concluded that M.BthKl methylates
at least the first C, and possibly even both Cs, in the sequence CGCG.
The restriction enzyme in the strain Res9 is analysed in the same manner as that described
above for HDlcryB. The eludon profile of a heparin/Sepharose affinity column in the case
of Res9 is the same as that of HDlcryB, i.e. BthKl is still produced and is therefore not
the cause of the reduced restriction barrier in the case of Res9. The mutation responsible
therefor thus still remains unexplained.
2.2 Methyladon-specific restriction
Specific methylation of adenine or cytosine is effected in E. coli K by Dam or Dcm
methylase. E. coli B strains are naturally dcm~. Restriction enzymes that recognise the
same sequence as the Dam or the Dcm methylase, can, according to their type, be
inhibited by this methyladon. These restricdon enzymes can therefore be used
diagnostically to clarify the methylation status of a specific DNA. Using this method it is
possible to show that both B. subtilis and B. thuringiensis exhibit neither Dam nor Dcm
methyladon.
Indications of the nature of the mutation in the strain Res9 can be obtained using DNA of
the shuttle vector pHY300PLK isolated from strains having a different ~/Dcm
phenotype. Table S shows the transformation frequencies of that DNA into lhe strains
HDlcryB and Res9. The ~/Dcm phenotype of the DNA has in each case been carefullyconf~rrned by digestion with the corresponding methylation-sensitive restricdon enzymes.
The results from Table S can be summarised and interpreted as follows.
Dam methylation of the DNA is responsible for the majority of the restriction barriers
observed in HDlcryB. Res9, on the other hand, is not influenced by the Dam phenotype
and accepts both Dam-methylated and unmethylated DNA equally well.
2049~8~3
- 27 -
The Dcm methylation thus evidently has no influence on the transforrnability of B.
thuringiensis HDlcryB. Since no isogenic strains were available, the relatively small, but
reproducible, differences between dhe individual host strains cannot be explained by a
single phenotype. There remains, however, the interesting observation that DNA from
different host strains has different transformabilities.
By means of in vitro methylation with commercially available Dam methylase it ispossible to confI~ that Dam methylation is in fact the cause of some of dhe restriction
barriers in HDlcryB (Tab. 6). In vitro methylation with three other methylases shows also
dlat it is not a general methylation but specifically Dam methylation that causes the
restricdon barriers.
Example 3: Further reduction of the restriction barriers bv means of specific methylation
Of the two restriction systems present in the Bacillus thuringiensis strain HDlcryB, the
main activity, which is based on a Dam-speci~lc restriction, can be inactivated by a
mutation in the derivative Res9. The residual activity that remains, which is still
responsible for a reduction in the transforrnation frequency of unmodified DNA by a
factor of 10-50 and is based on the restriction enzyme BthKI, can be significandy reduced
by specific methylation with the methylase M-FnuDII.
3.1 Specific methvlation of the shuttle vector pHY300PLK with M-FnuDII methvlaseThe medhylase M-FnuII supplied by New England Biolabs lNo. 224S, New England
Biolabs, 32 Tozer Road, Beverly, MA 01915-5599, USA] methylates specifically thesequence *CGCG and thus protects it against digesdon with FnuDII and its isoschizomers,
such as BthKI.
DNA of the shuttle vector pHY300PLK is isolated f~om Bacillus subtilis or E. coli and
methylated in the following mixture:
DNA pHY300PLK 0.3 ',lg
Tris-HCl [pH 7.5] 50 mM
EDTA 10 mM
~-mercaptoethanol 5 mM
S-adenosinemethionine 0.08 mM
M-FnuDII 2 UN~
20491~
- 28 -
~UN Methylatinn units. One unit corresponds to that amount of cnz~ me that is required fully to protect one ~lB of
~mbda DNA [in one hour/37C, in 10 111 of reaction volume] against digestion by FnuD~.
The methylation is carried out over a peIiod of one hour at a temperature of 37C. The
methylation reaction is discontinued by inactivating the methylase. For that purpose the
whole mixture is incubated once more, for 20 minutes at a temperature of 65C. The DNA
is then precipitated by the addition of ethanol and resuspended in 10 ~,11 of TE buffer. Each
mixture is reacted in triplicate, one aliquot in each case being used for the subsequent
transformation, another for testing the plasmid DNA for the absence of damage. The latter
test is carried out by means of agarose gel electrophoresis.
The effectiveness of the methylation is tested by digestion of the plasmid DNA with the
FnuDlI isoschizomer Thal. The results show that the DNA is protected against digestion
by Thal, that is to say it is fully methylated, whereas the non-methylated controls are
digested by the enzyme.
3.2 Combination of mutation and methvlation
The pHY300PLK plasmid DNA methylated hereinbefore in Section 3.1 is transformed in
accordance with the protocol described in Example 1.2.1 into the restriction-deficient B.
thuringiensis mutant Res9. The results are given in Table 7. The results show that a
combination of the methylation of the plasmid DNA by M-FnuDII and the use of Res9 as
host strain allows the restriction barriers that are to be found in ~te B. thuringiensis strain
HDlcryB to be completely overcome.
49~8~3
- 29 -
MEDIA AND BUFFERS
LB medium [g~l]
tryptone 10
yeast extract 5
NaCl 5
~ [g/l]
LB medium solidified with
AGAR 15
Antibiotic medium No. 3 (Difco Laboratories) [g/l]
beef extract 1.5
yeast extract 1.5
peptone S
glucose
NaCl 3-5
K2HP04 3.68
KH2P04 1.32
SCGY medium [g/l]
casamino acids
yeast extract 0.1
glucose 5
K2HP04 14
KH2P04 6
sodium citrate
(NH4)2S04 2
MgS04 7H20 0.2
GYS medium (Yousten & l~ogoff, 1969) [g/l]
glucose
yeast extract 2
(NH4)2S04 2
K2HP04 .S
~. . . ..
- 2C~49~L13~3
- 30-
MgSO4 7 H2O 0.2
CaCl2 2 H2O 0.08
MnSO4 H20
adjust pH to 7.3 before autoclaving.
PBS buffer [mM]
sucrose 400
MgCl2
phosphate buffer, pH 6.0 7
TBST buffer [mM]
Tween 20* 0.05% (w/v)
TrislHCI* (pH 8.0) 10
NaCl 150
Buffer High
[Maniatis et al (1982); page 1041 [mMl
NaCl 1oo
Tris/HCI* (pH 7.5) 50
MgCI2 10
dithiothreitol
Nick-translation buffer (lOx) [mMl
Tris/HCI* ~pH 7.2) 500
MgSO4 100
dithiothreitol
boYine serum albumin (BSA Pentax Fraction V) 500
llg/rnl
TE buffer ~mMl
Tris/HCl (pH 8.0) 10
EDTA
~Tweal 20 polyethoxysorbitan laurate
~Tris/HCI a,OC,a-~ris(hydroxyme~hyl)me~hylamino hydrochloride
.
2~49~L8!3
- 31 -
DEPOSIT
The microorganisms listed below, which are used within the context of the present
invention, have been deposited with the 'Deutsche Sammlung von Mikroorganismen
(German Collection of Microorganisms)' in Brunswick, Federal Republic of Germany, a
recognised international depository, in accordance with the requirements of the Budapest
Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes
of Patent Procedure.
Microorganism Date of Deposit Date Or
deposit number viability
certificate
Bacillus thuringiensis 4th March 1986 DSM 3667 5th March 1986
var. kurstaki HDl
Bacillus ~huringiensis 4th May 1988 DSM 4574 4th May 1988
var. kurstaki HDlcryB
var kurstakl HDl cryB 21st March 1990 DSM 5854 21st March 1990
Bacillus thuringiensis
var. kurstaki HDlcryB 4th May 1988 DSM 4572 4th May 1988
trans with pK61
_
Bacillus thuringiensis
var. kurstaki HDlcryB 4th May 1988 DSM 4571 4th May 1988
trans with pK93
Since a B. thuringiensis can be distinguished from a B: cereus using the selected criteria
on1y by its parasporal crystals, both the strain HDlcryB (DSM No~ 4574) and the
restriction-deficient strain Res9 (DSM No. 5854) can be classified as B. cereus.
This classification is possible because the strain HDlcryB is a plasmid-free, and therefore
a crystal-free, derivative of HDl (Stahly et al., 1978), which automatically results in its
classification as B. cereus. Owing to the known differences between the three
characterised strains, no taxonomic identity can therefore be expected. Since, however, the
2~9~! 8
- 32-
characteristics of the three strains shown in Section 1.5 do not conflict with their
demonstrated related origin, the strains HDlcryB and Res9 continue to be described as
B. thllringiensis within the context of the present invention.
204918~3
- 33 -
LlTERATURE
Asisbekjan, R. R. et al., Dokl. Akad. Nauk SSSR 274, 742-744, 1984
Bailey, C. R. and Winstanley, D. J., J. Gen. Microbiol. 132,2945-2947, 1986
Bernhard K et al., J. Bacteriol. 133, 897-903,1978
Bickle, T. A. et al., Nucl. Acids Res. _, 2561-2572, 1977
Bone, E. J. and Ellar, D. J., FEMS Microbiol. Letters 58, 171- 178, 1989
Bron., S. et al., Mol. Gen. Genet. 211, 186-189, 1988
Engel, P., Appl. Environm. Microbiol. 53,1-3,1987
Haima, P. et al., Mol. Gen. Genet. 209, 335-342,1987
Horinuchi andWeisblum, J. Bacteriol. 150, 815-825, 1982
Hoshino, T. et al., Agric. Biol. Chem. 44, 621-623, 1980
Kessler, C. and Holdce, H.-J., Gene 47, 1-153,1986
Kretz, P. L. et al., Nucl. Acids Research 17,5409, 1989
Lereclus, D. et al., FEMS Microbiol. Letters 60, 211-218, 1989
Macaluso, A., Abstr. Annu. Meet. Am. Soc. Microbiol., 309, 1989
MacNeil, D.J., J. Bacteriol. 170,5607-5612, 1988
MacNeil, D.J., Europ. Patent Appl., Case No. 8920 1140.4, 1989
Matsushima, P. et al., Mol. Gen. Genet. 206,393-400, 1987
Miller, J. F. et al., Proc. Nad. Acad. Sci. USA 85, 856-860, 1988
Orzech, K et al., J. Gen. Microbiol. 130, 203-208, 1984
Pirrotta, V. and Bickle, T. A., in: Meth. Enzymol. 65, 89-95, 1980
Raleigh, E. A. and Wilson, G., Proc. Nad. Acad. Sci. USA 83, 9070-9074, 1986
Schleif, R., in: Meth. Enzymol. 65, 19-23,1980
Schurter, W. et al., Molec. Genet. 218, 177-181, 1989
Sladek, T. L. et al., J. Bacteriol. 165, 219-225, 1986
Stahly DP et al~ Biochem. Biophys. Res. Comm., 84,581-588, 1978
Szybalski, W. et al., Gene 74, 279-280, 1988
Uozumi, T. et al., Mol. Gen. Genet. 152, 65-69, 1977
Vehmaanpera, J., FE~MS, Microbiol. Letters 4g, 101-105, 1988
Wirth R et al, J. Bacteriol. 165, 831-836,1986
204~L8~3
- 34 -
TABLES
Table 1. Restricdon balriers in the transformation of Bacillus
thuringiensis HDlcryB with shutde vector pHY300PLK
Transformation frequencies*
pHY300PLK isolated from: into strain HDlcryB
absolute relative
B.thuringiensis HDlcryB 1.2 x 107
E. coli HB101** 1.8 x 103 1.5 X 104
B.subtilis ISW1214*** 7.6xlOs 6.3x10-2
fot the tdative value ~e ftequency of dle plasmid isolated from dre
sttain HDlcryB is set a l.
[Arnetican Type Cultute Collection (ATCC), Rockville, Maryland, USA, sttain No.
33694]
[Toyobo Co. LTD, Osaka, 530 Jspan, Ordet No. PHY-001]
Table 2. Shuttle vectors that have been used for
the enrichment of restriction-deficient
mutants of HDlcryB.
Vector Selection gram+ replicon
(in HDlcryB)
1. pAM401 Cm pIP501
2. pHY300PLK Tc pAMal
3. pAM401 Cm pIPS01
4. pHP13 Em, Cm pTA1060
20491~
- 35 -
Table 3. Transformation behaviour of potential
restriction-deficient deri~atives of
B. thurin~iensis HDlcryB
Transformation frequencies* of pHY300PLK
isolated from:
B. thurin~. HDlcryB E. coli BZ234
transformed absolute I relative absolute relative
straln _
HDlcryB 9.5x106 9.0x103 9.5x104
ResS 1 .3x106 1 1 .Ox105 7.7x10-2
Res7 1 .5~C106 1 l.lxlOs 7.4xl0-2
Res8 6.0X104 1 2.0x104 3.3xlO-l
Res9 l.lx107 1 7.0x105 6.4xlO-2
* for calculation of the relative value the frequency of the
plasrnid isolated from the strain HDlcryB is set at 1
Table 4. Characterisation of the restriction barriers of
the strains HDlcryB and Res9.
I: Transforrnability of different recombinant
plasmids isolated from E. coli.
¦ Transformation frequencies into th~
strains
Plasmid isolated from HDlcryB Res9
I I . . .
p~204 ~ B.t. HDlcryB 1.3 x 106 l.S x 106
pXI204 E.c. HB101 < 8 x 10l 6.4 x 104
pX193 ~ B.t. HDlcryB 3.0 x 105 8.3 x 105
pXI93 E.c. HB101 4 1.3 x 104
I _ I ,
described in EP-A 0 342 633;
described in Schurter et al (1989) and EP-A 0 342 633; tbc intemal
reference pK entered in the deposit certificate issued by the DSM
has now been arnended to pXI in accordance with culrent
internadonal classification.
: . ' - ~ .:
.
:
~049
- 36 -
Table 5. Characterisadon of the restriction barriers of the
strains HDlcryB and Res9. II: Influence of the host
strain, especially its ~/Dcm phenotype, on the
transformability of the shuttle vector pHY300PLK.
relative* transformation frequency
into B. thuringiensis, strain:
phenotype
Plasmid isolated frot ~ Dam Dcm HDlcryB Res9
_
B. th. HDlcryB 1
B. th. Res9 1
B. subtilis ISW1214 9.1x10-2 8.9x10-2
E. coli BZ103 +/- 1.3x10-2 0.7x10-2
E. coli 3225 0.8x10-2 0.4x10-2
E. coli BL21 + 2.4x104 0.7x10-2
E. coli HB101 + + 8.2x104 2.1x10-2
E. coli W3110 + + 1.8x104 0.9x10-2
* pHY300PLK isolated from HDlcryB or Res9 transformed with same
frequency into strain HDlcryB and is set at 1. The transformation
frequencies of the other isolates are given in relation to that value.
The same applies also to transfonnations into strain Res9.
- .
204~3~8~3
- 37 -
Table 6. Sequence-specific in vitro methylation of pHY300PLK-DNA
isolated from the strain HDlcryB: Influence
on the transformation behaviour of HDlcryB and Res9.
relative+ transformation frequencies
into strain:
methylated
Methylase sequence HD 1 cryBRes9
.._
M.Hha I GC* GC 0.90.8
M.Hpa II CC* GG 0.40.6
M.Pst I CTGCAS G 0.7 0 7
Dam GA$ TC 3.7x10-3 0 8
+ the transformation frequencies into strain HDlcryB and Res9
were set at 1.
* S-methylcytosine
$ N6-methyladenine
Table 7. Transformation of B.thuringiensis HDlcryBRes9:
Effect of methylation of pHY300PLK-DNA by
the methylase M-FnuDII
~ __
Origin of the Treatment relative transfor-
plasmid of the DNA mation frequency
_
B.t. HDlcryB
B.s. ISW1214 0.06
B.s. ISW1214 control 0.02
B.s. ISW1214 M-FnuDII 0.58
E.coli TG1~ 0.0031
E.coli TGl* control 0.0029
E.coli TGl* M-FnuDII 0.32
_
* Componalt of in vilro Mulsgene6i6 Kit No. RPN1523 made b~ AMERSHAM, Buckingham hire
HP7 9NA, England
