DD283840A5 - METHOD FOR THE DIRECT, TARGETED AND REPRODUCIBLE GENETIC MANIPULATION OF B.THURINGIENSIS AND / OR B.CEREUS USING RECOMBINANT DNA TECHNOLOGY - Google Patents

Abstract

The invention relates to methods for the direct, targeted and reproducible genetic manipulation of B. thuringiensis and / or B. cereus using recombinant DNA technology. With the invention, a novel method is provided, are used in the simple, sometimes known process steps. The method consists of transforming B. thuringiensis and / or B. cereus with high efficiency by a simple transformation method using a recombinant DNA suitable for the intended genetic manipulation of B. thuringiensis and / or B. cereus. {Method; Manipulation; Thuringiensis; B cereus; recombinant DNA technology; Transformation method}

Description

For this 9 pages drawings

Field of application of the invention

The present invention describes a method which, for the first time, enables a direct and targeted genetic manipulation of Bacillus thuringiensis and the closely related B. coreus by means of recombinant DNA technology, based on an efficient transformation method for said Bacillus species.

Furthermore, the present invention relates to the construction of plasmids and "shuttle" vectors and the B. thuringiensis and / or B. cereus strains transformed therewith themselves.

Another object of the present invention relates to a method for the introduction and optionally expression of genes or other useful DNA sequences in Bacillus thuringiensis and / or Bacillus cereus, but especially ain method for the introduction and expression of Protoxingenen.

Also included in the present invention is a method for direct cloning, and optionally for expression and identification of novel genes or other useful DNA sequences in Bacillus thuringiensis and / or Bacillus cereus, thereby allowing, for the first time, libraries to establish and express directly in Bacillus ihuringien Is and / or Bacllluscereus.

Characteristic of the known state of the art

Baciilüs thuringiensis belongs to the large group of gram-positive, aerobic, endospores-forming bacteria. In contrast to the very closely related Bacillus species, B. cereus and B. anthracls, the majority of the previously known produces

In the course of its sporulation, for example, thuringiensis species has a paraspolar inclusion body, which is generally also referred to as a crystal body due to its crystalline structure. This crystal body is composed of insecticidally active crystalline protoxin proteins, the so-called δ-endotoxin.

These piotein crystals are responsible for the insect toxicity of E. thuringiensis. The δ-En'i Moxin develops its insecticidal activity, however, only after the oral uptake of the crystal body and its dissolution into, intestinal intestinal juice of target insects and the release of the actual toxic component from the protoxin by limited Proteolyso due to the action of proteases from the digestive tract Insects.

The δ-endotoxins of the various B, thurlnglensls strains are distinguished by their high specificity towards certain target insects, in particular against various Lepidopteran, Coleopteran and Diptera larvae and by their great effectiveness. Further advantages involved in the use of the B. thurlngler.sle δ-endotoxins are due to the apparent difficulty for the target insects to develop resistance to the crystalline protein, as well as the harmlessness of the toxins to humans, other mammals , Birds, fish or insects, with the exception of the target insects named ob9n.

The insecticidal potential of B. thurlnglensls protoxins was detected very early. Already since the late 20s

B. thuringiensls preparations used as Bioinsektizide for controlling various diseases caused by insects of crops. With the discovery of B. thurlngiensls var. Israelensis by üoldberg / 1 / and Margalit (1977) and B.thurlnglensls var. Tenebrionis by Krieg / 2 / et al (1983), the range of applications of B. thurlngiensls could even be applied to mosquito and beetle larvae be extended.

With the introduction of genetic engineering and the resulting new opportunities, the area of B.thurlnglensls- toxins has been given a new lease of life.

Thus, the cloning of δ-endotoxin genes in foreign host organisms such as in E. coli is already routine.

This has led to the fact that now the DNA sequences of a whole series of δ-Endotoxingenen are known (eg.

Schnepf, H.E./3/ and Whiteley, H.R., 1981; Klier, A./4/ et al, 1982; Geiser, M./5/ et al, 1986; Haider, M.Z./6/ et al, 1987).

Most B, thuringlensis species contain several genes that encode an insecticidally active protein. These genes, which are only expressed during the sporulation phase, are located in the majority of cases on large transmittable plasmids (30 to 150 mM) and can therefore very easily be located between the various B.thurglnglsl strains and between B.thurglnglsls and B. cereus, if compatible (Gonzalez, JM / 7 / et al, 1982).

The protoxigenic genes of B. thurlnglenslt var. Kurstaki belong to a family of related genes, several of which have already been cloned and sequenced. This work was carried out primarily in an E. coli cloning system.

The cloning of B. thurlnglensls genes thus far has been essentially limited to a few and exclusively hot-terired host systems, of which the E. coli system is the best studied and understood.

Meanwhile, reports have also become known about successful cloning and expression of protoxin genes in other host systems, such. B. in B. subtilis (Klier, A./4/ et al, 1982), Pt »ei: domonat f luorenszens (Obukowicz, M.G. / 8 / et al, 1986), and Saccharomyces cerevlslao (EP 0238441). The incorporation and the expression of the δ-endotoxin gene in plant host cells have also been successful (EP 0292435).

When cloning in E. coil, the fact is exploited that some protoxi igeno in addition to the gram-positive promoters coincidentally also contain an E. coli -like promoter. These promoter-like DNA sequences make it possible to express the 'j.thurglgeniens protoxin genes also in heterologous host systems, provided that they are able to recognize the control sequences mentioned above.

The expressed protoxin proteins may then be isolated and identified after disruption of the host cells by known methods.

However, it has been shown in the meantime that not all cases E.coli-like promoters in protoxins are present (Donovan / 9 / et al, 1988), so that so far only very specific Protoxingene that meet the above requirements, in heterologous Host systems are expressible and thus identifiable.

The cloning of genes outside the natural host organism and the use of these strains in practice as bioinsecticides are thus associated with a number of serious disadvantages:

a) Expression of B. thurlngienls protoxin genes is possible only in certain cases.

b) There is generally no or only a slight secretion of expressed foreign proteins.

c) Correct folding of the δ-endotoxins is not always guaranteed in the reducing environment of heterologous host cells, which may result in an undesirable change in the specific activity or the host range of the toxins.

d) If expression takes place at all, the expression rates of the cloned foreign genes among the native expression sequences are usually only small.

Schnepf / 3 / and Whitley / 10 / (1981; 1985) estimate that E.coli-cloned B. thuringiensis toxin constitutes only 0.5% to 1% of the total cell protein of E. coli, whereas the crystal protein in B thuringiensis is between 30% and 40% of the dry weight of sporulating cultures. These serious differences in rates of exposition may be due to the lack of sporulation-specific control signals in the heterologous host systems as well as difficulties in recognition of the B. thuringiensis promoters and / or problems in the post-translational modification of the toxin molecule by the foreign host.

e) Many of the host strains commonly used for expression are not as toxicologically toxic as B. thuringiensis and B. cereus.

f) B. thuringiensis and B. cereus are a major natural constituent of the microbial soil flora, which is not true for most of the common host strains used for expression.

These aforementioned problems and difficulties could be overcome if it were possible to mimic said B. thurlngiensis genes directly in the homologous host system, where the natural gram-positive promoters of the protoxin genes can be used for expression.

However, there is no method that makes B. thuringiensis, which is a commercially important bacterium, susceptible of direct genetic modification, thus enabling, for example, efficient reintroduction of a cloned protoxin gene into a B. thuringiensis strain.

The reason for this is primarily to be seen in the fact that it has not yet been possible, an efficient transformation system for

B. thuringiensis and the closely related B. cereus to develop, which ensures sufficiently high transformation rates and thus allows the application of already established rDNA techniques on B. thuringiensis.

The previously used methods for the production of new B. thuringiensis strains with new insecticidal properties are based primarily on the conjugal transfer of plasmid-encoded protoxin genes.

Successful reinsertion of a cloned B. thuringiensis crystal protein gene in B. thuringiensis has so far only been described in a single case (Klier, A./11/ et al, 1983), but here too, in the absence of a suitable transformation system for B. thuringiensis, to which conjugal transfer between B. subtilis and B. thuringiensis had to be resorted to. Also in this method described by Klier et al., E. coli is used as an intermediate host.

However, the conjugal transfer ancestors have a number of serious disadvantages that make them inappropriate for routine application to the genetic modification of B, thuringlensis, and / or B. cereus.

a) The conjugated transf Br of plasmid-encoded protoxin genes is only possible between B. thurlnglensls strains or between B. cereus and B.Ihurhgiensls strains, which are compatible with each other.

b) Conjugal plasmid transformation between more distant stems often results in a low transfer frequency.

c) There is no possibility to regulate or modify the expression of the protoxin genes.

d) There is no way to modify the gene itself.

e) In the presence of multiple protoxin genes in a strain, the expression of individual genes may be greatly reduced due to the so-called gene dose effect.

f) Instabilities may occur due to possible homologous recombination of related protoxin genes.

Alternative methods of preparation, which are routinely used, for example, in many gram-positive organisms in the mid-range, proved to be unsuitable for both B. thuringlensis and B. cereus.

To the aforementioned methods belongs z. B. the direct transformation of bacterial protoplasts by means of the polyethylene glycol treatment, in many Streptomyces strains (Bibb, JJ / 12 / et al, 1978) and B, subtllls (Chang, S., and Cohen, SN, / 3/1979 ), B. mogaterlum (Brown, BJ, and Carlton, BC, / 14/1980), Streptococcus lactis (Kondo, JK, and McKay, LL, / 15/1984), S. faecalis (Wirth, R./16/ et al.), Corynebacterium glutamicum (Yoshihama, M./17/ et al., 1985), as well as numerous other gram-positive bacteria.

For the application of this method, the bacterial cells must first be protoplastized, i. h., the cell walls are digested with the help of lytic enzyme.

Another prerequisite for the successful application of this direct transformation method is the expression of the newly introduced genetic information and the regeneration of the transformed protoplasts on complex solid media, before a successful transformation z. B. can be detected by means of a selection marker.

This transformation process proved unsuitable for B. tliuringlensls and the closely related B. cereus. Due to the high resistance of B. thurlnglensls cells to lysozyme and the inadequate regenerability of the protoplasts to intact cell wall-containing cells, the achievable transformation rates remain low and difficult to reproduce (Alikhanian, SJ / 18 / et al, 1981; Martin, PA / 19 / et al., 1981; Fischer, H.-M./20/ et al, 1984).

With this method, therefore, at most very simple plasmids which are unsuitable for working with recombinant DNA can be introduced at low frequency into B. thuringlensis or B. cereus cells.

Individual reports of satisfactory transformation rates, which could be obtained using the method described above, are based on the development of very complex optimization programs, which, however, are only concretely applicable to a specific B.thurlnglensls strain and involve a great deal of effort and costs (Schall, D./21/, 1986). These methods are therefore unsuitable for routine industrial scale use.

Object of the invention

The invention provides a novel method for the direct, targeted and reproducible genetic manipulation of B. thuringlensis and / or B. cereus. In the method, simple, sometimes known process steps are used.

Explanation of the essence of the invention

The object of the invention is to develop new methods which make B. thuringlensis or the closely related B. cereus amenable to direct genetic modifiability and thus allow, for example, a cloning of protoxin genes in the natural host system. However, so far it has not been possible to solve the existing difficulties and problems satisfactorily.

At present there are neither suitable transformation methods available which allow a fast efficient and reproducible transformation of B.thuringlensis and / or R.cereus with a sufficiently high transformation frequency, nor are suitable cloning vectors available that are suitable for other bacterial host systems already established recombinant DNA techniques also on B. thuringiensls allow. This is equally true of B. cereus.

Surprisingly, this object has now been achieved in the context of the present invention by the use of simple, partly known method measures.

Thus, according to the invention, a novel method is used which, based on recombinant DNA technology, makes possible for the first time a direct, targeted and reproducible genetic manipulation of B. thuringlansis and of B. cereus by using Bacillus thuringlensis and / or Bacillus cereus with the aid of a simple transformation method high efficiency, using a recombinant DNA suitable for the intended genetic manipulation of Bacillus thuringiensls and / or Bacillus cereus.

Furthermore, the present invention relates to a method for the introduction, cloning and expression of genes or other useful DNA sequences, but in particular of protoxigenic, in B. thuringiensls and / or B. cereus, which is characterized in that

a) isolating said genes or DNA;

b) operably linking the isolated genes or DNA to expression sequences operable in Bacillus thuringiensls and / or B. cereus;

c) transforming the genetic constructions of section b) into Bacillus thuringlensis and / or B. cerous cells using suitable vectors

d) optionally expressing a corresponding gene product and, if desired, isolated.

Also encompassed by the present invention is a direct method for cloning, expression and identification of genes or other useful DNA sequences, but especially of protoxigenic genes, in B. thuringiensls and / or B. cereus, which is characterized in that

a) the total DNA of Bacillus thurlnglensls digested by means of suitable Restruktionsenzyme;

b) isolated from the resulting restriction fragments of suitable size;

c) splicing said fragments into a suitable vector;

d) Bacillus thurlnglensls and / or B. cereus cells are transformed with said vector and

e) detects and optionally isolates new DNA sequences from the transformants using suitable screening methods. In addition to structural genes, of course, any other useful DNA sequences can be used in the inventive method, such as. B. non-coding DNA sequences which have a regulatory function, such as. B. 'anti-sense DNA.

The method according to the invention thus opens up a multiplicity of new possibilities, which are of great interest both from a scientific as well as a commercial point of view.

For example, it is now possible for the first time to obtain information on the regulation of the endotoxin synthesis of Ö, in particular with regard to sporulation, on the genetic level.

Also, the question as to where the toxin molecule is located and the section (s) responsible for insect toxicity, and to what extent it is also related to host specificity, should now be clarified to let. The knowledge of the molecular organization of the various toxin molecules and of the toxin gene coding for these molecules from the various B. thurlglensls species is of extraordinary practical interest for a targeted genetic manipulation of these genes, which is now possible for the first time with the aid of the method according to the invention. The new method according to the invention, in addition to a targeted modification of the δ-endotoxin genes themselves, also permits the manipulation of the regula'.oric DNA sequence which controls the expression of these genes, as a result of which both the specific properties of the δ-endotoxins, such as. B. ihi β host specificity, their absorption behavior u. a. selectively changed as well as their production rates can be increased, for. J. by the incorporation of stronger and more efficient promoter sequences.

Targeted mutation of selected genes or subgenes in vitro leads to new B.thuringlensis and / or B. cereus variants.

Another possibility for the construction of new B.thuringlensis and / or B.cereus variants consists in the splicing together of genes or Ganabschnitten derived from different B. Thurlnglensls sources, thus B.thuringlensis and / or B, cereus strains with arise an extended range of applications. Also synthetically or semi-synthetically produced toxin genes can be used in this way for the construction of new B. thurlngiensls and / or B. cereus varieties.

Moreover, due to the large increase in the transformation frequency and the simplification of the method, the method according to the invention makes it possible for the first time to generate gene banks and to rapidly screen modified and new genes in B.thuringlensis and / or B.cereus.

In particular, the method according to the invention now makes it possible for the first time to directly express oenbanks in 3.thuringiensls and / or B. cereus and to identify new protoxin genes in B.thuringlensis by means of known, preferably immunological or biological methods.

The subject of the present invention is thus a method based on a stai Ken increasing the efficiency of B. thuringlensls- / B. cereus transformation over previously known methods makes possible for the first time a direct genetic modification of the B.thuringlensis and / or B.cereus genome.

In particular, the present invention relates to a method for transforming B.thuringlensis and / or B. cereus by introducing recombinant DNA, in particular plasmid and / or vector DNA, into B.thuringlensis and / or B.cereus cells Help of electroporation.

A method for the transformation of B.thuringlensis and / or B.cereus with DNA sequences coding for δ-endotoxin and DNA sequences which code for a protein which essentially has the insect-toxic properties of said B.thuringlensis toxins is preferred.

Another object of the present invention relates to the expression of DNA sequences which encode a δ <ndotoxin or a protein which has at least substantially the insect-toxic properties of B. Thurlnglensls toxin, in transformed B.thuringlensis and / or B. cereus cells.

Also included in the present invention is a method for producing bifunctional vectors, so-called "shuttle" vectors, for B. thuringiensis and / or B. cereus and the use of said "shuttle" vectors for the transformation of B. thuringiensis and / or B. cereus cells.

Preference is given to the construction of bifunctional vectors which, except in B. thuringiensis and / or B. cereus, replicate in one or more other heterologous host systems, but in particular in E. coli cells.

In particular, the present invention relates to a method of producing "shuttle" vectors for B.thuringlensis and / or B.cereus which contain a DNA sequence encoding a δ-endotoxin polypeptide naturally present in B. thuringiensis , or at least one polypeptide which is substantially homologous thereto, ie which has at least substantially the insect-toxic properties of the B.thuring ionsis toxin. Also included in the present invention is the use of these "shuttle" vectors to transform B. thuringiensis and / or B. cereus cells and the expression of the DNA sequences present on said "shuttle" vectors, in particular those DNA sequences which code for a δ-endotoxin from B. thuringiensis or at least for a protein, which has essentially the insecticidal properties of B. thuringiensis toxins.

Likewise encompassed by the present invention is the use of B.thuringlensis and / or B. cereus as general host organisms for the cloning and expression of homologous and in particular also heterologous DNA or a pimer combination of homologous and heterologous DNA.

A further subject of this invention concerns <previously characterized plasmids and "shuttle" vectors themselves, their use for the transformation of B. thuringiensis and / or B. cereus and the B. thuringiensis and B.cereus cells themselves transformed therewith.

Particularly preferred in the context of this invention are the bifunctional ("shuttle") vectors pXI61 (= pk61) and pXI93 (= pk93), which, transformed into B.thuringiensis var. Kurstaki HD1 cryB and B.cereus 569K, respectively, are designated as International Depository Center recognized "German Collection of Microorganisms" (Braunschweig, FRG) according to the provisions of the Budapest Treaty under the number DSM4573 (DSM 4572, transformed into B.thuringiensis var. Kurstaki

HD 1 cryB) or DSM4571 (pXI93, transformed into B. thurlnglensls var. Kurstaki HD 1 cryB) and DSM4573 (pXI 93, transformed into B. cereus 569K) have been deposited.

More particularly, the present invention relates to novel B.thurglnglansls and B.coreus varieties transformed with a DNA sequence encoding and expressing a δ-endotoxin from B.thurglnglen "1" or at least one protein substantially has the toxic properties of B. thuringlonsls toxins.

The transformed B.thurlnglensls and B.cereus cells, as well as the toxins produced by them, can be used for the preparation of insecticidal agents which are a further object of the present invention.

Also included are methods and agents for controlling insects using transformed B.thurglnlsls and / or B.cereus cells previously described, and cell-free crystalloid (δ-endotoxin) preparations containing the protoxins produced by said transformed Bacillus cells contain.

The following is a brief description of the figures will be given.

Figure 1: Transformation of E. coli HB101 with pBR322 (o) and * B.thurlnglensls HD1 cryB with pBC 16 (·) (Δ number of surviving »HD1 cryB cellon).

Figure 2: Influence of the age of a B.thurglglisl HD1 cryB culture on the transformation frequency. Figure 3: Influence of the pH of the PBS buffer solution on the transformation frequency. Figure 4: Influence of the sucrose concentration of the PBS buffer solution on the transformation frequency. Figure 5: Dependence between the number of transformants and the amount of DNA used per transformation. Figure 6: Simplified restriction region of the "shuttle" vector * pXI 61. The shaded bar marks the of the

gram-positive pBC 16 -derived sequences, the remainder being derived from the gram-negative plasmid pUC 8. Figure 7: Simplified restriction map of * pXI93. The hatched bar indicates the protoxin structural gene (arrow, Kurhd 1) and the 5 'and 3' non-coding sequences. The remaining non-Herta part is from the

"Shuttle" vector \ oXI61

 Figure 8: SDS (sodium dodecylsulfate / polyacrylamide gel electrophoresis of extracts of sporulating cultures of B.thurlngiensls HD 1 cryB, B.cereus 569K and their derivatives.

[1: HD1 cryB (pXI93), 2: HD1 cryB (pXI61), 3: HD1 cryB, 4: HD1, LBGB-1449, 5: B.cereus 569K (pXI937.6:

569K)

a) Comassie-stained, M: molecular weight standard, MW: molecular weight (dalton), arrow: position of the 130000-

Dalton protoxin.

b) Western blot of the same gel spiked with polyclonal antibodies against the K-1 crystal protein of B.thuringlensis HDL

Positive bands were detected using labeled anti-goat antibodies. Arrow: Position of the 130-00C DaItOn PrOtoxmS. Other gangrenous degradation products of the protoxin.

Figure 9: Transformation of B. subtills LBG B-4468 with pBC 16 plasmid DNA using the electroporation method optimized for B.thurlnglensls

 (o: transformant plasmid DNA; ·: live germ count / ml)

* The internal name pK chosen in the priority document for the naming of the plasmids has been replaced by the dia officially recognized name pXI for the foreign version.

The designation for the asporogenic B.thuringls HD1 mutant used in the exemplary embodiments was also changed from cryß to cryB.

An essential aspect of the present invention relates to a novel transformation process for B.thurlnglensls and

B. ce reu s, which is based on the introduction of plasmid DNA in B. thurlngiensis and / or B. cereus cells using the known electroporation technology.

After all attempts to transfer the already established in other bacterial host systems transformation method on B. thuringiensls and the closely related B. cereus up to the time of the present invention failed, could now in the context of this invention by the Anv / ending of the electroporation technology and accompanying Measures are achieved a surprising success.

This success has to be regarded as surprising and unexpected, especially in view of earlier electroporation attempts by a Soviet group (Shivarova, N./22/ et al., 1983)

B. thuringiensis protoplasts were performed, but the transformation frequencies achieved were so low that this method was subsequently considered useless for a B. thuringiensis transformation and consequently found no further consideration.

Based on investigations of the process parameters critical for electroporation of B. thuringiensis and / or B. cereus cells, it has now surprisingly been possible to develop a transformation method which is ideally adapted to the needs of B. thuringlensis and B. cereus and to transformation rates that leads in one area

between 10 ⁶ and 10 ⁸ cells / pg plasmid DNA, but in particular in a range between 10 ⁶ and 10 ⁷ cells / pg plasmid DNA.

Approximately the same high transformation rates with values between 10 ² and a maximum of 10 ⁶ Transformanten ^ g plasmid DNA, could previously be achieved only with the described in Schall / 21 / (1986) PEG (polyethylene glycol) transformation method. However, high transformation rates remain limited to those B. thuringiensis strains for which the PEG method has been specifically adapted in very time-consuming optimization studies, making this method unsuitable for practical application.

In addition, in practice, these methods prove in many cases as not or only poorly reproducible.

In contrast, the present process according to the invention is a transformation process applicable in principle to all B.thurlngiensls and B.cereus strains, which is less time-consuming, more efficient and thus more efficient than the traditional PEG transformation methods.

Thus, for example, whole, intact cells can be used in the ancestor according to the invention, which eliminates the time-consuming and for B.thuringlensls and B. cereus critical protoplasting and the subsequent regeneration on complex culture media.

In addition, when using the PEG ancestor, the implementation of the necessary procedural measures can take up to one week, while with the transformation method of the invention, the transformed cells within a few hours (usually overnight) are available.

A further advantage of the method according to the invention relates to the number of B.thurlngiensls and / or B.cereus cells which can be transformed per unit time.

While in traditional PEG procedures only small aliquots can be plated simultaneously to avoid inhibition of regeneration by too dense growth of the cells, large amounts of B. thurl nglensis and / or B. cereus cells can be obtained using the electroporation technique be plated at the same time.

This allows the detection of transformants even at very low transformation frequencies, which is not possible with the above-described methods or only with considerable effort.

In addition, DNA quantities in the nanogram range are already sufficient to obtain at least some transformants.

This is particularly important when a very efficient transformation system is needed, such as when using DNA material from E. coli, which due to a strong restriction system in B.thuringlensls cells compared to B.thurlnglensls DNA to a Reduction of the transformation frequencies by a factor of 10 ³ can lead.

The transformation process of the invention, which is based essentially on the electroporation technology known per se, is characterized by the following specific procedures:

a) preparing a cell suspension with a suitable cell density in a culture medium suitable for the growth of B.thurglnglenels cells and with aeration sufficient for the growth of the cells;

b) separating the cells from the cell suspension and resuspending in an inoculation buffer suitable for subsequent electroporation;

c) adding a DNA sample in a concentration suitable for electroporation;

d) introduction of the approach described under point b) and c) into an electroporation apparatus;

e) one or more short-duration discharges of a capacitor across the cell suspension for short-term generation of high electric field strengths for a time sufficient to transform B.thuringlensls and / or B.cereus cells with recombinant DNA;

f) optionally incubating the electroporierton cells;

g) plating out the electroporated cells on a suitable selection medium and h) reading out the transformed cells.

In a specific and preferred in this invention embodiment of the inventive method, the B.thuringlensh cells are first incubated in a suitable nutrient medium with adequate ventilation and at a suitable temperature, preferably from 3O ⁰ C to 35 ° C, until an optical density (OD ₅₅₀ ) of 0.1 to 1.0 is reached.

The age of the Bacillus cultures intended for electroporation has a marked influence on the transformational equivalency. Therefore, an optical density of the Bacillus cultures of 0.1 to 0.3, in particular of 0.2, is particularly preferred. It should be noted, however, that good transformation frequencies can also be achieved with Bacillus cultures from other growth phases, in particular also with overnight cultures (see FIG. 2).

The starting material used is usually fresh cells or spores, but it can just as well be used on frozen cell material. These are preferably cell suspensions of B. thuringlensis and / or B, cereus cells in suitable liquid media, to which advantageously a certain proportion of an "antifreeze" is added.

Suitable antifreeze agents are primarily mixtures of osmotically active components and DMSO in water or a suitable buffer solution. Other suitable components that may be considered for use in antifreeze solutions include sugars, polyhydric alcohols, such as. Glycerol, sugar alcohols, amino acids and polymers, e.g.

Polyethylene glycol.

Assuming B. thuringlensis spores, they are first inoculated in a suitable medium and incubated overnight at a suitable temperature, preferably from 25 ⁰ C to 28 ⁰ C, and sufficient aeration. This approach is then diluted and treated further in the manner described above.

For the induction of sporulation in B. thurlngiensls all media can be used which cause such sporulation. Preferred in the context of this invention is a GYS medium according to Yousten, A.A., and Rogoff, M.H./23/, (1969).

The oxygen input into the culture medium is usually carried out by moving the cultures, for. On a shaker, with rotational speeds between 50 rpm and 300 rpm being preferred.

The cultivation of B. thuringionsis spores and vegetative microorganism cells in the context of the present invention is carried out according to known, commonly used methods, wherein for reasons of practicability preferably liquid nutrient media are used.

The composition of the nutrient media may vary slightly depending on the B. thuringiensis or B. cereus strain used.

Generally, complex media with poorly defined, readily assimilable carbon (C) and nitrogen (N) sources, as commonly used for the cultivation of aerobic Bacillus species, are preferred.

In addition, vitamins and essential metal ions are required, but are usually contained in the complex nutrient media used in sufficient concentration as components or impurities.

Optionally, said components such. Essential vitamins, as well as Na ⁺ , K ⁺ , Cu ²⁺ , Ca ²⁺ , Mg ²⁺ , Fe ³⁺ , NH ₄ ⁰ , PO ₄ ³ "-, SO ₄ ² " -, Cl "-, CO ₃ ² " ions and the trace elements cobalt and manganese, zinc, etc. may be added in the form of their salts.

Particularly suitable nitrogen sources are, in addition to yeast extracts, yeast hydrolysates, yeast autolysates and yeast cells, especially soybean meal, maize meal, oatmeal, edamine (enzymatically digested lactalbumin), peptone, casein hydrolyzate, corn liquor and meat extracts, without the subject of the invention should be limited by this exemplary listing in any way.

The preferred concentration for the said N sources is between 1.0 g / l and 20 g / l.

As C sources are primarily glucose, lactose, sucrose, dextrose, maltose, starch, cerelose, cellulose and malt extract in question. The preferred concentration range is between 1.0 g / l and 20 g / l.

In addition to complex nutrient media, it is of course also possible to use semisynthetic or fully synthetic media which contain the above-described nutrients in suitable concentration.

In addition to the LB medium which is preferably used in the context of the present inventions, all other culture media suitable for the cultivation of B. thurl nglensls and / or B. cereus can also be used, such as, for example, B. Antibiotic Medium 3, SCGY-Madium et al. The storage of sporulated B. Thurlnglensls cultures is preferably carried out on GYS media (slant agar) at a temperature of 4 ° C.

After the cell culture has reached the desired cell density, the cells are harvested with caps of centrifugation and suspended in a suitable buffer solution, which is previously preferably cooled with ice.

The temperature proved to be non-critical in the course of the investigations and is therefore freely selectable over a wide range. Preferred is a temperature range from 0 ° C to 35 ⁰ C, preferably from 2 ° C to 15 ° C and most preferably from 4 ⁰ C. The incubation time of the Baclllus cells before and after electroporation only minor influences the achievable frequency of transformation (see Table 1). Only an overlong incubation leads to a decrease of the transformation frequency. An incubation time of 0.1 to 30 minutes, in particular 10 minutes, is preferred. The temperature proved to be non-critical in the course of the investigations and is therefore freely selectable over a wide range. Preference is given to a temperature range from ⁰ ° C. to 35 ^° C., preferably from 2 ^° C. to 15 ^° C., and very particularly preferably from 4 ^° C. This process can be repeated once to several times. Particularly suitable buffer solutions in the context of this invention are osmotically stabilized Phosphatpuffei, as a stabilizing agent sugar such. As glucose or sucrose, or sugar alcohols, such as. Mannitol and adjusted to pHs of 5.0 to 8.0. Very particular preference is given to phosphate buffer of the PBS type having a pH of 5.0 to 8.0, preferably of pH 5.5 to 6.5, the sucrose as stabilizing agent in a concentration of 0.1 M to 1, 0M, but preferably from 0.3 M to 0.5 M (see Figures 3 and 4). '

Aliquots of the suspended Bacillus cells are then transferred to cuvettes or any other suitable vessels and probed with a DNA sample for a suitable period of time, preferably for a period of 0.1 to 30 minutes, but more preferably 5 to 15 minutes at a suitable temperature, preferably at a temperature of 0 ⁰ C to 35 ⁰ C, but especially at a temperature of 2 ° C to 15 ⁰ C and most preferably at a temperature of 4 ⁰ C, incubated together.

When working at low temperatures, it is advantageous to use already pre-cooled cuvettes or any other suitable and precooled vessels.

There is a linear relationship between the number of transformed cells and the DNA concentration used in electroporation, with the number of transformed cells increasing with increasing DNA concentration (see Figure 5). The preferred in the context of this invention DNA concentration is in a range between 1 ng

and 20pg. Particularly preferred is a DNA concentration of 10ng to 2pg.

Thereafter, the entire batch containing B.thuringlensls and / or B.cereus cells as well as plasmid DNA or another suitable DNA sample is subjected to electroporation and electroporation, i. H. briefly exposed to an electrical pulse.

Electroporation apparatuses which are suitable for use in the method according to the invention are already offered by various manufacturers, such as. Bio Rad (Richmond, Calif., USA, Gene Pulser Apparatus ¹ ), Biotechnologies and Experimental Research, Inc. (San Diego, Calif., USA; BTXTransfector 100), Promiga (Madison, WI, USA "X-Cell 2000 Electroporation System"), etc.

Of course, in the method according to the invention, any other suitable device can be used.

It can be used different pulse shapes, such. B. rectangular pulses or exponentially decaying pulses.

The latter are preferred in the context of this invention. They are due to the discharge of a capacitor and are characterized by an initially very rapid increase in voltage and by a subsequent exponential decay phase as a function of resistance and capacitance. The time constant RC gives a measure of the length of the exponential decay time. It corresponds to the time required to let the voltage decay to 37% of the output voltage (V ₀ ).

A decisive parameter for influencing the bacterial cell relates to the strength of the electric field applied to the cells, which is calculated from the ratio of the applied voltage and the distance of the electrode plates.

Also of great importance in this context is the exponential decay time, which depends on both the configuration of the apparatus used (eg the capacitance of the capacitor) and on other parameters, such as eg. B. the relationship of the buffer solution or the volumes used for the intended electroporation cell suspension. For example, in the course of the investigations, it has been shown that a reduction of the volume of the cell suspension provided for the electroporation by the hides leads to an increase in the transformation frequency by a factor of 10.

An extension of the exponential decay time via an optimization of the buffer solution used also results in a significant increase in the transformation frequency.

All measures which lead to an extension of the exponential decay time and consequently to an increase in the transformation frequency are therefore preferred in the context of this invention.

The cooldown preferred in the context of the method according to the invention is between about 2 ms and about 50 ms, but in particular between about 8 ms and about 20 ms. Most preferred is an exponential decay time of about 10ms to about 12ms.

In the context of the present invention, the bacterial cells are briefly subjected to very high electric field strengths by short-term discharge (s) of a capacitor via the DNA-containing cell suspension, as a result of which the permeability of the B. thuringiensis cells is increased at short notice and reversibly. The electroporation parameters are matched to one another in the course of the process according to the invention in such a way that an optimal uptake of the DNA present in the electrolysis buffer into the Baclllus cells is ensured.

In the context of this invention, the capacitance setting on the capacitor is advantageously 1 pF to 250 μΡ, but in particular 1 pF to 5OpF and very particularly preferably 25 pF. The choice of the output voltage is not critical in many areas and therefore freely selectable. Preferably, an output voltage V ₀ of 0.2 kV to 5OkV, but in particular from 0.2kV to 2.5kV and

most preferably from 1.2 kV to 1.8 kV. The distance of the electrode plates depends i.a. starting from the dimensioning of the electroporation apparatus. It is advantageously between 0.1 cm and 1.0 cm, preferably between 0.2 cm and 1.0 cm. Particularly preferred is a plate spacing of 0.4cm. From the abs' <ind of the electrode plate and the output voltage set at the capacitor, the field strength values result, which act on the cell suspension. These advantageously lie in a range between 100V / cm and 50000V / cm. Particularly preferred are field strengths of 100V / cm to 10000 V / cm, but in particular from 3000V / cm to 4500V / cm.

The fine-tuning of the freely selectable parameters, such. Capacitance, output voltage, plate spacing, etc., depends to some extent on the architecture of the equipment used and therefore may vary within certain limits from case to case. The specified limit values can therefore also be exceeded or fallen below in certain cases, if this is necessary to achieve optimum field strength values.

The actual electroporation process can be repeated once or several times until an optimum transformation frequency for the respective system has been reached.

Following the Elektropcrntion may advantageously be a post-incubation of the treated Baclllus-Zellon be carried out, preferably for a period of 0.1 to 30 minutes, at a temperature of from ⁰ C to 35 ⁰ C, preferably from 2 ° C to 15 ⁰ C. . Subsequently, warden, the electroporated Zeil m diluted with a suitable medium and again for a suitable period, preferably from 2 to 3 hours, with adequate ventilation, and at a suitable temperature, preferably from 20 ° C to 35 ⁰ C, inkubien.

Subsequently, the B.thurlnglensls cells are plated out on solid media which contain an agent suitable for the selection of the DNA sequences newly introduced into the bacterial cell as an additive. Depending on the type of DNA used, it may be in said agents z. B. to antibiotically active compounds to dyes u.a. act. Particularly preferred in the context of this invention for the selection of Bacillus thuringlensls and / or B.ceroue cells are antibiotics selected from the group consisting of tetracycline, kanamycin, chloramphenicol and erythromycin.

Also preferred are chromogenic substrates, such. As X-gal (ö-bromo ^ -chloro-S-indolyl-ß-D-galactoside), which can be detected by a specific color reaction.

Other phenotypic markers are known to those skilled in the art and may also be used within the scope of this invention.

It can be used any suitable for the cultivation of B.thurlnglensls cells nutrient media to which one of the commonly used solidification media, such as. Ag, r, agarose, gelatin and the like. is added. The process parameters previously described in detail for B. thuringlensls are equally applicable to B. cereus cells.

The previously described method according to the invention for the transformation of B. thuringiensis or B. cereus does not, as has hitherto been the case in the prior art, remain restricted to the use of certain plasmids occurring naturally in B. thuringiensis and / or B. cereus but is applicable to all types of DNA. Thus, it is now possible for the first time to specifically transform B.thungiensis and / or B.cereus, in which case homologous, d. H. naturally occurring in B. thuringiensis or the closely related B.cereus plasmid DNA, also plasmid DNA of heterologous origin can be used.

This may be both plasmid DNA naturally occurring in an organism other than B. thuringiensis or the closely related B. cereus, such as, for example, the plasmids pUB110 and pC194 from Staphylococcus aureus (Horinouchi, S., and Weisblum, B ./24/, 1982; Polak, J., and Novick, RP / ⁰ V, 1982) and the plasmid plMI3 from B.subtillsfMahler, J. and Halvorson, HO / 26 /, 1980), which are capable of in B. thuringiensis and / or B. cereus, as well as hybrid plasmid DNA isolated by recombinant DNA technology from homologous plasmid DNA or from heterologous plasmid DNA or from a combination of homologous and heterologous plasmid DNA. DNA is constructed. The latter hybrid plasmid DNA is more suitable for working with recombinant DNA than the natural isolates. By way of example, the plasmids pBD64 (/ 27 / Gryczan Tet al, 1980), pBD347, pBD348 and pUB1664 may be mentioned here, without, however, in any way restricting the subject matter of the present application.

Of particular importance for carrying out cloning experiments in different B. thuringiensis or B. cereus strains are likely to be the already established B.subtllis cloning vectors such as pBD64. In addition to plasmid DNA, any other DNA can also be transformed into B. thuringiensis or B. cereus in the context of the present invention. The transfornated DNA can either replicate autonomously or integrated in the chromosome. This may be, for example, a vector DNA, which is derived not from a plasmid, but from a phage.

Another object of the present invention relates to the construction of bifunctional vectors ("shuttle" vectors).

Particularly preferred in the context of this invention is the construction and use of bifunctional (hybrid) plasmid vectors, which are capable of being, except in B. thuringiensis or the closely related B. cereus, in one or more species several heterologous host organisms and that are identifiable in both homologous and heterologous host systems.

In the context of this invention, heterologous host organisms are all those organisms which do not belong to the group B. thuringiensis / B. cereus and are capable of stably maintaining a self-replicating DNA. According to the above definition, both prokaryotic and eukaryotic organisms can thus function as heterologous host organisms. By way of example, at this point as representatives of the group of prokaryotic host organisms, individual representatives of the genera Bacillus, such. B. B. subtillis or B. megaterium, Staphylococcus, such as. B. S. aureus, Streptococcus, such as. B. Streptococcus faecalis, Streptomyces, such as. B. Stroptomyces spp., Pseudomonas, such as. B. A.tumefaciens orA.rhlzogenes, Salomonella.Erwinia, etc. mentioned. Of the group of eukaryotic hosts are primarily yeasts and animal and plant cells to call. This exemplary list is not exhaustive and is not intended to limit the scope of the present invention in any way. The person skilled in the art is familiar with further suitable representatives from the group of prokaryotic and eukaryotic host organisms.

Particularly preferred in the context of this invention are B. subtilis or B. megaterium, Pseudomonas spp. and in particular E. coli from the group of prokaryotic hosts and yeasts and animal or plant cells from the group of eukaryotic hosts.

Very particular preference is given to bifunctional vectors which are capable of being produced both in B.thuringlensls and / or B.cereus cells. as well as in E, to replicate coil.

Also included in the present invention is the use of said bifunctional vectors to transform B.thuringlensls and B.cereus.

The construction of "shuttle" vectors is carried out using recombinant DNA technology, wherein plasmid and / or vector DNA homologous (B.thuringlensls, B. cereus) and heterologous origin are cut first by means of suitable restriction enzymes and then those DNA fragments which contain the essential functions for replication in the respective desired host system functions are recombined in the presence of suitable enzymes.

The source of plasmid and / or vector DNA of heterologous origin may be the aforementioned heterologous host organisms.

The linking of the various DNA fragments must be done in a manner that preserves the essential functions for replication in the various host systems.

In addition, it is of course also possible to use plasmid and / or vector DNA of purely heterologous origin for the construction of "shuttle" vectors, but at least one of the heterologous fusion partners must contain DNA segments which can be replicated in the homologous B.thurglnnisis / B. cereus host system.

As a source of plasmid and / or vector DNA of heterologous origin, which is nevertheless capable. in the B.thuringlensls / B.cereus host system, some representatives of the group of eggs gram-positive bacteria may be mentioned at this point, selected from the group consisting of the genera Staphylococcus, such as. B. Staphylococcus aureus, Streptococcus, such as. B. Streptococcus faecaüs, Bacillus, such as. B. Bacillus megaterium or B. subtilis, Streptomyces, such as. B.

Streptomyces spp. etc. There are a number of other organisms which can be used in the process according to the invention and are known to the person skilled in the art, by way of example of the representatives of the group of Gram-positive bacteria listed here by way of example.

A further subject of the present invention thus relates to a process for the preparation of bifunctional vectors which are suitable for the transformation of B.thurlngiensis and / or B. cereus, characterized in that plasmid DNA of homologous and heterologous origin is first of all

a) with the help of suitable restriction enzymes broken into fragments and

b) subsequently those fragments which contain the essential functions for replication and selection in the respective desired host system functions, in the presence of suitable enzymes together again in a way that the essential for replication and selection in the various host systems functions are retained ,

This produces bifunctional plasmids which, in addition to the functions required for replication in B. thuringeinsis or B. cereus, contain further DNA sequences which ensure replication in at least one other heterologous host system.

In order to ensure fast and efficient selection of the bifunctional vectors in both homologous and in the (heterologous) host system (s), it is advantageous to provide said vectors with specific selection markers which are present in both B.thuringlensls and / or B.cereus as well as being useful in the heterologous host system (s), d. H. enable fast and uncomplicated selection. Particularly preferred in the context of this invention is the use of antibiotic resistance encoding DNA sequences, in particular of DNA sequences selected for resistance to antibiotics selected from the group consisting of kanamycin, tetracycline, chloramphenicol, erythromycin u.a. encode.

Also preferred are genes encoding enzymes with a chromogenic substrate, such as. X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). The transformed colonies can then be detected very easily by means of a specific color reaction.

Other phenotypic marker genes are known to those skilled in the art and can also be used within the scope of this invention.

Particularly preferred in the context of this invention is the construction of. In addition to DNA sequences which allow replication in B. thurliensis or B. cereus or in both host systems, shuttle vectors also contain those DNA segments which are suitable for replication in other bacterial host systems, for example in B. subtilis , B. megaterium, Pseudomonasspp., E.coli, etc. are not definitive.

Also preferred are "shuttle" vectors, which replicate both in B. thuringiensis or B. cereus or in both, on the one hand, and on the other hand in eukaryotic host systems selected from the group consisting of yeast, animal and plant cells u.a.

Especially preferred is the construction of "shuttle" vectors which, in addition to DNA sequences necessary for the replication of said vectors in B. thuringlensls or B. cereus or in both systems, also contain such DNA sequences which replicate them Enable "shuttle" vectors in E.coU.

Examples of such starting plasmids for the construction of "shuttle" vectors for the B. thuringiensis S. cerei s / E. coli system, which must not be considered limiting in any way, are the B. cereus plasmid pBCM 6 as well as the plasmid pUC8 derived from E. coli plasmid pBR322 (Vieira, J., and Messing, J., 28/1982).

A further subject of the present invention relates to bifunctional ("shuttle") vectors which, in addition to the functions essential for replication and selection in homologous and heterologous host systems, additionally contain one or more genes in expressible form or other useful DNA sequences of this invention are methods of making these vectors which are characterized by splicing said genes or other useful DNA sequences into these bifunctional vectors with the aid of suitable enzymes.

With the aid of the "shuttle" vectors according to the invention and the transformation method described above, it is thus now possible for the first time to synthesize DNA sequences which have been cloned in a foreign host system in B. thuringiensis and / or B. with high efficiency outside of B. thuringiensls cells. to transform cereus cells.

For the first time, genes or other useful DNA sequences, especially those with a regulatory function, can be stably introduced into B. thuringiensis or B. cereus cells and optionally expressed there, whereby B. thuringiensis or B. Cereus cells with new and desirable properties arise.

As genes that can be used in the context of the present invention, both homologous and come

heterologous gene (s) or DNA as well as synthetic gene (s) or DNA according to the definition made in the context of the present invention as well as combinations of said DNA's.

The coding DNA sequence can be constructed exclusively from genomic, cDNA or synthetic DNA. Another possibility is the construction of a hybrid DNA sequence consisting of both cDNA and genomic DNA and synthetic DNA or a combination of these DNA's.

In this case, the cDNA may be from the same gene as the genomic DNA or both the cDNA and the genomic DNA may be from different genes. In this case, however, both the genomic DNA and / or the cDNA, each alone, may be made from the same or different genes. If the DNA sequence includes portions of more than one gene, these genes can be derived either from one and the same organism, from several organisms, from different strains, or from organisms belonging to more than one genus of the same or another taxonomic unit.

In order to ensure the expression of said structural genes in the bacterial cell, the coding gene sequences must first in operable manner with in it are linked B. cereus cells functional expression sequences B.thuringiensls · and / oc!.

The hybrid gene constructions in the frame: of the present invention thus contain, in addition to the structural gene (s), expression signals which include both promoter and terminator sequences as well as further regulatory sequences of the 3 'and 6' untranslated regions.

Particularly preferred in the context of this invention are the natural expression signals from B. thuringiensls and / or B, cereus itself as well as mutants and variants thereof, which are substantially homologous to the natural sequence. In the context of this invention, a DNA sequence of a second DNA sequence is substantially homologous when at least 70%, preferably at least 80%, but especially at least 90%, of the active portions of the DNA sequence are homologous. As defined herein, the term "substantially homologous" is considered to be homologous to two different nucleotides in a DNA sequence of a coding region when the replacement of one nucleotide with the other represents a silent mutation.

Very particular preference is given to the use of sporulation-dependent promoters from B. thuringiensls which ensure expression as a function of speculation.

Particularly preferred for the transformation of B. thuringiensls or B. cereus in the context of this invention is the use of DNA sequences which code for a δ-endotoxin.

The coding region of the chimeric gene of the present invention preferably contains a nucleotide sequence encoding a polypeptide naturally occurring in B. thuringiensls, or a polypeptide substantially homologous thereto, d. h., Which has at least substantially the toxicity properties of a crystalline δ-endotoxin protein of B. thuringlensis. In the context of the present invention, by definition, a polypeptide has substantially the toxicity properties of the B. thuringiensis δ-endotoxin protein when it is insecticidal to a similar insect larvae as Jas crystalline protein of a subspecies of B. thuringiensis. Some suitable subtypes are, for example, those selected from the group consisting of kurstaki, berliner, alesti, tolworthi, sotto, dendrolimus, tenebrlonis and israelensis. The preferred subspecies for Lepidoptera larvae is kurstaki and especially kurstaki HDI. The coding region may be a region naturally occurring in B. thuringiensls. Alternatively, however, the coding region may optionally also contain a sequence different from the sequence in B. thuringiensis, but which is equivalent to it due to the degeneracy of the genetic code.

The coding region of the chimeric gene may also encode a polypeptide that differs from a naturally occurring crystalline δ-endotoxin protein, but which still has substantially the insect toxicity properties of the crystalline protein. Such a coding sequence will usually be a variant of a natural coding region. By a "variant" of a natural DNA sequence is meant, by definition, a modified form of a natural sequence which still fulfills the same function, but which variant may be a mutant or a synthetic DNA sequence and is essentially the corresponding one In the context of this invention, a DNA sequence of a second DNA sequence is substantially homologous when at least 70%, preferably at least 80%, but especially at least 90%, of the active portions of the DNA sequence are homologous By definition of the term "substantially homologous", two different nucleotides in a DNA sequence of a coding region are considered to be homologous if the exchange of one nucleotide for the other represents a silent mutation.

Thus, any chimeric gene encoding an amino acid sequence that has the insecticidal properties of a B. thuringium oil endotoxin and that meets the disclosed and claimed requirements can be used in the present invention. Particularly preferred is the use of a nucleotide sequence which is substantially homologous to at least the portion or portions of the natural sequence responsible for the insecticidal activity and / or host specificity of the B. thuringiensis toxin.

The polypeptide expressed by the chimeric gene also usually shares at least immunological properties with a natural crystalline protein because it has at least in part the same antigenic determinants. Accordingly, the polypeptide encoded by said chimeric gene is preferably structurally related to the δ-endotoxin of the crystalline protein produced by B. thuringiensis. B. thuringiensis produces a crystalline protein having a subunit corresponding to a protoxin having a molecular weight (MW) of about 130,000 to 140,000. This subunit may be cleaved by proteases or by alkali into insecticidal fragments of MW 70000 and possibly even less.

For the construction of chimeric genes whose coding portion comprises such fragments of the protoxin or even smaller parts thereof, the fragmentation of the coders may proceed as long as the fragments or parts of these fragments still have the requisite insecticidal activity. The protoxin, insecticidal fragments of the protoxin and insecticidal portions of these fragments can be linked to other molecules such as polypeptides and proteins. Coding regions suitable for use in the method of the invention may be obtained from B. thuringiensis genes encoding the crystalline toxins (Whiteley et al. PCT application WO 86/01536 and US patents 4448885 and 4467036). A preferred nucleotide sequence encoding a crystalline protein is between nucleotides 153 and 3623 in formula I or is a shorter sequence encoding an insecticidal fragment of such a crystalline protein (Geiser et al / 5/, 1986, and EP 238,441 ).

Formula I

10 20 30 40 50 60 GTTAACACCC TGGGTCAAAA ATTGATATTT AGTAAAATTA GTTGCACTTT GTGCATTTTT

70 80 90 100 110 120 TCATAAGATG AGTCATATGT TTTAAATTGT AGTAATGAAA AACAGTATTA TATCATAATG

130 ILO 150 160 170 180 AATTGGTATC TTAATAAAAG AGATGGAGGT AACTTATGGA TAACAATCCG AACATCAATG

190 200 210 220 230 240 AATGCATTCC TTATAATTGT TTAAGTAACC CTGAAGTAGA AGTATTAGGT GGAGAAAGM

250 260 270 280 290 300 TAGAAACTGG TTACACCCCA ATCGATATTT CCTTGTCGCT ¹ AACGCAATTT CTTTTGAGTG

310 320 330 340 350 360 AATTTGTTCC CGGTGCTGGA TTTGTGTTAG GACTAGTTGA TATAATATGG GGAATTTTTG

370 380 390 400 410 420 GTCCCTCTCA ATGGGACGCA TTTCTTGTAC AAATTGAACA GTTAATTAAC CAAAGAATAG

430 440 450 460 470 480 AAGAATTCGC TAGGAACCAA GCCATTTCTA GATTAGAAGG ACTAAGCAAT CTTTATCAAA

490 500 510 520 530 540 TTTACGCAGA ATCTTTTAGA GAGTGGGAAG CAGATCCTAC TAATCCAGCA TTAAGAGAAG

550 560 570 580 590 600 AGATGCGTAT TCAATTCAAT GACATGAACA GTGCCCTTAC AACCGCTATT CCTCTTTTG

610 620 630 640 650 660 CAGTTCAAAA TTATCAAGTT CCTCTTTTAT CAGTATATGT TCAAGCTGCA AATTTACATT

670 680 690 700 710 720 TATCAGTTTT GAGAGATGTT TCAGTGTTTG GACAAAGGTG GGGATTTGAT GCCGCGACTA

730 740 750 760 770 780 TCAATAGTCG TVATAATGAT TTAACTAGGC TTATTGGCAA CTATACAGAT CATGCTGTAC

790 800 810 820 830 840 GCTGGTACAA TACGGGATTA GAGCGTGTAT GGGGACCGGA TTCTAGAGAT IGGATAAGAT

850 860 870 880 890 900 ATAATCAATT TAGAAGAGM TTAACACTAA CTGTATTAGA TATCGTTTCT CTATTTCCGA

910 920 930 940 950 960 ACTATGATAG TAGAACGTAT CCAATTCGAA CAGTTTCCCA ATTAACAAGA GAAATTTATA

970 980 990 1000 1010 1020 CAAACCCAGT ATTAGAAAT TTTGATGGTA GTTTTCGAGG CTCGGCTCAG GGCATAGAAG

1030 1040 1050 1060 1070 1080 GAAGTATTAG GAGTCCACAT TTGATGGATA TACTTAACAG TATAACCATC TATACGGATG

1090 1100 1110 1120 1130 1140

CTCATAGAGG AGAATATE TGGTCAGGGC ATCAAATAAT GGCTTCTCCT GTAGGGTTTT

1150 1160 1170 1180 1190 1200

CGGGGCCAGA ATTCACTTTT CCGCTATATG GAACTATGGG AAATGCAGCT CCACAACAAC

1210 1220 1230 1240 1250 1260

GTATTGTTGC TCAACTAGGT CAGGGCGTGT ATAGAACATT ATCGTCCACT TTATATAG / υΛ

1270 1280 1290 1300 1310 1320

GACCTTTTAA TATAGGGATA AATAATCAAC AACTATCTGT TCTTGACGGG ACAGAATTTG

1330 1340 1350 1360 1370 1380

CTTATGGAAC CTCCTCAAAT TTGCCATCCG CTGTATACAG AAAAAGCGGA ACGGTAGATT

1390 1400 1410 1420 1430 1440

CGCTGGATGA AATACCGCCA CAGAATAACA ACGTGCCACC TAGGCAAGGA TTTAGTCATC

1450 1460 1470 1480 1490 1500

GATTAAGCCA TGTTTCAATG TTTCGTTCAG GCTTTAGTAA TAGTAGTGTA AGTATAATAA

1510 1520 1530 1540 1550 1560

GAGCTCCTAT GTTCTCTTGG ATACATCGTA GTGCTGAATT TAATAATATA ATTCCTTCAT

1370 1580 1590 1600 1610 1620

CACAAATTAC ACAAATACCT TTAACAAATE CTACTAATCT TGGCTCTGGA ACTTCTGTCG

1630 1640 1650 1660 1670 1680

TTAAAGGACC AGGATTTACA GGAGGAGATA TTCTTCGAAG AACTTCACCT GGCCAGATTT

1690 1700 1710 1720 1730 1740

CAACCTTAAG AGATE ACTGCACCAT TATCACAAAG ATATCGGGTA AGAATTCGCT

1750 1760 1770 1780 1790 1800

ACGCTTCTAC CACAAATTTA CAATTCCATA CATCAATTGA CGGAAGACCT ATTAATCAGG

1810 1820 1830 1840 1850 1860

GGAATTTTTC AGCAACTATG AGTAGTGGGA GTAATTTACA GTCCGGAAGC TTTAGGACTG

1870 1880 1S90 1900 1910 1920

TAGGTTTTAC TACTCCGTTT AACTTTCA ATGGATCAAG TGTATTTACG TTAAGTGCTC

1930 1940 1950 1960 1970 1980

ATGTCTTCAA TTCAGGCAAT GAAGTTTATA TAGATCGAAT TGAATTTGTT CCGGCAGAAG

1990 2000 2010 2020 2030 2040

TAACCTTTGA GGCAGATE GATTTAGAAA GAGCACAAAA GGCGGTGAAT GAGCTGTTTA

2050 2060 2070 2080 2090 2100

CTTCTTCCAA TCAAATCGGG TTAAAAACAG ATGTGACGGA TTATCATATT GATCAAGTAT

2110 2120 2130 2140 2150 2160

CCAATTTAGT TGAGTGTTTA TCTGATGAAT TTTGTCTGC-A TGAAAAAAAA GAATTGTCCG

2170 2180 2190 2200 2210 2220 AGAAAGTCAA ACATGCGAAG CGACTTAGTG ATGAGCGGAA TTTACTTCAA GATCCAAACT

2230 2240 2250 2260 2270 22ÜO TTAGAGGGAT CAATAGACAA CTAGACCGTG GCTGGAGAGG AAGTACGGAT ATTACCATCC

2290 2300 2310 2320 2330 2340 AAGGAGGCGA TGACGTATTC AAAGAGAATT ACGTTACGCT ATTGGGTACC TTTGATGAGT

2350 2360 2370 2380 2390 2400 GCTATCCAAC GTATTfATAT CAAAAAATAG ATGAGTCGAA ATTAAAAGCC TATACCCGTT

2410 2420 2430 2440 2450 2460 ACCAATTAAG AGGGTATATC GAAGATAGTC AAGACTTAGA AATCTATTTA ATTCuCTACA

2470 2480 2490 2500 2510 2520 ATGCCAAACA CGAAACAGTA AATGTGCCAG GTACGGGTTCCTTATGGCCG CTTTCAGCCC

2530 2540 2550 2560 2570 2580 CAAGTCCAAT CGGAAAATGT GCCCATCATT CCCATCATTT CTCCTTGGAC ATTGATGTTG

2590 2600 2610 2620 2630 2640 GATGTACAGA CTTAAATGAG GACTTAGGTG TATGGGTGAT ATTCAAGATT AAGACGCAAG

2650 2660 2670 2680 2690 2700 ATGGCCATGC AAGACTAGGA AATCTAGAAT TTCTCGAAGA GAAACCATTa GTAGGAGAAG

2710 2720 2730 2740 2750 2760 CACTAGCTCG TGTGAAAAGA GCGGAGAAAA AATGGAGAGA CAAACGTGAA AAATTGGAAT

2770 2780 2790 2800 2810 2820 GGGAAACAAA TATTGTTTAT AAAGAGGCAA AAGAATCTGT AGATGCTTTA TTTGTAAACT

28SO 2840 2850 2860 2870 2880 CTCAATATGA TAGATTACAA GCGGATACCA ACATCGCGAT GATTCATGCG GCAGATAAAC

2890 2900 2910 2920 2930 2940 GCGTTCATAG CATTCGAGAA GCTTATCTGC CTGAGCTGTC TGTGATTCCG GGTGTCAATG

2950 2960 2970 2980 2990 3000 CGGCTATTTT TGMGAATTA GAAGGGCGTA TTTTCACTGC ATTCTCCCTA TATGATGCGA

3010 3020 3030 3040 3050 3060 GAAATGTCAT TAAAATGGT GATTTTAATA ATGGCTTATC CTGCTGGAAC GTGAAAGGGC

3070 3080 3090 3100 3110 3120 ATGTAGATGT AGAAGAACAA AACAACCACC GTTCGGTCCT TGTTGTTCCG GAATGGGAAG

3130 3140 3150 3160 3170 3180 CAGAAGTGTC ACAAGAAGTT CGTGTCTGTC CGGGTCGTGG CTATATCCTT CGTGTCACAG

3190 3200 3210 3220 3230 .3240 CGTACAAGGA GGGATATGGA GAAGGTTGCG TAACCATTCA TGAGATCGAG AACAATACAG

3250 3260 3270 3280 3290 3300 ACGAACTGAA GTTTAGCAAC TGTGTAGAAG AGGAAGTAiA TCCAAACAAC ACGGTAACGT

3310 3320 3330 3340 3350 3360 GTAVTGATTA TACTGCGACT CAAGAAGATE ATGAGGGTAC GTACACTTCT CGTaATCGAG

3370 3380 3390 3A00 3410 3420 GATATGACGG AGCCTATGAA AGCAATTCTT CTGTACCAGC TGATTATGCA TCAGCCTATG

3430 3440 3450 3460 3470 3480 AAGAAAAAGC ATATACAGAT GGACGAAGAG ACAATCC7TG TGAATCTAAC AGAGGATATG

3490 3500 3510 3520 3530 3540 GGGATTACAC ACCACTACCA GCTGGCTATG TGACAAAAGA ATTAGAGTAC TTCCCAGAAA

3550 3560 3570 3580 3590 3600 CCGATAAGGT ATGGATTGAG ATCGGAGAAA CGGAAGGAaC ATTCATCG7G GACAGCGTGG

3610 3620 3630 3640 ₍ 3650 3660 AATTACTTCT TATGGAGGAA TAATATATGC TTTATAATGT'aAGGTGTGCA. UTAAAGAATE

3670 3680 3690 3700 3710 3720 GATTACTGAC TTGTATTGAC AGATAAATAA GGAAATTTT ATATGAATAA AAAACGGGCA

3730 3740 3750 3760 3/70 3780 TCACTCTTAA MGAATGATG TCCCTTTiT GTATGATTTA ACGAGTGATA TTTAAATGTT

3790 3800 3810 3820 3830 3840 TTTTTTGCGA AGGCTTTACT TAACGGGGTA CCGCCACATG CCCATCAACT TAAGAATTTG

3850 3860 3870 3880 3890 3900 CACTACCCCC AAGTGTCAAA AAACGTTATT CTTTCTAAAA AGCTAGCTAG AAAGGATGAC

3910 3920 3930 3940 3950 3960 ATTTTTATG AATCTTTCAA TTCAAGATGA ATTACAACTA TTTTCTGAAG AGCTGTATCG

3970 3980 3990 4000 4010 4020 TCATTTAAC CCTTCTCTTT TGGAAGAACT CGCTAAAGAA TTAGGTTTTG TAAAAAGAAA

4030 4040 4050 4060 4070 4080 ACGAAAGTTT TCAGGAAATG AATTAGCTAC CATATuTATC TGGGGCAGTC AACGTACAuC

4090 4100 4110 4120 4130 4140 GAGTGATTCT CTCGTTCGAC TATGCAGTCA ATTACACGCC GCCACAGCAC TCTTATGAGT

4150 4160 4170 4180 4190 4200 CCAGAAGGAC TCAATAAACG CTTTGATAAA AAAGCGGTTG AATTTTTGAA ATATATTTTT

4210 4220 4230 4240 4250 4260 TCTGCATTAT GGAAAAGTAA ACTTTGTAAA ACATCAGCCA TTTCAAGTGC AGCAC ~ ACG

4270 4280 4290 4300 4310 4320 TATTTTCAAC GAATCCGTAT TTTAGATGCG ACGATTTTCC AAGTACCGAA ACATTTAGCA

4330 4340 4350 4360 CATGTATATC CTGGGTCAGG TGGTTGTGCA CAAACTGCAG

The coding region defined by nucleotides 156b's 3623 of Formula I encodes a polypeptide of the formula

Formula Il

Met Asp Asn Asn Pro Asn He Asn Gius Cys 10

He Pro Tyr Asn Cys Leu Ser Asn Pro GIu 20

VaI GIu VaI Leu GIy GIy GIu Arg He GIu 30

Thr GYy Tyr Thr Pro He Asp He Ser Leu AO

Ser Leu Thr Gin Phe Leu Leu Ser GIu Phe 50

VaI Pro GIy Ala GIy Phe VaI Leu GIy Leu 60

VaI Asp He He Trp GIy He Phe GIy Pro 70

Ser GIn Trp Asp Ala Phe Leu Vai Gin lie ₍ 80

GIu GIn Leu He Asn GIn Arg He GIu GIu 90

Phe AIa Arg Asn Gin Ala He Ser Arg Leu 100

GIu GIy Leu Ser Asn Leu Tyr Gin He Tyr 110

Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp 120

Per Thr Asn Pro AIa Leu Arg GIu GIu Met 130

Arg He GIn Phe Asn Asp Met Asn Ser AIa 140

Leu Thr Thr Ala He Pro Leu Phe Ala VaI 150

GIn Asn Tyr Gin VaI Pro Leu Lau Ser VaI 160

Tyr VaI Gin Ala Ala Asn Leu His Leu Ser 170

VaI Leu Arg Asp VaI Ser VaI Phe GIy GIn 180

Arg Trp Giy Phe Asp Ala Ala Thrhe Asn 190

Ser Arg Tyr Asn Asp Leu Thr Arg Leu He 200

GIy Asn Tyr Thr Asp His Ala VaI Arg Trp 210

Tyr Asn Thr GIy Leu GIu Arg VaI Trp GIy 220

Pro Asp S'jr Arg Asp Tip He Arg Tyr Asn 230

GIn Phe Arg Arg Glu Leu Thr Leu Thr VaI 240

Leu Αερ He VaI Ser Leu Phe Pro Asn Tyr 250

Asp Ser Arg Thr Tyr Pro Hg Arg Thr VaI 260

Ser GIn Leu Thr Arg Glu He Tyr Thr Asn 270

Pro VaI Leu GIu Asn Phe Asp GIy Ser Phe 280

Arg GIy Ser Ala Gin GIy He GIu GIy Ser 290

He Arg Ser Pro Leu Met Asp He Leu 300

Asn Ser Arg Gly He Met Pro GIu Met GIy VaI Ala Thr Leu Phe Asn Ser VaI GIy Thr Tyr Arg Asp GIu Pro Pro Ser His Ser Asn Pro Met GIu Phe He Thr Asn Leu GIy Pro Arg Arg Leu Arr, Gin Arg Ser Thr He Asp Phe Ser Leu Gin Phe Thr Ser Sec Phe Asn Arg He Phe GIu Gin Lys Ser Asn Thr Asp Leu VaI Leu Asp

He Thr GIu Tyr Ala Ser Phe Thr Asn Ala Gin Leu Ser Ser He GIy Leu Asp Ser Ser Lys Ser He Pro Arg Gin VaI Ser Ser Ser Phe Ser Asn Asn Gin He Giy Ser Giy Phe Thr Ser VaI Asn Tyr Arg Thr Asn GIy Arg AI Thr Ser Gly Thr Pro VaI Phe Ser Gly GIu Phe Ala GIu Ala Vai Gin He Tyr His GIu Cys GIu Lys

He Tyr Thr Tyr Trp Ser Pro VaI Gly Phe Pro Leu Ala Pro GIn Gly Gin GIy Thr Leu Tyr He Asn Asn Gly Thr GIu Asn Leu Pro Gly Thr VaI Pro Gin Asn Gly Phe Ser Met Phe Arg VaI Ser He Trp He His He He Pro Pro Leu Thr Gly Thr Ser Thr Gly Gly Pro Gly GIn He Thr AIa VaI Arg He Leu GIn Phe Pro He Asn Met Ser Ser Phe Arg Phe Asn Phe Thr Leu Ser Asn GIu VaI VaI Pro AIa Tyr Asp Leu Asn GIu Leu Gly Leu Lys He Asp Gin Leu Ser Asp Lys Giu Leu

Asp Ala His Gly His Gin Phe Ser Gly Tyr Gly Thr Gin Arg He VaI Tyr Arg Arg Arg Pro Gin Gin Leu Phe Ala Tyr Ser Ala VaI Asp Ser Leu Asn Asn VaI His Arg Leu Ser Gly Phe He Arg AIa Arg Ser AIa Ser Ser GIn Lys Ser Thr VaI VaI Lys Asp He Leu He Ser Thr Pro Leu Ser Arg Tyr AIa His Thr Ser GIn Gly Asn Gly Ser Asn Thr VaI Gly Ser Asn Gly Ala His VaI Tyr He Asp GIu VaI Thr GIu Arg AIa Phe Thr Ser Thr Asp VaI Va.l Ser Asn GIu Phe Cys Ser Glu Lys

310 320 330 3A0 350 360 370 380 390 AOO AlO 420 A 30 AAO A 50 A60 A70 A80 A90 500 510 520 530 5AO 550 560 570 580 590 600 610 620 630 6A0 650 660 670

VaI Lys His Ala Lys Arg Leu Ser Asp Glu 680

Arg Asn Leu Leu Gin Asp Pro Asn Phe Arg 690

GIY He Asn Arg GIn Leu Asp Arg GIy Trp 700

Arg GIy Ser Thr Asp He Thr He Gin GIy 710

GIy Asp Asp VaI Phe Lys GIu Asn Tyr VaI 720

Thr Lei Lei GIy Thr Phe Asp Giu Cys Tyr 730

Pro Thr Tyr Leu Tyr Gin Lys He Asp GIu 740

Ser Lys Leu Lys AIa Tyr Thr Arg Tyr GIn 750

Leu Arg GYy Tyr He GIu Asp Ser Gin Asp 760

Leu GIu He Tyr Leu He Arg Tyr Asn AIa 770

Lys His GIu Thr VaI Asn VaI Pro GIy Thr 780

GIy Ser Leu Trp Pro Leu Ser AIa Pro Ser '790

Pro He Gly Lys Cys Ala His His Ser His 800

His Phe Ser Leu Asp He Asp Vai GIy Cys 810

Thr Asp Leu Asn Giu Asp Leu Giy VaI Trp 820

VaI He Phe Lys He Lys Thr Gin Asp GIy 830

His Ala Arg Leu GIy Asn Leu Giu Phe Leu 840

GIu Giu Lys Pro Leu Vai GIy GIu AIa Leu 850

Ala Arg VaI Lys Arg Ala Glu Lys Lys Trp 860

Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 870

Thr Asn He VaI Tyr Lys GIu AIa Lys GIu 880

Ser VaI Asp Ala Leu Phe VaI Asn Ser GIn 890

Tyr Asp Arg Leu Gin Ala Asp Thr Asn He 900

Ala Met He His Ala Ala Asp Lys Arg Val 910

His Ser He Arg Giu Ala Tyr Leu Pro GIu 920

Leu Ser VaI He Pro GIy VaI Asn Ala Ala 930

He Phe GIu Giu Leu GIu Giy Arg He Phe 940

Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn 950

Vai He Lys Asn Giy Asp Phe Asn Asn GIy 960

Leu Ser Cys Trp Asn VaI Lys Giy His VaI 970

Asp VaI GIu Giu Gin Asn Asn His Arg Ser 980

VaI Leu VaI VaI Pro GIu Trp GIu Ala GIu 990

VaI Ser Gin GIu VaI Arg VaI Cys Pro GIy 1000

Arg GIy Tyr He Leu Arg VaI Thr Ala Tyr '.

Lys GIu GIy Tyr GIy GIu GIy Cys VaI Thr 1020

He His Glu He Glu Asn Asn Thr Asp Glu 1030

Leu Lys Phe Ser Asn Cys VaI GIu GIu GIu 1040

VaI Tyr Pro Asn. \ Thr Thr VaI Thr Cys Asn 1050

Asp Tyr Thr Ala Thr Gin Glu Giu Tyr Glu 1060

GIy Thr Tyr Thr Ser Arg Asn Arg GIy Tyr 1070

Asp GIy Ala Tyr Glu Ser Asn Scr Ser VaI 1080

Pro Ala Asp Tyr Ala Ser Ala Tyr GIu GIu 1090

Lys Ala Tyr Thr Asp Arg Arg Arg Asp Asn 1100

Pro Cys GIu Ser Asn Arg GIy Tyr GIy Asp 1110

Tyr Thr Pro Leu Pro Ala GIy Tyr VaI Thr 1120

Lys Giu Leu Giu Tyr Phe Pro GIu Thr Asp 1130

Lys VaI Trp Hey GIu He GIy GIu Thr GIu HAO

GIy Thr Phe He VaI Asp Ser VaI GIu Leu 1150

Leu Leu Met GIu GIu End 1156

In order to transform a chimeric gene by means of the method according to the invention into B.thurlnglensls or B.cereus cells, the Ge. preferably first incorporated into a vector. Particularly preferred is the incorporation into the bifunctional vectors of the invention.

If the gene of interest is not available in an amount sufficient to be introduced into Bacillus cells, the vector may be first amplified by replication in a heterologous host cell. Most suitable for the amplification of genes are bacterial or yeast cells. When a sufficient amount of the gene is available, it is introduced into the Bacillus cells. The introduction of the gene into B. thurlngiensls or B.cereus cells can be done with the same vector. Particularly suitable are the bifunctional vectors of the invention.

Some examples of bacterial host cells suitable for chimeric replication include bacteria selected from the genera Escherlchla, such as E. coil, Agrobacterium, such as A. rhlzogenes, Pseudomonas, such as Pseudomonas spp. Bacillus, such as B. magneterium or B. subtills, etc. Through the transformation process according to the invention, it is now possible for the first time in the context of this invention to use B.thurlglensls or B.cereus themselves as host cells. Methods of cloning heterologous genes into bacteria are described in U.S. Patents 4,237,224 and 4,468,464.

The replication of genes in E. coil that encode the crystalline protein of B.thuringlensls is described by Wong et al., 29 (1983).

Some examples of yeast host cells suitable for replicating the genes of the invention include those selected from the genus Saccharomyces (European Patent Application EP 0238441).

Any vector in which the chimeric gene can be incorporated and which replicates in a suitable host cell, such as bacteria or yeast, can be used to amplify the genes of the invention. The vector can be derived, for example, from a phage or a plasmid. Examples of phage-derived vectors that can be used in this invention are vectors derived from M13 and λ phage. Some suitable vectors derived from M13 phages include M13mp18 and M13mp19. Some suitable λ vectors derived from λ phages include Xgt11, \ gt7 and X-Charon4.

Of the vectors which are derived from plasmids and are particularly suitable for replication in bacteria, examples which may be mentioned here are pBR 322 (Bolivar et al / 30/, 1977), pUC18 and pUC 19 (Norrander et al / 31/, 1983 ) and Ti plasmids (Bevan et al.

1983) without, however, limiting the scope of the invention in any way. The preferred vectors for amplifying genes in bacteria are pBR322, pUC18 and pUC19.

For cloning directly in B. thuringiensis and / or B. cereus, primarily direct cloning vectors should be mentioned, e.g. pBD347, pBD348, pBD64 and pUB 1664, as well as, in particular, "shuttle" vectors, which have been previously described in detail, but are not limited thereto.

Particularly preferred in the context of this invention are the bifunctional ("shuttle") vectors pXI61 (= pK61) and pXI93 (= pK93) which, transformed in B. thuringiensis var. Kurstaki HD 1 cryB and B. cereus 569K, respectively, in US Pat International Reproduction Agency recognized "German Collection of Microorganisms" (Braunschweig, FRG) in accordance with the provisions of the Budapest Treaty under the number DSM 4 573 (DSM 4 572, transformed into B.thuringlensis var.

kurstaki HD1 cryB) resp. DSM4571 (pXI93, transformed into B. thuringiensis var. Kurstaki HD 1 cryB) and DSM4573 (pXI93, transformed into B. cereus 569 K).

In order to construct a chimeric gene suitable for bacterial replication, a promoter sequence, a 5 'untranslated sequence, a coding sequence and a 3' untranslated sequence are inserted into a vector or assembled in the correct order in one of the vectors described above , Suitable vectors according to the invention are those which are able to replicate in the host cell.

The promoter, the 5 'untranslated region, the coding region and the 3' untranslated region may optionally be first combined outside the vector in one unit and then inserted into the vector.

Alternatively, however, parts of the chimeric gene can also be inserted individually into the vector.

In the case of the B. thuringiensis or B. cereus cloning vectors, this process step can be omitted since the entire unit isolated from B. thuringlensis, consisting of a 5'-mediated region, the coding region and a 3'-untranslated region into which vector can be spliced.

IL ti

The vector also preferably contains a cognate gene, which gives the host cell a property by means of which it is possible to recognize the cells transformed with the vector. Preferred are marker genes encoding an antibiotic resistance. Some examples of suitable antibiotics are ampicillin, chloramphenicol, erythromycin, tetracycline, H / gromycin, G418 and kanamycin.

Also preferred are marker genes containing enzymes with a chromogenic substrate, such as. X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). The transformed colonies can then be detected very easily by means of a specific color reaction.

The insertion or assembly of the gene in the vector is carried out by standard methods, for example, the use of recombinant DNA (Maniatis et al., 33/1982) and the use of homologous recombination (Hinnen et al., 34, 1978).

The methods of recombinant DNA technology rely on first cutting the vector and inserting the desired DNA sequence between the cut pieces of the vector; the ends of the desired DNA sequence are then linked to the corresponding ends of the vector.

The vector is preferably cut with appropriate restriction endonucleases. For example, some restriction endonucleases are those that form blunt ends, such as Sma I, Hpa I and EcoR V, as well as those that form cohesive ends, such as EcoR I, Sacl and BamHI.

The desired DNA sequence normally exists as part of a larger DNA molecule, such as a chromosome, a plasmid, a transposon or a phage. The desired DNA sequence in these cases is excised from its original source and optionally modified so that its ends can be joined to those of the cut vector. For example, when the ends of the desired DNA sequence and the cut vector are blunt-ended, they can be ligated with ligands specific for blunt ends, such as T4 DNA ligase; be connected to each other.

The ends of the desired DNA sequence may also be joined in the form of cohesive ends to the ends of the cut vector, in which case a cohesive-end-specific ligase, which may also be T4 DNA ligase, is used. Another suitable ligase specific for cohesive ends is, for example, E. coli DNA ligase.

Cohesive ends are conveniently formed by cutting the desired DNA sequence and the vector with the same restriction endonuclease. In this case, the desired DNA sequence and the cut vector have cohesive ends which are complementary to one another.

The cohesive ends can also be constructed by attaching complementary, homopolymeric tails to the ends of the desired DNA sequence and the cut vector by means of the terminal deoxynucleotidyl transferase. Alternatively, cohesive ends can also be prepared by attaching a synthetic oligonucleotide sequence recognized by a particular restriction endonuclease known as linker, and cleaving the sequence with the endonuclease (see, for example, Maniatis et al. , 1982).

For the first time within the scope of this invention, it is therefore possible to genetically modify B. clones of genes of the genus, in particular DNA sequences coding for δ-endotoxin, to genetically modify them outside of B. thurlnglensls, and then to clone them in B. thuringlensis and / or B. cereus genes. Cells where said δ-endotoxin genes (in a homologous bacterial host system) can be expressed.

This means that now the genome of B. thuringiensis can be targeted genetically manipulated by first large amounts of plasmid material are obtained in a foreign cloning system and this is then transformed into B. thuringiensis.

Of particular interest is the possibility of modifying the δ-endotoxin genes as well as the control sequences regulating the expression of these genes.

In addition to chimeric genes, it is of course also possible to introduce any other chimeric genetic construct into Bacillus thuringlensls and / or Bacillus cereua cells using the method according to the invention.

For example, using the method of the invention, it is conceivable to introduce antisense DNA into the genome of a Bacillusthuringlensls and / or Bacillus cereus cell so that in the course of expression of said antisense DNA, an RNA encoding the expression of the antisense DNA is transcribed In this way it is possible to specifically suppress the expression of certain unwanted genes in Bacillus thuringiensis and / or Bacillus cereus.

In addition, it is now also possible, in addition to the provision of improved, well-defined B.thurlngiensis strains for the production of improved bioinsecticides, to use B.thuringiensis as a general host for the cloning and optionally expression of heterologous and / or homologous genes.

Moreover, in a specific and preferred embodiment of the method according to the invention, it is now possible for the first time to obtain new genes, but especially new protoxin genes directly in the natural host, i. in B. thuringiensis or B. cereus.

In the search for new protoxin genes, a gene library of B. thuringiensis is first created.

In a first process step, the total DNA of a protoxin-producing B. thuringiensis strain is isolated by means of known methods and then broken down into individual fragments. The B. thurlngiensis DNA can be fragmented either mechanically, for example under the action of shear forces, or preferably by digestion with suitable restriction enzymes. Depending on the choice of enzymes, a partial or complete digestion of the DNA sample is obtained. Particularly preferred in the context of this invention is the use of restriction enzymes which contain quaternary recognition parts and / or lead to a partial digestion of B. thuringlensis DNA, such as. The restriction enzyme SaulllA, but is not limited thereto. Of course, all other suitable restriction enzymes can also be used in the process according to the invention.

The restriction fragments obtained in the manner described above are then separated according to their size by means of methods known per se. For the size-dependent separation of DNA fragments are usually used centrifugation method, such. Sucrose gradient centrifugation or electrophoretic methods such as agarose gel electrophoresis or a combination of these methods.

Those fractions containing fragments of the correct size, d. H. Fragments capable of encoding a protoxin due to their size are pooled and used for further processing steps.

First, the previously isolated fragments are transformed into suitable cloning vectors using standard techniques

spliced and then introduced by means of the transformation method of the invention directly into Bacillus thuringlensis or B. cereus, but preferably in per oxinfreie strains of Bacillus thurlnglonsls. As vectors both gram-positive Plosmide such. PBC 16, pUB 110, pC 194, as well as the previously described "shuttle" vectors are particularly preferred in the context of this invention, the shuttle vector pXI 200, which is described in detail below (see Example 9.1).) Suitable vectors preferably contain DNA sequences which ensure easy identifiability of the transformed, vector-containing clones from the myriad of untransformed clones, Particularly preferred are DNA sequences which code for a specific marker which leads to an easily selectable trait when expressed By way of example, resistance to ampicillin, chloramphenicol, erythromycin, tetracycline, hygromycin, G 418 or kanamycin may be mentioned here as well as marker genes which contain enzymes with a chromogenic substrate, such as, for example, X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) .The transformed colonies can then be easily determined using egg ner specific color reaction can be detected.

No. ι electroporation, the treated Bacillus thuringlensis- be transmitted or B. cereus cells in a selective sporulation medium and there until full sporulation at a temperature of 10 ° C to 40 ⁰ C, but preferably at a temperature of 2O ⁰ C to 35 ⁰ C and most preferably incubated at a temperature of 29 ⁰ C to 31 ⁰ C. The sporulation medium contains as selective substance preferably one of the abovementioned antibiotics, depending on the vector used, as well as a suitable solidifying agent, such as. As agar, agarose, gelatin, etc. In the course of sporulation it comes to the autolysis of the speculative cells, which is for the subsequent screening of great procedural advantage, since an artificial disruption of the cells is eliminated. For clones containing the desired protoxin gene and expressed under the control of their natural promoter, the crystal proteins formed are freely available in the medium. These crystal proteins freely present in the medium can then be fixed, for example, by means of membrane filters or by other suitable measures. Suitable membrane filters are, for example, nylon or nitrocellulose membranes. Membranes of this type are freely available.

The crystal proteins fixed in this way can then be easily detected and identified within the scope of a suitable screening.

Preferred within the scope of this invention is immunological screening using protoxin-specific antibodies. The methods of immunoscreening are known and described in detail, for example, in / 35 / Young et al., 1983. Particularly preferred in the context of the method according to the invention is the use of monoclonal antibodies which specifically recognize a specific part of the protein molecule. These antibodies can be used either singly or in the form of a mixture. In addition, of course, polygonal antisera can also be used for immunoscreening. Mixtures based on monoclonal and polyclonal antibodies are also conceivable.

Methods for producing monoclonal antibodies against Bacillus thuringlensis protoxin proteins are known and e.g. For example, in / 36 / Huber-Lukafi, (1984) or in / 37 / Huber-Lukacetal., (1986) described in detail. These procedures can also be applied in the present case.

The immunological screening method based on antibodies is a part of the present invention. In addition, within the scope of this invention, of course, other suitable screening methods for detecting new DNA sequences in B. thuringiensls and / or B. cereus can be used.

Bacillus thuringiensis and B. cereus cells which have been transformed by the method described above, as well as the toxins produced by these transformed Bacillus cells, are excellently suited for the control of insects, but especially for the control of insects from the orders Lepidoptera, Diptera and Coleoptra. Another object of the present invention thus relates to a method for controlling insects, which is characterized in that insects or their habitat with

a) treating B.thuringiensis or B.cereus cells or with a mixture of both transformed with a recombinant DNA molecule containing a structural gene coding for a δ-endotoxin polypeptide naturally present in B. thuringlensis or a polypeptide substantially homologous thereto; or b) with cell-free crystal body preparations containing a protoxin produced by said transformed Bacillus cells.

Also included in the present invention are insecticidal agents, in addition to the commonly used carrier vehicles, distributing agents or carriers and distributing agents

a) B. thuringiensis or B.cereus cells or a mixture of said Bacillus cells transformed with a recombinant DNA molecule containing a structural gene coding for a δ-endotoxin polypeptide naturally present in B thuringiensis or a polypeptide substantially homologous thereto; or but

b) cell-free crystal body preparations containing a protoxin produced by said transformed Bacillus cells.

For use as insecticides, the transformed microorganisms containing the recombinant B. thuringiensis toxin gene will preferably be transformed live or dead B. thuringiensis or B. cereus cells, including mixtures of live and dead B. thuringiensis and B. cereus Cells, as well as the toxin proteins produced by said transformed cells in unmodified form or preferably together with the usual in the art formulation Hiifstoffe, used and formulated in a conventional manner, for. To suspension concentrates, spreadable pastes, directly sprayable or dilutable solutions, wettable powders, soluble powders, dusts, granules, and also encapsulates in e.g. polymeric substances.

The application methods such as spraying, misting, dusting, scattering, brushing or pouring are chosen in the same way as the type of means according to the desired goals and the given conditions.

Of course, insecticidal mixtures consisting of transformed, live or dead B. thuringiensis and / or B. cereus cells, as well as cell-free crystal body preparations containing a protoxin produced by said transformed Bacillus cells, may of course also be used.

The formulations, d. H. the transformed live or dead Bacillus cells or mixtures thereof and the toxin proteins produced by said transformed Bacillus cells, and optionally solid or liquid adjuvants

containing agents or preparations are prepared in a known manner, for. B. by intimately missing the transformed cells and / or toxin proteins with solid carriers and optionally surface-active compounds (surfactants).

As solid carriers, for. For dusts and dispersible powders, ground natural minerals are generally used, such as calcite, talc, kaolin, montmorillonite or attapulgite. To improve the physical properties, it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. As granular adsorptive granules are porous types such. As pumice, broken brick, sepiolite or bentonite, as non-sorptive support materials such. As calcite or sand in question. In addition, a variety of pregranulated materials of inorganic or organic nature, in particular dolomite or crushed plant residues can be used.

Suitable surface-active compounds are nonionic, cationic and / or anionic surfactants having good dispersing and wetting properties. Among surfactants, surfactant mixtures should also be used.

Suitable anionic surfactants may be both so-called water-soluble soaps and water-soluble synthetic surface-active compounds.

As soaps are the alkali, alkaline earth or unsubstituted or substituted ammonium salts of higher fatty acids (Cio-Cjj), such as. As the no or K salts of oleic or stearic acid or of natural fatty acid mixtures, the z. B. can be obtained from coconut or tallow oil, called. Furthermore, the fatty acid methyl-taurine salts are to be mentioned, such as. For example, the sodium salt of cis -t-methyl-S-octadecenylamino-1-ethanesulfonic acid (content in formulations preferably about 3%).

More often, however, so-called synthetic surfactants are used, in particular Fettsulfonato, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates or fatty alcohols, such as. B. 2,4, /, 9-tetramethyl-5-decyne-4,7-diol (content in formulations, preferably at about 2%).

The fatty sulfonates or sulfates are generally present as alkali metal, alkaline earth metal or optionally unsubstituted ammonium salts and have an alkyl radical having 8 to 22 carbon atoms, wherein alkyl also includes the alkyl moiety of acyl radicals, for. As the Na or Ca salt of lignin, the dodecyl sulfate or a fatty alcohol sulfate mixture prepared from natural fatty acids. This subheading also covers the salts of sulfuric acid esters and sulphonic acids of fatty alcohol-ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and a fatty acid residue having 8-22 C atoms. Alkylarylsulfonates are z. As the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid or a naphthalenesulfonic acid-formaldehyde condensation product.

Furthermore, also corresponding phosphates, we come z. As salts of the phosphoric acid ester of a p-nonylphenol (4 to 14) -ethylene oxide adduct, in question.

Suitable nonclinic surfactants are primarily polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, saturated or unsaturated fatty acids and alkylphenols, which may contain 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon radical and 6 to 18 carbon atoms in the alkyl radical of the alkylphenols.

Further suitable nonionic surfactants are the water-soluble polyethylene oxide adducts containing from 20 to 250 ethylene glycol ether groups and from 10 to 100 propylene glycol ether groups with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol having from 1 to 10 carbon atoms in the alkyl chain. The compounds mentioned usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Examples of nonionic surfactants which may be mentioned are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene / polyethylene oxide adducts, tert-butylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Also suitable are fatty acid esters of polyoxyethylene sorbitan, such as the polyoxyethylene sorbitan trioleate.

The cationic surfactants are, above all, quaternary ammonium salts which contain at least one alkyl radical having 8 to 22 carbon atoms as N-substituent and which have unsubstituted or halogenated lower-alkyl, -benzyl or lower hydroxyalkyl radicals as further substituents. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. The stearyltrimethylammonium chloride or the benzyldi (2-chloroethyl) ethylammonium bromide.

The surfactants commonly used in formulation technology are u. a. described in the following publications:

/ 38/1986 International McCutcheon's Emulsifiers 81 Detergents, The Manufacturing Confectioner Publishing Co., Glen Rock, NJ, USA; Helmut Stäche "Tensid-Taschenbuch" Carl Hanser-Verlag Munich / Vienna 1981. The agrochemical compositions generally contain 0.1 to 99%, in particular 0.1 to 95%, of the transformed, live or dead Bacillus cells or mixtures thereof or the toxin proteins produced by said transformed Bacilliis cells, 99.9 to 1%, in particular 99.8 to 5%, of a solid or liquid additive and 0 to 25%, in particular 0.1 to 25%, of one surfactant.

While merchandise tends to be more concentrated, the end user typically uses dilute formulations.

Such agents may contain other additives such as stabilizers, defoamers, viscosity regulators, binders, adhesives and fertilizers or other agents to achieve special effects.

The transformed live or dead Bacillus cells or mixtures thereof containing the recombinant B.thuringlensis toxin genes as well as the toxin proteins produced by said transformed Bacillus cells themselves are highly suitable for the control of noxious insects. Preferably, plant-destroying insects of the order Lepldoptei a be mentioned, especially those of the genera Plerls, Hellothis, Spodoptera and Plutella, such as Pieris brassicae, Heliothis virescens, Hellothis zea, Spodoptera llttoralis and Plutella xylostella.

Other harmful insects that can be controlled with the aid of the insecticidal preparations described above, for example, beetles of the order Coleoptera, especially those of the family Chrysomelidae, such as. B. Diabrotica undecimpunctata, D. longicornis, D. virgitera, D. undecimpunctata howardi, Agelastica alni, Leptinotarsa decemlinoata, etc. as well as insects of the order Diptera, such as Anopheles sergentii, Uranatenla ungulculata, Culex univittatus, Aedes aegypti, Culex piplens, etc.

The application rates in which the Bacillus cells or the toxin proteins produced by them are used depend on the respective conditions, such as, for example, the weather conditions, the soil condition, the plant growth and the time of application.

Formulloruiiflsbelspiele for B. Thuringlensls Toxln containing material

In the following formulation examples, the term "Bacillus cells" is to be understood as meaning those B.thurglnlsls and / or B.cereus cells which contain a recombinant B. thuringlensis gene according to the invention. (The data are by weight percentages throughout .)

F1. granules a) b) Bacillus cells and / or from this produced toxin protein 5% 10Ή kaolin 94% - Highly dispersed silicic acid 1% - attapulgite - 90 ° / i

The Bacillus cells and / or produced by this toxin protein are first suspended in methylene chloride, then the suspension is sprayed onto the support material and then the suspending agent is evaporated in vacuo.

F 2. Dusting agent a) b) Bacillus cells and / or from this produced toxin protein 2% ' 5% Highly dispersed silicic acid 1% 5% talc 97% - kaolin _ 90%

Ready-to-use dusts are obtained by intimately mixing the excipients with the Bacillus cells and / or the toxin protein produced therefrom.

F 3. Injection powder Bacillus cells and / or toxin protein produced by them Sodium lignin sulfonate Sodium lauryl sulfate Sodium diisopropylnaphthalenesulfonate Octylphenol polyethylene glycol ether (7-8 moles of ethylene oxide

Highly dispersed silica kaolin

The Bacillus cells and / or toxin protein produced by them are thoroughly mixed with the additives and the mixture obtained is then ground well in a suitable mill.

This gives wettable powders which can be diluted with water to give suspensions of any desired concentration.

a) b) C) 25% 50% 75% 5% 5% - 3% - 5% - 6% 10% _ 2% _ 5% 10% 10% 62% 27% _

F4. Extruder granules Bacillus cells and / or toxin protein produced by them Sodium lignosulfonate Carboxymethylcellulose Kaolin

10% 2% 1% 87%

The Bacillus cells and / or toxin protein produced by them are mixed with the excipients, carefully pre-ground and moistened with water. This mixture is extruded and then dried in a stream of air.

F5. Envelope granules Bacillus cells and / or toxin protein produced by them Polyethylene glycol 200 kaolin

3% 3% 94%

The homogenously mixed Bacillus cells and / or toxin protein produced by them are uniformly applied in a mixer to the kaolin moistened with polyethylene glycol. In this way, dust-free coating granules are obtained.

F6. suspension concentrate 40% Bacillus-ΖοΙΙθπ and / or from 10% this produced toxin protein Ethylenglykot 6% Nonylphenolpolyethylenglykol (15 moles of ethylene oxide) 3% Alkylbenzolsulfonsäure- 1% triethanolamine * carboxymethylcellulose 0.1% Silicone oil in the form of a 75% 39% aqueous emulsion water

* Alkyl is preferably linear with 10 to 14, especially 12-14 carbon atoms such as n-Dodecylbenzolsulfonsiluretriethanolaminsalz.

The homogeneously mixed Bacillus cells and / or produced by this toxin protein are intimately mixed with the Zusatzst open. This gives a suspension concentrate from which suspensions of any desired concentration can be prepared by dilution with water.

embodiments General recombinant DNA techniques

Since many of the recombinant DNA techniques used in this invention are routine to those skilled in the art, a brief description of the commonly used techniques will be given below, so that these general details may be dispensed with in the concrete embodiment. Unless specifically stated, all of these are described in the reference of / 33 / Maniatis et al., 1982.

A. Cutting with restriction endonucleases

Typically, about 50 μg / ml to 100 μg / ml of DNA are included in the reaction mixture in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA. For jsdespg DNA, add 2 to 5 units of restriction endonucleases and incubate the reaction amine at the manufacturer's recommended temperature for one to three hours. The reaction is terminated by heating at 65 ° C for 10 minutes, by extraction with phenol, followed by precipitation of the DNA with ethanol. This technique is also described on pages 104-106 of the / 33 / Maniats et al. Reference.

B. Treatment of the DNA with polymerase to produce blunt ends

50 μg / ml to 500 μg / ml DNA fragments are added to a reaction mixture in the buffer recommended by the manufacturer, New England Biolabs. The reaction mixture contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. After addition of a suitable DNA polymerase, the reaction is carried out at 15 ° C. for 30 minutes and is then terminated by heating at 65 ° C. for 10 minutes. For fragments obtained by cleavage with restriction endonucleases which generate 5 'protruding ends, such as EcoRI and BamHI, the large fragment, or Klenow fragment, of the DNA polymerase is used. For fragments obtained by endonucleases that generate 3'-protruding ends, such as Pst I and Sac I, the T4 DNA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of the / 33 / Maniatis et al.

C. Agarose gel electrophoresis and purification of DNA fragments from gel contaminants

The agarose gel electrophoresis is performed in a horizontal apparatus as described on pages 150-163 of the / 33 / Maniatis et al. Reference. The buffer used corresponds to the Tris-Boat or Tris acetate buffer described therein. The DNA fragments are stained by 0.5 μg / ml ethidium bromide, which is either present in the gel or in the tank buffer during electrophoresis, or optionally added after electrophoresis. The DNA is visualized by illumination with long-wave ultraviolet light. When the fragments are to be separated from the gel, agarose is used which can be stored at low temperature and purchased from Sigma Chemie, I, St. Louis, Missouri. After electrophoresis, the desired fragment is excised, placed in a plastic tube, heated to 65 ^° C. for about 15 minutes, extracted three times with phenol, and precipitated twice with ethanol. This procedure is opposite to that of / 33 / Maniatis et al. on page 170 slightly changed.

Alternatively, the DNA can also be isolated from the agarose gel using the "Geneclean Kit" (Bio 101 Inc., La JoIIa, CA, US).

D. Removal of 5'-terminal phosphates from DNA fragments

During the plasmid cloning steps, treatment of the vector plasmid with phosphatase reduces vector recircularization (discussed on page 13 of the / 33 / Maniatis et al. Reference). After cleaving the DNA with the correct restriction endonuclease, one unit of calf intestinal alkaline phosphinease, which can be purchased from Boehringer-Mannheim, Mannheim, is added. The DNA is incubated for one hour at 37 ° C and then extracted twice with phenol and precipitated with ethanol.

E. Linking the DNA fragments

When fragments with complementary cohesive ends are to be linked together, approximately 100 ng of each fragment is incubated in a 20 μΙ to 40 μΙ reaction mixture with approximately 0.2 unit T4 DNA ligase from New England Biolabs in the manufacturer's recommended buffer. The incubation is carried out at 15 ^° C. for 1 to 20 hours. When DNA fragments are to be linked to gated ends, they are incubated as described above, but the amount of T4 DNA ligase in this F- ¹ II is increased to 2 to 4 units.

F. The transform of DNA into E. coil

E. coli strain HB101 is used for most experiments. DNA is introduced into E. coli by the calcium chloride method as described by / 33 / Maniatis et al., Pages 250 to 251.

G. Screening of E. coli for plasmids

After transformation, the resulting colonies of E. coli are checked for the presence of the desired plasmid by a rapid plasmid isolation procedure. Two common methods are described on pages 366 to 369 of the / 33 / Maniatis et al reference.

H. Large scale isolation of plasmid DNA

Methods for the large scale isolation of plasmids from E. coli are described on pages 88 to 94 of the / 33 / Maniatis et

«..- reference described. ι

Media and buffer solutions (G / l) LB medium 10 tryptone 5 yeast extract 5 NaCl (G / l) Antibiotic Medium No. 3 (Difco Laboratories) 1.5 Cattle meat extract 1.5 yeast extract 5 peptone 1 glucose 3.5 NaCl 3.68 K ₂ HPO ₄ 1.32 KH ₂ PO ₄ (G / l) SCGY medium 1 Casaminoacids 0.1 yeast extract 5 glucose 14 K ₂ HPO ₄ 6 KH ₂ PO ₄ 1 Na ₃ citrate 2 (NH ₄ I ₂ SO ₄ 0.2 MgSO ₄ -7H ₂ O (G / l) GYS medium (/ 22 / Yousten & Rogoff, 1969) 1 glucose 2 yeast extract 2 (NH ₄ J ₂ SO, 0.5 K ₂ HPO ₄ 0.2 MgSO ₄ -7H ₂ O 0.08 CaCl ₂ -2H ₂ O 0.05 MnSO ₄ .H ₂ O Set pH to 7.3 before autoclaving. iMM) PBS buffer 400 sucrose 1 MgCl ₂ 7 Phosphate buffer, pH 6.0 (MM) TBST buffer 0.05% (w / v) Tween 20 * 10 Tris / HCl "(pH 8.0) 150 NaCl

* Tween 20 polyethoxysorbitan laurate

Tris / HCl α, α-tris-hydroxymethyl-methylamino hydrochloride

The internal designation pK chosen in the priority document for the naming of the plasmids has been replaced by the officially recognized symbol pXI for the foreign version.

Also, the designation for the asporognous B. thuringlonsis HDt mutant used in the examples of the embodiments was changed from cryβ to cryB.

Example 1: Transformation of B.thurlngiensis by means of electroporation

Example 1.1: 10 ml of an LB medium (tryptone 10 μg / l, yeast extract 5 g / l, NaCl 5 g / l) were mw spores of B.thurlnglonsl var. Kurstaki HDIcryB (/ 39 / Stahly DP et al., 1978) of a plasmid-free variant of B.thuringlensls var. kurstaki HD1, inoculated. This Ansaü is incubated overnight at a temperature of 27 ° C on a rotary shaker at 50Upm. Thereafter, the pg / ml culture is diluted 100 times in 100 ml to 400 ml LB medium and cultured further at a temperature of CO ⁰ C on a rotary shaker at 250 rpm until an optical density (ODc ₆₀ ) of 0.2 is reached. The cells are harvested by centrifugation and suspended in Vw volumes of an ice-cold PBS buffer (40 mM sucrose, 1 mM MgCl ₂ , 7 mM phosphate pH 6.0). Centrifugation and subsequent suspension of the harvested β-thuringiensis in PBS buffer is repeated once.

The thus pretreated Ze'ien can then either directly electroporated or after addition of glycerol in the buffer solution (20% [w / v]), stored at -2O ⁰ C to -70 ° C and used at a later date. 800μΙ aliquots of the iced cells are then transferred to precooled cuvettes, then 0.2 μg of pBC16 plasmid DNA (/ 40 / Bernhard K. et al., 1978) (20 μg / ml) is added and the entire batch is incubated at 4 When using frozen cell material, a suitable aliquot of frozen cells is first thawed in ice or at room temperature, followed by treatment using fresh cell material, and the cuvette is placed in an electroporation apparatus and in the suspension present B.thuringlensis cells are subjected to electroporation, so that they are subjected to voltages of between 0.1 kV and 2.5 kV by discharging once a capacitor.

The capacitor used here has a capacitance of 25pF, the cuvettes have an electrode spacing of 0.4 cm, resulting in a discharge depending on the setting to an exponentially decreasing field strength with initial peak values of 0.25kV / cm to 6.25kV / cm , Disexponentielle cooldown is in a range between 10ms and 12ms. For the electroporation experiments described, for example, a Bio Rad electroporation machine may be used ("Gene Pulser Apparatus", # 165-2075, Bio Rad, 1414 Harbor Way South, Richmond, CA & 4804, USA) the method according to the invention can be used. According to a further lOminütigen incubation at 4 ⁰ C, the cell suspension is diluted with 1.2 ml of LB medium and incubated for 2 hours at a temperature of 3O ⁰ C on a rotary shaking machine at 250Upm.

Subsequently, suitable dilutions are plated on LB agar (LB medium solidified with agar, 15 g / l) which contains an antibiotic suitable for the selection of the newly obtained plasmid as an additive. In the case of pBC 16, this is tetracycline, which is added to the medium in a concentration of 20 mg / l.

The transformation frequencies peculiar to B.thuringienslsHD 1cryB and -pBC 16 depending on the applied output voltage at given plate spacing are shown in Figure 1.

The expression of the introduced DNA can be detected by the occurring tetracycline resistance. As early as 2 hours after the transformation of pBC 16 into B. thuringiensls, a complete phenotypic expression of the newly introduced tetracycline resistance takes place (see Table 2).

Example 1.2: The transformation of B. thuringiensis cells is performed in exactly the same way as Example 1.1. with the exception that the volume of the cell suspension intended for electroporation is in this case 400 μΙ. This procedural measure can increase the transformation frequency by a factor of ten.

Example 2: Transformation of B. thuringlensis HD1 cry B with a variety of plasmids Most of the experiments are performed with the plasmid pBC 16, a naturally occurring plasmid from B. Cereus. In addition, other naturally occurring plasmids can be successfully introduced into B.thurlnrjlensls- cells such as PUB1 ¹ O (/ 25 / Polack, J., and Novik, RP, 1982), pC194 (/ 24 / Horinouchi, S., and Weisblum, B., 1982) and plM 1i (/ 20 / Mahler, I., and Halvorson, HO, 1980) (see Tabello 3).

Also variants of these plasmids, which are more suitable for the Aiueiten with lekombinanter DNA as the natural isolates can be transformed using the method of the invention in the B. thuringiensls · strain HD 1 cryB, such. B. the B. subtilis cloning vector pBD64 (/ 27 / Gryczan, T., et al., 1980) and the plasmid pBD347, pBD348 and pUB1 664 (see Table 3; the plasmid pBD347, pBD348 and pi IB1164 can be obtained from Dr. W Schurter, CIBA-GEGY AG, Basel). The transformation results of Table 3 make it clear that regardless of the plasmid DNA used when using the transformation method according to the invention transformation frequencies are achieved, which are all in the range between 10 ^s to 10 ⁷ with one exception.

Example 3: Construction of a shuttle vector for Bacillus thuringiensis

Existing bifunctional vectors for E. coil and B. subtilis such. PHV33 (/ 41 / Primrose, S.B., and Ehrlich, S.D., Plasmid, 6:

193-201, 1981) are not suitable for B. thuringiensis HD 1 cryB (see Table 3).

For the construction of a potent bifunctional vector, the large EcoRI fragment of PBC16 is first inserted by means of T4 DNA ligase into the EcoRI site of plasmid pUCP 728 / Vieira, J., and Messing, J., 1982). Subsequently, E. coli cells are transformed with this construct. A construction recognized as correct by means of a restriction analysis is called pXI62.

This is followed by removal of the EcoRI site distal to the pUC8 polylinker region. By partial EcoRI digestion, pXI 62 is linearized. The EcoRI cohesive ends are filled in with Klenow polymerase and reincorporated with T4 DNA ligase. After transformation into E. coil, a construction recognized as correct by means of a restriction analysis is selected and named pXI61. A map of pX61 with the restriction enzyme sites intersecting pXI61 only once is shown in Figure 3.

This construction can be transformed directly into B.thuringls' HD1 cryB using the transformant described in Example 1.

Due to strong restriction barriers in B.thurlnglensis strains, the transformation rates in this case are lower when using E. coli-derived pXI61 DNA than when using plasmid DNA derived from B.thurlglensl HD1 cryB (see Table 3). Nevertheless, pXI61 proves to be so suitable for carrying out cloning experiments in B.thurlglensls.

Example 4 Intake of the Kurhdi delta-endotoxins In strains of B. thurl nglensls and B. cereus The DNA sequence used in the context of this invention for the introduction and expression in B. thurl nglensls and B. cereus, respectively, which is suitable for a short-term delta- Endotoxin protein is derived from the plasmid pK36, the date of March 4, 1986 at the recognized as International Depository German Collection of microorganisms, Germany, in accordance with the requirements of the Budapestor Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patenting deposited under deposit number DSM3668.

A detailed description of the methods for the identification and isolation of the δ-endotoxin genes and the construction of the plasmid pK36 is contained in the European patent application EP 0238441 and in the form of a reference a part of the present invention.

pK36 plasmid DNA is completely digested with the restriction enzymes Pst 1 and BamHI and the 4.3 Kb fragment Fragmsnt, which contains the Kurhd 1 delta-endotoxin gene (see formula I), is isolated from an agarose gel. This fragment is then injected into pXI61, which was previously digested with Pst 1 and BmH 1 and treated with calf intestinal alkaline phosphatase. After the transformation of E. coll HB101, a construction identified as correct by means of a rust analysis analysis, which is named pXI93, is isolated. A restriction map of pXI 93 is shown in Figure 7. pXI93 can be introduced in two different ways in B. thurlngiensls HD 1 cryB.

a) B. Thurlnglensls cells are directly transformed with a pXI93 isolate from E. coil using the transformation method according to the invention described in Example 1.

b) pXI93 is first transformed into B. snbtllis cells as described by Chang and Cohen, 1979. From a transformant containing the complete and intact pXI93 plasmid DNA, this is isolated and then transformed into B.thurglglgsl HD1 cryB using the electroporation method described in Example 1.

The application of both methods leads to transformants containing the intact pXI93 plasmid, which can be demonstrated by restriction analysis.

Example 5: Detection of the Expression of the Delta Endotoxin Gene in B. Thuringiens Sporulating cultures of B. thuringiensis HD1 cryB, HD1 cryB (pXI61) and HD1 cryB (pXI93) and HD1 are compared in phase-contrast microscopy at 400X magnification. Only in pXI93 and HD1-containing strain can the typical bipyramidal protein crystals be detected. Extracts from the same cultures are electrophoresed on an SDS polyacrylamide gel. Only for the plasmid containing pXI93 and HD 1 could a protein band of 130000 daltons be detected on the gel, which corresponds to the Kurhd 1 gene product (Figure 8a).

In the "Western blot" analysis (Figure 8b), this 130000 dalton protein and its degradation products react specifically with polyclonal antibodies which had previously been tested against crystal protein from B.thurlnglensis var. Kurstoki HD 1 according to / 42 / Huber-Luksc, H. A detailed description of this process can be found in European Patent Application EP 238.4 A1, which is a part of this invention in the form of a reference: Plasmid pXI93 is located upstream of the toxin kc-gating region a 156-bp DNA segment containing the above-described sporulation-dependent J.-i Toi'.dem promoter (/ 29 / Wong, HC, ot al, 1983) This sequence is sufficient for high expression of the dibi indotoxin gene in B. thuringiensis HD1 cryB and B.cereus 569K.

EXAMPLE 6 Evidence of the Toxic Activity of the Vekombinant B. Thuringl H01 cryB (pXI93) B.thuringlensbHDI cryB and HD1 cryB (nX! 93) are grown at 25 ° C in spinal medium (GYS medium). After complete sporulation in the phase-contrast microscope is controlled, spores and (if any) protoxin crystals are harvested by centrifugation and spray-dried. The resulting powder is added at various concentrations to the diet of L-1 larvae of He'Iothls virescens (tobacco budworm). The mortality of the larvae is determined after six days.

As expected, the protoxin-free Sta.r.m HD 1 cryB is not toxic to Heliothis virescens, while the strain transformed with plasmid pXI93 causes a dose-dependent mortality of H. virescens (Table 4). This shows that recombinant strains prepared by the electroporation method can actually be used as the bioinsecticide.

Example 7: Elaboration of various B. thuringienols and R. spec. Strains The transformation protocol for β-thuringiensis HD 1 cryB described in Example 1 can also be applied to other strains.

All tested strains of B.thurlnglensi j var. Kurstaki lasser can be transformed easily and efficiently with this method (section 5).

With a B.cereus laboratory strain excellent transformation frequencies can also be achieved. The same is true of the B. thuringiens var si ken (var israelensis, var kurstaki). B. subtilis, on the other hand, can only be transformed very poorly by the Eiekiroporiitic.

By contrast, the application of the prochoplast-dependent: PEG-Methoc s with B. subtilis transformation rates of 4 χ 10 ⁶ / μρ plasmid DiMA could be achieved.

The low transmissivity rates of B.subtilis using electroporation technique are not related to selected parameters, e.g. As an inappropriate voltage or with a high mortality rate, caused by ele .'- 'rische pulses, as shown in the Fig. 9 can be seen.

Example 8: Transformation of B. thuringia HD 1 cry ^p with the β-galactosidase gene 8.1. Incorporation of a BamHI Restrlctlonsschnlttstelle immediately before the first AUQ - codon of B.thurlngiensls Protoxlngens Before the ß-G & lactosidase gene from the Plaomid piWiTh5 (available from Dr. M. Geiser, CIBA-GEIGY AG, Basel, Switzerland) with the promoter of Kurhd 1 δ-endotoxin gene from B. thurlngiensls can be linked, first the located in the vicinity of the AUG start codon DNA sequence of the protoxin gene must be modified.

This modification is performed by oligonucleotide-mediated mutagenesis using the single stranded phage M 13mp8 containing the 1.8 kb Hincll-HindIII fragment from the δ-endotoxin gene comprising the B 'region of the toxin gene.

Zunächs; 3 μg of the plasmid pK36 (see Example 4) are digested with the restriction enzymes HindIII and Hindi. The resulting 1.8kb fragment is purified by agarose gel electrophoresis and then isolated from the gel.

In parallel, 100 ng of M13 mp8 DNA: B-DNA (Biolab, Tozer Road, Beverly MA, 01915, USA or any manufacturer) are digested with the restriction enzymes Smal and HindIII, subjected to phenylation and precipitated by the addition of ethanol. The thus treated phage DNA is then mixed with 200r> g of the previously isolated protoxin fragment and linked to it by the addition of T4 DNA ligase.

Following transfection of E. coli JM103, 6 white plaques are picked and analyzed by restriction mapping.

An isolate in which the link between de, n ß-galactosidase gene and the promoter of the Kurhd 1 δ-endotoxin gene from B.thurli.glensls has been done correctly, is drawn and receives the name M13 mp8 / Hinc-Hind.

Using a DNA synthesizer ("APPLIED BIOSYSTEM DNA SYNTHESIZER"), an oligonucleotide is synthesized which has the following sequence:

(5 ¹ ) GTTCGGATTGGGATCCATAAG <3 ')

This synthetic oligonucleotide is complementary to a region on the M 13mp8 / Hinc-Hind DNA which extends from position 153 to position 173 of the Kurdh 1 δ-endotoxin gene (see formula I). However, the oligonucleotide sequence set forth above has a "mismatch" in position 162 and 163 over the sequence defied in Formula I, which results in the formation of a BamHI restriction site The general procedure for mutagenesis is described in JM Zoller and M. Smith (/ 43 / JM Zoller and M.Smith;.. 19) described about 5pg single M 13mp18 / Hinc-Hind phage DNA is mixed with 0 ^ g phosphorylated Obligonukleotiden in a total volume of 40μΙ This mixture is heated to 65 ⁰ C for 5 minutes then zuniichst cooled to 5O ⁰ C and then cooled gradually to 4 ^0C. Thereafter, buffer, nucleotide triphosphates, ATP, T4 DNA ligase and large fragment of DNA polymerase are added and incubated overnight at 15 ⁰ C, as described ( / 43 / JM Zoller and M. Smith) After agarose gel electrophoresis, circular double-stranded DNA is purified and transfected into E. coli strain JM103 ' Alternatively, the E.coll strain JM107 can be used.

The resulting plaques are probed for sequences that hybridize with ³² P-labeled oligonucleotide; the phages are examined by DNA restriction endonuclease analysis.

A phage containing a correct construction in which h has a BamHI site just prior to the first AUG codon of the protoxin gene is designated M ISmpe / iHinc-Hind / bam.

8.2 Linkage of the β-galactosidase gene with the δ-endotoxin promoter

8.2.1: The δ-endotoxin promoter is located on a 162 bp EcoRI / BamHI fragment of M13mp8 / Hinc-Hind / Bam phage DNA. RF phage DNA is digested with the restriction enzyme BamHI. The overhangs resulting from this at the 5 'ends are removed by treatment with "Mung Bean" nuclease (Biolabs) according to the manufacturer's instructions.

Thereafter, the DNA is digested with the restriction endonuclease EcoRI and the fragment comprising 162 bp isolated from the agarose gel after carrying out an agarose gel electrophoresis.

The β-galactosidase gene is isolated from the plasmid piWiTh 5. piWiTh 5 DNA is first cut at the unique HindIII site. The 3 'recessed ends are filled in with the help of the Klenow fragment of DNA polymerase (see / 33/ Maniatis et al., 1983, pages 113-114) and the modified DNA is then digested with the restriction enzyme Sail. The DNA fragment containing the β-galactosidase gene is isolated by means of agarose gel electrophoresis.

The vector pXI61 (see Example 3) is digested with the restriction enzymes EcoRI and Sail and the two previously isolated fragments are spliced into the vector pXI61.

After transformation of this ligation mixture into the E. coli strain HB101 or JM107, the correctly linked cell clones are prepared by restriction analysis and their β-galactosidase activity against the chromogenic substrate X-gal (S-bromo-chloro-Sindolyl). β-D-galactoside). A clone that contains a correct genetic construct is called pXI80.

8.2.2 .: In an alternative embodiment, the 162 bp EcoRI / BamHI fragment containing the δ-endotoxin promoter is isolated by cutting M13mp8 / Hinc-Hind / Bam with EcoRI and BamHI and subsequent gel electrophoretic separation.

The β-galactodase gene is also isolated in this case from the plasmid piWiTh 5 (see Example 8.1.). The plasmid DNA was digested with the resuItion enzymes BamHI and BGIII and the large fragment was eluted from the agarose gel after gel electrophoresis.

The vector phY300 PLK (# PHY-001 Toyobo Co., Ltd., 2-8 Dojima Hama 2-Chome, Kita-ku, Osaka, 530 Japan), which can be obtained commercially (see Example 9.1.), Is assigned with digested the restriction enzymes EcoRI and BgIII. The two ruvor isolated fragments are then spliced into the vector pHY300 PLK.

The entire ligation mixture is then transformed into E. coli strain JM107 (Bethesda Research Laboratories [BRL], 411 N, Stonestreet Avenue, Rockville, MD 20850, USA). A clone having β-galactosidase activity is further analyzed by restriction digests. A clone containing a correct genetic construct is called PX1101.

8.3. Transformation of the plasmid pXI80 or pXI101 In B.subtilis and S.thurlnglensis pXieobzw. pX1101 Plasmid DNA is first transformed into B. subtilis protoplasts according to a known protocol described by Chang and Cohen (/ 13 / Chang and Cohen, 1979).

A correct clone is read, the DNA to be transformed isolated by standard methods and transformed via electroporation (see example DinB.thurlnglensisHDI cryB cells.

The transformed B. thuringiens cells are plated on GYS agar (sporulation medium) containing X-gal as an additive.

Correctly transformed clones turn blue upon onset of sporulation.

A B.thurlnglenslt HD 1 cryB strain transformed with the pXI61 vector, on the other hand, remains white under the same conditions.

The restriction analysis shows that in correctly transformed clones an intact pXI80bzw. pX1101 plasmids

B. thuringiensls cells present.

3.4, β-glactosylase gene under the control of a sporulation-dependent promoter

B. thurlrigiens1 HD 1 cryB cells containing the pXI f 0 and pX1101 plasmids, respectively, are cultured on GYS medium as previously described. At different times wä! During the growth phase (both during the vegetative growth phase and during the sporulation phase) a β-galactosidase assay is performed according to the method described in /44 / J.H. Miller's Experiments Protocol ("Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, 1972, Experiment 48 and 49).

The differences to the experimental protocol mentioned above relate to the use of X-gal as the chromogenic substrate and the measurement of the colored hydrolysis product formed by the cells after about 1 hour

The cells are then removed by centrifugation and the optical density of the supernatant is determined at a wavelength of 650 nm (OD ₆₆₀ ).

It can be seen that the increase in optical density is dependent on sporulation. The untransformed B. thuringiensis cells can not hydrolyze the chromogenic substrate X-gal.

Example 9: Construction of Genebanks In Bacillus thuringiensls

9.1. Construction of pXI200

The plasmid pXI200 is a deriva; of the plasmid pHY300 PLK manufactured by Toyobo Co., Ltd. (# PHY-001, Toyobo Co., Ltd., 2-8 Dojima Hama 2-Chome, Kita-ku, Osaka, 530 Japan) can be obtained commercially. Plasmid pHY300, whose construction is described in European Patent Application EP 162725, contains both an ampicillin (amp ^R ) and a tetrazyclin (tetr ^R ) radical.

The plasmid pHY300 PLK is completely digested with BgII and Pvul. The resulting restriction fragments are then separated by means of agarose gel electrophoresis. The 4.4 Kb fragment is isolated from the agarose gel, purified and then religated with T4 DNA ligase.

The entire ligation mixture is transformed into E. coli HB101. After incubation of the transformed E. coli HB101 cells at 37 ⁰ C UUF a selective L-agar containing 20 pg / ml tetracycline, the tetracycline-resistant (Tc ¹⁾ transformants are selected. An ampicillin-sensitive (Ap ^s ) clone (100 μg / m ampicillin) can then be used to isolate a piasmide that has lost the PstI site in the Ap' gene together with the 0.3 Kb Pvul / BglI fragment. This plasmid is called pXI200.

9.2. Cloning of protoxins from Bacillus thuringiensis var. Kurstaki HDI into Bacillus thurlgiensis HDI cryß

The total DNA (50 μg) of Bacillus thuringiensis var. Kurstaki HDI is completely digested by incubation with the restriction enzyme PstI and Hpal. The restriction fragments obtained in this way are transferred to a continuous sucrose gradient (5% (w / v) - 23% [w / v]) and fractionated there by density gradient centrifugation according to their size and collected in fractions of 500 μl each TST 41 rotor (Kontron swing) 10 ⁵ g performed. at 2.4 x max over a period of 16 hours and at a temperature of 15 ⁰ C. Subsequently, aliquots of each 50μΙ for determining the fragment size (on a 0.8% agarose gel I'.v / v] agarosein tris acetate EDTA or Tris borate EDTA; see / 33 / Maniatis et al., 1982.) Those fractions containing fragments between 3Kb and 6Kb are pooled and concentrated by ethanol precipitation to a volume of 10μΙ.

5 μg of the in Example 9.1. The "pXI200" shuttle vectors are digested to completion with the restriction enzyme PstI and Sma 1. The 5 'phosphate groups of the resulting restriction fragments are then removed by treatment with calf intestinal alkaline phosphatase.

0.2 μg to 0.3 μg of the excessively isolated HDI DNA are then mixed with 0.5 μg of pXI200 vector DNA and 0.1 μl (so-called "white units") are added, one unit of T4 DNA ligase corresponds to one enzyme activity that is sufficient 1 nM ^[32 P] pyrophosphate at a temperature of 37 ⁰ C and over a period of 20 minutes to convert it into a Norit-absorbable material) of T4 DNA ligase incubated overnight at 14 ° C. the entire ligation mixture is followed by electroporation (see example 1). transformed directly in Bacillus thuringiensis HDI cryB cells. the electroporated cells are B. thuringiensis then to a selective Sporulationsagar containing 20 .mu.g / ml tetracycline as selection means, and plated out at a temperature of 25 ⁰ C incubated until complete sporulation.

9.3. Preparation of monoclonal antibodies against B. thuringiensis protoxin protein

The production of monoclonal antibodies against δ-endotoxin from Bacillus thuringiensis var. Kurstaki HDI is carried out analogously to the description in / 36 / Huber-Lukai, (1984) or in / 37 / Huber-Lukac et al. (1986).

Hybridoma cells used for antibody production are fusion products of Sp2 / 0-Ag myeloma cells (described in / 45 / Shulman et al., 1978, available from the Americn Type Culture Collection of Rockville, Maryland, USA and spleen lymphocytes from BALB / c mice previously immunized with δ-endotoxin and B. thuringiensis kurstaki HDI.

In this way it is possible to obtain monoclonal antibodies which are specifically directed against the δ-endotoxin of B. thuringiensis. Particular preference is given to monoclonal antibodies which either bind specifically to an epitope in the K- intrinsic half of the protoxin protein (eg, Huber-Lukaö et al., Antibody 54.1, 1986, reference), or an epitope in the b'-lepidopteran gene. active prototoxins constant part of the protein, the C-terminal half (eg, antibody 83.16 of Huber-Lukaö et al., 1986 reference) recognize.

However, other monoclonal or even polyclonal antibodies can also be used for the subsequent immunoscrimming (see Example 9.4.).

-39- 283 ό40

9.4. Immunological screening

For immunological screening, those according to Example 9.3. prepared or other suitable monoclonal

Antibodies used.

First, the crystal proteins freely present after sporulation of B. thuringiensis cells are bound by transfer membranes (eg, Pail Biodyne Transfer Membrane, Pail Lutidine Filtration Corporation, Glen Cove, NY), adding them for a period of about 5 Minutes are placed on the plates. The filters are then washed for 5 minutes with TBST buffer (0.05% [w / v] Tween 20.1 mM Tris / HCl IpH 8.0), 15 mM NaCl in H ₂ O bidist.) And then for 15 to 30 minutes to Blocking non-specific binding in a mixture of TBST Puffor and 1% (w / v) skim milk incubated.

The filters prepared in this way are then incubated overnight with the protoxin-specific antibodies (antibody mixture from 54.1 and 83.1, / 37 / Huber-Lukai et al., (1986)). The unbound antibodies are removed by washing the filter three times with TBST buffer for 5 to 10 minutes each time. To detect the antibody-bound protoxin, the filters are incubated with another antibody. The secondary antibody used here is an antibody labeled with alkaline phosphatase which can be obtained commercially, for example, from Bio-Rad (catalog # 170-6520, goat anti-mouse IgG | H + L] -alkaline phosphatase conjugate). After an incubation period of 30 minutes, unbound secondary antibodies are removed by washing three times (5 to 10 minutes each) with filter with TBST buffer as described above. Subsequently, the filters with a substrate mixture consisting of NBT Cp-nitro blue tetrazolium chloride; Nitro-blue tetrazolium chloride) and BCIP (δ-bromo-chloro-S-indolyl phosphate-p-toluidine salt). The enzymatic reaction is carried out according to the instructions of the manufacturer (Bio-Rad, 1414 Harbor Way South, Richmond CA, 94804, USA).

carried out. .

Positive, i. Protoxin-containing clones can be easily recognized by their purple color. This is due to the enzymatic reaction of Alkalischon Phosphatass with the aforementioned substrate mixture. From the example given in Example 9.2. described transformation with the ligation there indicated resulted in between 800 and 1000 transformants. Of these, 2 colonions clearly show positive signals in the previously described enzyme reaction.

From positive clones, in which expression of the protoxin gene could be detected on the basis of the described enzyme reaction, plasmid DNA is isolated. By means of the restriction analysis u id by comparison with known restriction maps, the cloned protoxin genes can be further characterized and finally identified.

Both clones contain a recombinant plasmid with an insert of 4.3 Kb. The following restriction digests with HindIII, Pvull, EcoRI and XbaI allow identification of the gene on the insert by comparison with the known restriction endotoxin genes from B. thuringiensis var kurstaki HD1. In both cases it is the kurhdi gene, also known as 5.3 Kb Protoxi: .qen and described in / 5 / Geiser et al., 1986.

This gene, directly cloned in B. thuringiensls and identified by means of immunological screening, moreover hybridizes to a BamHI / HindIII fragment of the 5.3Kb gene comprising 1847 bp in plasmid pK36 / 5 / Geiser et al., 1986). In SDS / PAGE, both clones show a band of 130000 daltons typical of the protoxin, which is described in Western Blot / 46 / Towbin et al.,

1979) specifically with the previously described (see Example 9.4.) Monoclonal antibodies.

tables Table 1:

Influence of incubation time at 4 ⁰ C before and after electroporation on the transformation frequency. B, thuringiensis HD1 cryB was transformed by electroporation with 0.2 μg pBC 16 per batch

sample 1 0 2 5 3 4 5 6 7 8th Pre-incubation * (minutes) 20 20 10 20 20 20 20 20 Nachinku bation ** (minutes) 20 20 0 5 10 20

Transformation frequency (transforma-

Plasmid DNA)

2.6 x 10 ⁶

2.1 x 10 ⁶

2,2x10 ^e

2.3x10 ⁶

2.5 x 10 ⁶

1.9x10 ⁶

3,3x10 "

1,7x10 "

* Incubation at 4 ^C C between the addition of DNA and electroporation

* * Incubation at 4 ⁰ C between the electroporation and the beginning of the expression period.

Table 2:

Expression of tetracycline resistance of pBC 16 after transformation into B. thuringiensisHDIcryB. B. thurlnglensis HD1 cryB was transformed with pBC16 plasmid DNA using the electroporation protocol of the present invention. After different incubation times in LB medium at 30 ° C, the transformed cells are sanctioned by plating on LB agar containing 20μ9 / ηιΙ tetracycline.

Expression time transformation number living

the tetracycline frequency (trans cells

resistance (hours) formants / Mg DNA)

0.5 0 4 x 10 "

1 1.6 x 10 ⁶

2 8.8x10 ^e 1.4 χ

3 8 XiO ¹ 1.6 x

Table 3:

Transformation of B. thurlnglensis strain HD1 cryB with different plasmids

plasmid ancestry Resistance marker " gram positive B. cereus _ tc pUB110 - Km, Cm "Shuttle" vectors Cm Transformation ²¹ reference gram negative naturally occurring plasmids Staphylococcus Km ₁ BIe replicon Amp, Tc tc frequency aureus - plMI 3 repli - Cm Amp pBC16 S. aureus - Cm con. 1.9x10 ^B pUB110 B. subtilis - em plMI 3 repli - Em. Cm 3,3x10 ⁶ * modified plasmids / cloning vectors con, pC194 pUB110 replica - Cm, Em 6 χ 10 ⁶ · plM13 con, 1.8 χ 10 ⁶ pBD64 pBR322 / pC194, pUC8 / pBC16 5x10 ⁶ pBD347 2,9x10 ⁵ pBD348 1.1 xiO ⁵ PUB1664 3,5x10 ⁴ pHV33 <50 * PK61 2,8x10 ⁴

1 Tc: tetracycline; Km: Kanamycin; BIe: bleomycin; Cm: chloramphenicol; Em: erythromycin.

2 All plasmid DNA is from B.thuringls HDI cry8 except * isoated by B. subtills LBG4468.

Table 4:

Biotest of B.thuringiensis HD1 cryB and HD1 cryB (pXI 93) against Heliothis virescens.

Spray dried sporulated cultures (spores and [if present] protoxin crystals) were added in the indicated amounts to the diet of L-1 larvae of Heliothis virescens.

Concentration of mortality (%) of H. virescens

Spores and protoxin caused by:

crystals ^ g / g diet)

HDIcryB HD1 cryB (pXI93)

200 0 57

100 0 43

50 3 27

25 0 10

12.5 0 0

Table 5:

Transformability of strains of B. thuringlensis, B. cereus and B. subtllls. All strains were transformed according to the electroporation method described in Example 1 with the plasmid pBC16.

tribe

Transformation "Frequency

B.thurlnglonsl's var. Kurstaki

HDIcryB

HD1 dipel

HD1-9

HD 73

HD191

B. thuringlensl var. Thurigiensis

HD2-D6-4

B.thurlnglensls var. Israelensis

LBG B-4444

B. cereus

569 K

B.subtllls

LBG B-4468

1 .

0.25

0.9

0.1

0.5

13.8 2.6 7.5 0.0002 Relative values relative to the transformation frequency, designated as 1, obtained with B. thuringlensis var. Kurstaki HDI cryB.

Deposit of microorganisms

Of each of the microorganisms listed below, which are used in the present invention, a culture at the "German Collection of Microorganisms" recognized in Braunschweig, Federal Republic of Germany, as corresponding to the requirements of the Budapest Treaty for the International Recognition of Deposit Of microorganisms for the purpose of patenting, a statement on the viability of the deposited samples were made by the said International Depository.

Deposit of microorganisms

microorganisms Deposit depository date of date number viability Certificate HB101 (PK36) March 4, 1986 DSM 3668 March 7, 1986 (E.coliHB101 transformed mitpK36 Plasmid DNA) • HD1 cry May 4, 1988 DSM 4574 May 4, 1988 (Bacillusthu- rlngienslsvar. kurstaki HD1 cry • HD1cryß (* PK61) May 4, 1988 DSM 4572 May 4, 1988 (B.thuringlensls HD1 cryss transfor with »pK61 Plasmid DNA) • HD1cryß ( "pK93) May 4, 1988 DSM 4571 May 4, 1988 (B.thuringlensls HD1 cryss transfor with * pK93 Plasmid DNA) 569 K May 4, 1988 DSM 4575 May 4, 1988 (Bacillus cereus 569K) 569K (* pK93) May 4, 1988 DSM 4573 May 4, 1988 (B. cereus 569 K transformed with PK93 plasmid DNA)

The internal name pK chosen for the naming of the plasmids in the priority dokumer.t has been replaced by the officially recognized name pXI for the foreign version.

The designation for the asporogenic B. thurlglensl HD1 Mutanto used in the exemplary embodiments was also changed from cryß to cryB.

bibliography

1 Goldberg, L., and Margarlit, J., Mosquito News, 37: 355-358, 1977

2 Krieg, A., et al., Z. Ang. Ent, 96: 500-508, 1983

3 Schnepf, H.-E., and Whiteley H.-R., Proc. Natl. Acad. ScI, USA, 78: 2893-2897, 1981

4 Klier, A., et al .. The EMBO J, 1: 791-799, 1982

5 Geiser, M., et al., Gene, 48: 109-118, 1986

6 Haider, M.-Z., et al., Gene, 52: 285-290, 1987

7 Gonzalez, J.-M., et al., Proc. Natl. Acad. USA, 79: 6951-6955, 1982

8 Obukowicz, M.-G., et al., J. Bacterlol., 168: 982-989, 1986

9 Donovan, L-P., Et al., Mol. Genet., 214: 365-372, 1988

10 Schnepf, H.-E., and Whitely, H.-R., J. Biol. Chem., 260: 6273, 1985

11 Kiisr, A., et al., Mol. Gen. Genet., 191: 257-262, 1983

12 Bibb, J.-J., et al., Nature, 274: 398-400, 1978

13 Chang, S., and Cohen S.-N., Molac. Gen. Genet., 168: 111-115, 1979

14 Brown, B.J., and Carlton B.C., J. Bacterlol, 142: 508-512, 1980

15 Kondo, J.-K., and McKay, L-L, Appl.Environ.Mlcrobiol., 48: 252-259, 1984

16 Wirth., R., et al., J. Bacteriol., 165: 831-836, 1986

17 Yoshihama, M., et al., J. Bacterl., 162: 591-597, 1985

18 Alikhanian, S.-J., et al., J. Bacteriol., 1 <t6: 7-9,1981

19 Martin, P.A., et al., J. Bacterlol., 145: 980-983, 1981

20 Fischer, H.-M., Arch. Microbiol., 139: 213-217, 1984

21 Sound, D "Gene transfer between isolates of Bacillus thuringiensis by protoplast transformation and fusion. Dissertation, University of Tübingen, 1986

22 Shivarova, N., Zeltschr.Allgem.M »': oblol., 23: 595-599, 1983

23 Youston, A.A., and Rogoff, M.H., J. Bacteriol., 100: 1229-1236, 1996

24 Horinouchi, S., and Weisblum, B., J. Bacterlol., 150: 815-825, 1982

25 Polak, J., andNovick, R.P., Plasmid, 7: 152-162, 1982

26 Mahler, J., and Halvorson, H.-O., J. Gen. Mlcrobiol., 120: 259-263, 1980

27 Gryczan, T., et al., J. Bacterlol., 141: 246-253, 1980

28 Vieira, J., and Messing, J., Gene, 19: 259-268, 1982

29 Wong, et al., J. Biol. Chem., 258: 1960-1967, 1983

30 Bolivar, et al., Gene 2: 95-113, 1977

31 Norrander Valley, Gene, 26: 101-1904, 1933

32 Bevan, et al., Nature, 304: 184-187, 1983

33 Maniatis et al. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, 1982

34 Hinnen et al, Proc. Natl. Acad. See, USA, 75: 1929-19J3, 1978

35 Young RA et al, Proc Natl. Acad. Sci., USA, 80: 1194-1198, 1983

36 Huber-Lucfii, M., "On the Interaction of the Bacillus thuringiensis delta-endotoxin with Monoclonal Antibodies and Lipids," Dissertation No. 7547, ETH Zurich, 1984

37 Huber-Lucai, M., et al. Infect. Immunol., 54: 228-232, 1986

McCutcheon's, 1986 McCutcheon's International Emulsifiers & Detergents, The Manufacturing Confections Publishing Co., Glen Rock, NJ, USA

39 Stahly, D. P., et al. Biochem. Biophys.Res.Comm, 84: 581-588, 1978

40 Bernhard, K., et al., J. Bacteriol., 133: 897-903, 1978

41 Primrose, S.-B., Ehrlich, S.-D., Plasmid 6: 193-201, 1981

42 Huber-Lukac, H., Dissertation, Swiss Federal Institute of Technology, Zurich, Switzerland, No.7050,1982

43 Zoller, J.-M. and Smith, M., Nucl. Acids Res., 10: 6487, 1982

44 Miller, J.-H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972

45 Shulman et al., Nature, 276: 269, 1978

Towbin, H., et al., Proc. Kind. Acad. Sci., USA, 73: 4350-4354, 1979

Patent Literature EP 162,725 EP 238,441 WO 86/01536 US-P 4,448,885 US-P 4,447,036 US-P 4,237,224 US-P 4,468,464

Claims (68)

1. A method for the direct, targeted and reproducible genetic manipulation of B. thuringiensis and / or B. cereus using recombinant DNA technology, characterized in that B. thuringiensis and / or B. cereus by means of a simple transformation method with high efficiency transformed using a rokombiniorten DNA suitable for the intended genetic manipulation of B. thuringiensis and / or B. cereus. 2. The method according to claim 1, characterized in that one carries out the transformation of B. thuringiensis and / or B. cereus by means of an electroporation. 3. The method according to claim 2, characterized in that the transformation of B.thuringiensis and / or B.cereus comprises the following method measures: a) preparing a cell suspension with a suitable cell density in a for the cultivation of B. iringiensis and / or B.cereus cells suitable culture medium and sufficient for the growth of the cells ventilation; b) separating the cells from the cell suspension and resuspending in an inoculation buffer suitable for subsequent electroporation; c) adding a DNA sample in a concentration suitable for electroporation; d) introduction of the approach described under point b) and c) into an electroporation apparatus; e) One or more short-duration discharges of a capacitor across the cell suspension for short-term generation of high electric field strengths over a time sufficient for transformation of B. thuringiensis and / or B. cereus cells with recombinant DNA; f) optionally re-incubation of the electro-oorated cells; g) plating the electroporated cells on a suitable selection medium; and h) reading the transformed cells. 4. The method according to claim 3, characterized in that one uses spores for the production of the cell suspension B.thuringiensis mentioned under point 3a). 5. The method according to claim 3, characterized in that in the mentioned under item 3e) electroporation frozen Bacillus thuringiensis and / or Bacillus cereus cells used. 6. The method according to claim 3, characterized in that for the cultivation of B. thuringiensis cells a) using complex nutrient media with readily assimilable carbon and nitrogen sources commonly used in the cultivation of Dacillus aerobic species; ai) if appropriate, in addition to the media mentioned under a) added vitamins and essential metal ions; or b) uses fully or semi-synthetic nutrient media, the b,) a complex or a defined readily assimilable carbon and Nitrogen source or a combination of both and b ₂ ) contain essential vitamins and metal ions. A method according to claim 3, characterized in that said Bacillus cell suspension has an optical density of 0.1 to 1.0. 8. The method according to claim 3, characterized in that the under b) inoculation buffer is an osmotically stabilized phosphate buffer. 9. The method according to claim 8, characterized in that said phosphate buffer contains as stabilizing agent sugar or sugar alcohols. 10. The method according to claim 9, characterized in that said stabilizing agent is sucrose, which is present in a concentration of 0.1 M to 1.0 M. 11. The method according to claim 8, characterized in that said phosphate buffer has a pH of pH 5.0 to pH 8.0. 12. The method according to claim 3, characterized in that the incubation of the Bacillus cells takes place both before, during and after the electroporation at a temperature between 0 ⁰ C and 35 ⁰ C. 13. The method according to claim 3, characterized in that the incubation of the Bacillus cells takes place both before, during and after the electroporation at a temperature between 2 ° C and 15 ° C. 14. The method according to claim 3, characterized in that the concentration of the added DNA sample is 1 ng to 20 μρ. 15. The method according to claim i4, characterized in that said DNA sample is vector DNA. 16. The method according to claim 3, characterized in that in the course of electroporation, the bacterial cells durc ¹ ! short-term discharges of a capacitor via the DNA-containing cell suspension in the short term with a very high electric field strength beau ', whereby the permeability of B. thuringiensis and / or B.cereus cells is increased in such a way that the uptake of the DNA in suspension into the Bacillus cells. 17. ancestor according to claim 16, characterized in that the field strength values are 100 V / cm to 50,000 V / cm. 18. The method according to claim 16, characterized in that the field strength values are 100 V / cm to 10000 V / cm. 19. The method according to claim 3, characterized in that the exponential Ablmgzeit the pulse with which the Bacillus cell suspension is applied, in a range of about 2ms and about 50ms lieyt. 20. The method according to claim 3, characterized in that the electroporated cells are plated after a suitable Nachinkubationsphase on solid media containing an appropriate for the selection of the transformed Bacillus cells additive. 21. The method according to claim 20, characterized in that said addition is a suitable for the selection of B. thuringiensis and / or B.cereus antibiotic selected from the group consisting of tetracycline, kanamycin, chloramphenicol, erythromycin. 22. The method according to claim 20, characterized in that said addition is a suitable for the selection of B.thurintjiensis and / or B.cereus chromogenic substrate. 23. The method according to claim 1, characterized in that the recombinant DNA used for the transformation of B. thuringiensis and / or B. cereus homologous or heterologous origin or is a combination of homologous and heterologous DNA. 24. The method according to claim 1, characterized in that said recombinant DNA contains one or more structural genes and 3 'and 5' flanking, in Bacillus thuringiensis or Bacillus cereus or in both functional regulatory sequences operably connected to the (the ) Structural gene (s) are linked and thus ensure the expression of said structural gene (s) in Bacillus thuringiensis or Bacillus cereus or in both. A method according to claim 1, characterized in that said structural gene encodes a δ-endotoxin polypeptide naturally occurring in B. thuringiensis, or a polypeptide substantially homologous thereto, d. H. which at least substantially exhibits the toxicity properties of a crystalline δ-endotoxin polypeptide from B. thuringiensis. A method according to claim 25, characterized in that said δ-endotoxin-encoding DNA sequence is substantially homologous to at least the portion or particles of the natural 6-endotoxin coding sequence responsible for the insecticidal activity. , 27. The method according to claim 23, characterized in that said DNA is vector DNA. 28. The method according to claim 27, characterized in that said vector DNA is derived from plasmid DNA. 29. The method according to claim 27, characterized in that said vector derives DNA from phage DNA. 30. The method according to claim 1, characterized in that one uses for the transformation bifunctional vectors that are capable except in B. thuringiensis or the closely related B.cereus or in both at least in eir.em or in several replicate to other heterologous host organisms and that are identifiable in both the homologous and heterologous host systems. 31. The method according to claim 30, characterized in that in said heterologous host organisms to a) prokaryotic organisms selected from the group consisting of the genera Bacillus, Staphylococcus, Streptococcus, Streptomyces, Pseudomonas, Escherichia, Agrobacterium, Salmonella, Erwinia, etc., or b) eukaryotic organisms selected from the group consisting of yeast, animal and plant cells, etc. 32. The method according to claim 31, characterized in that said heterologous host organism is E. coli. 33. The method according to claim 30, characterized in that nian B.thuringiensls and / or B. cereus is transformed with a bifunctional vector prepared by a process characterized by the following steps: a) Fragmentation of plasmid DNA of homologous and heterologous origin by means of suitable restriction enzymes; and b) subsequently linking those fragments which contain the essential functions for the replication as well as the selection in the respective desired host system, in the presence of suitable enzymes in such a way that the essential for replication and selection in the various host systems functions are retained. 34. The method according to claim 33, characterized in that one pure plasmid DNA Origin. 35. The method according to any one of claims 33 or 34, characterized in that it is said a) homologous plasmid DNA is DNA which is naturally present in Bacillus thuringiensis or B. cereus or which is essentially homologous, b) heterologous plasmid DNA is DNA naturally occurring in bi) prokaryotic organisms selected from the group consisting of the genera Bacillus, Staphylococcus, Streptococcus, Streptomyces, Pseudomonas, Escherichla, Agrobacterium, Salmonella, Erwinia, etc., b ₂ ) eukaryotic organisms selected from the group consisting of yeast, animal and plant cells, etc.
is present or this is substantially homologous. 36. The method according to claim 35, characterized in that said heterologous plasmid DNA is a DNA that is naturally present in E. coli or that is substantially homologous. 37. The method according to claim 33, characterized in that said bifunctional vectors in addition to the essential for replication and selection in homologous and heterologous host systems functions additionally have one or more gene (s) in expressible form or other useful DNA sequences that one spliced into said bifunctional vectors with the aid of suitable enzymes. 38. The method according to claim 37, characterized in that said genes or other useful DNA sequences homologous, heterologous or synthetic origin or consist of a combination of said genes or DNA sequences. 39. Method according to claim 37, characterized in that said genes consist of one or more structural genes as well as 3 'and 5' flanking, in B.thuringiensis or B.cereus or in both functional regulatory sequences operably linked to the structural gene and thus ensure the expression of said structural gene in B. thuringiensis or B. cereus, or in both. 40. The method according to claim 39, characterized in that said regulatory sequences are expression signals that include both promoter and terminator sequences and other regulatory sequences of the 3 'and 5' untranslated regions together. 41. The method according to claim 40, characterized in that said expression signals occur naturally in B. thuringiensis or in B.cereus or in both or that they are mutants and variants of these natural expression signals which are substantially homologous to the natural sequence. 42. The method according to claim 41, characterized in that said expression signals include a sporulation-dependent promoter from B. thuringiensis. 43. The method according to claim 39, characterized in that said structural gene encodes a δ-endotoxin polypeptide naturally occurring in B. thuringiensis or a polypeptide substantially homologous thereto, d. H. which at least substantially exhibits the toxicity properties of a crystalline δ-endotoxin polypeptide from B. thuringiensis. 44. Method according to claim 39, characterized in that said structural gene is a DNA sequence naturally occurring in B. thuringiensis and encoding δ-endotoxin, or at least one variant of a natural DNA sequence corresponding to the corresponding natural sequence is substantially homologous. 45. The method according to claim 43, characterized in that said polypeptide is a δ-endotoxin polypeptide from suitable subspecies of B. thuringiensis, selected from the group consisting of kurstaki, berliner, alesti, sotto, tolworthi, dendrolimus, tenebrionis and israelensis substantially is homologous. 46. The method according to claim 44, characterized in that said δ-F.ndotoxin-encoding DNA sequence is substantially homologous to at least the portion or portions of the natural δ-endotoxin coding sequence responsible for its insecticidal activity ( are). 47. The method according to claim 43, characterized in that said DNA sequence coding for δ-endotoxin is a DNA fragment from B. thuringiensis var. Kurstaki HDI, which is located between nucleotides 156 and 3623 in formula I or else Any shorter DNA fragment that still encodes a polypeptide with insect-toxic properties: Formull 10 20 30 AO 50 60 GTTAACACCC TGGGTCAAAA ATTGATATTT AGTAAAATTA GTTGCACTTT GTGCATTTTT 70 80 90 100 110 120 TCATAAGATG AGTCATATGl TTTAAATTGT AGTAATGAAA AACAGTATTA TATCATAATG 130 140 150 160 170 180 AATTGGTATC TTAATAAAAG AGATGGAGGT AACTTATGGA TAACAATCCG AACATCAATG 190 200 210 220 230 240 AATGCATTCC TTATAATTGT TTAAGTAACC CTGAAGTAGA AGTATTAGGT GuAGAAAGAA 250 260 270 280 290 300 TAGAAACTGG TTACACCCCA ATCGATATTT CCTTGTCGCT AACGCAATTT CTTTTGAGTG 310 320 330 340 350 360 AATTTGTTCC CGGTGCTGGA TTTGTGTTAG GACTAGTTGA TATAATATGG GGAATTTTTG 370 380 390 400 410 420 GTCCCTCTCA ATGGGACGCA TTTCTTGTAC AAATTGAACA GTTAATTAAC CAAAGAATAG 430 440 450 460 470 480 AAGAATTCGC TAGGAACCAA GCCATTTCTA GATTAGAAGG ACTAAGCAAT CTTTATCAAA 490 500 510 520 530 540 TTTACGCAGA ATCTTTTAGA GAGTGGGAAG CAGATCCTAC TAATCCAGCA TTAAGAGAAG 550 560 570 580 590 600 AGATGCGTAT TCAATTCAAT GACATGAACA GTGCCCTTAC AACCGCTATT CCTCTTTTG 610 620 630 6A0 650 660 CAGTTCAAAA TTATCAAGTT CCTCTTTTAT CAGTATATGT TCAAGCTGCA AATTTACATT 670 680 690 700 710 720 TATCAGTTTT GAGAGATGTT TCAGTGTTTG GACAAAGGTG GGGATTTGAT GCCGCGACTA 730 740 750 760 770 780 TCAATAGTCG TTATAATGAT TTAACTAGGC TfATTGGCAA CTATACAGAT CATGCTGTAC 790 800 810 820 830 840 GCTGGTACAA TACGGGATTA GAGCGTGTAT GGGGACCGGAi TTCTAGAGAT TGGATAAGAT 850 860 870 880 890 900 ATAATCAATT TAGAAGAGAA TTAACACTAA CTGTATTAGA TATCGTTTCT CTATTTCCGA 910 920 930 940 950 960 ACTATGATAG TAGAACGTAT CCAATTCGAA CAGTTTCCCA ATTAACAAGA GAAATTTATA 970 980 990 1000 1010 1020 CAAACCCAGT ATTAGAAAT TTTGATGGTA GTTTTCGAGG CTCGGCTCAG GGCATAGAAG 1030 1040 1050 1060 1070 1080 GAAGTATTAG GAGTCCACAT TTGATGGATA TACTTAACAG TATAACCATC TATACGGATG 1090 1100 1110 1120 1130 1140 CTCATAGAGG AGAATATE TGGTCAGGGC ATCAAATAAT GGCTTCTCCT GTAGGGTTTT 1150 1160 1170 1180 1190 1200 CGGGGCCAGA ATTCACTTTT CCGCTATATG GAACTATGGG AAATGCAGCT CCACAACAAC 1210 1220 1230 1240 1250 1260 GTATTGTTGC TCAACTAGGT CAGGGCGTGT ATAGAACATT ATCGTCCACT TTATATAGAA 1270 1280 1290 1300 1310 1320 GACCTTTTAA TATAGGGATA AATAATCAAC AACTATCTGT TCTTGACGGG ACAGAATTTG VIIIOIDOVO IWOIOODOO WWDVDOVO VWOVIlIVO IV1W0VD00 VOIIIDDWI OVOZ 0C03 OZOc 0102 0003 0661 OWOVDOODD II0I1IW0I IWODIVOVI VIVIIlOWO IWDOOVDII WDIIDIOIV 0861 0L61 0961 0561 0V61 0C61 DIDOIOWII ODVIIIVIOI 0WD1V00IV WOIIIIDW III0DDIDV1 DVI1I100VI 0Z61 0161 006 068 0981 0L21 0IDV00V1II DOWOODDIO VDVIIIWlO VOOOIOVIOV 01V1DWD0V DIIIIlWOO 0981 0581 0V81 0C81 0281 0181 DOVDIWIlV IDDVOWOOD VOIIWDIVD VIVDDIlWD VIIIWVDVD DVIDIIDODV 0081 06Π 08Π OLLI 09LI OSU IDODllWOV V1000D1VIV OVWDVDIVl 1VDDVD0IDV I1V1VWI0V OWIlDDWD O '? Ll OZLl OZLl OUl 00Π 0691 1I1V0VDD00 IDDVDlIDW OWODIlDIl VIVOVOOVOO VDV1I1V00V DDVOOWVlI 0891 Oi9l 0991 0591 0 ^ 91 0C91 0D10ID1IDV V001DID001 1D1WIDVID IWWDWlI IDDVlVWDV DVIIVWDVD 0Z91 0191 0091 0651 0851 IVDIIDD11V VIVlWIWI IIW0I0010 VI0DIVDV1V 00IIDID1I0 1V1DD1D0V0 0951 0551 0 * 751 0C51 0251 0151 WlWIVlOV VI010VI0VI WIOVIlIOO 0VDII0D1II 01VVDII101 VDDOWIIVO 0051 06Vl O8 »7l OL *? L 09Vl 05Vl D1VD10VI1I VOOWDOOVl DDVDD010DV VDWlWOVD VDDODOVIW V01V001000 OVVl OCVl GZVl OlVl 0OVl 06ei J.1V0V100DV VODDOVWVV 0VDVIV101D 0DD1VDD011 IWVDlDDlD DWOOIVlID 08Cl OLiI 09Cl OStI OVEl OCCl IWOOIIWV WOIOOWVO VOVOVOOlW VWVOVOOOO VOWWOIOI OOIOOVIOVO 09LZ OGLZ O *? LZ OZLZ OZLZ OILZ OWOVOOVIO VIIVOOVWO VOWOOlOII IWOVlOlW VOOVIOVOW OOIVOOOOIV OIL 0692 0892 0L9Z 0993 0592 OWOOOVOV IIVOVOllV IVOIOOOIVI OIOOVIIOVO OVOiVWIIO VOVOVIOIVO 0V92 0Γ92 0292 0192 0092 0652 0II0IV0I1V OVOOIIOOIO 1IIV01V000 IIVOIVOOOO IOIWWOOO IWOOlOWO 0852 OLGZ 0952 0552 0V52 C ^C G2 0000V01I10 000001VII0 0I10000VI0 OVOOOIOIW VIOVOVWOO VOVWOOOIV 0252 0152 OO'J2 06V2 08V? OLH VOVIOOOIIV VIIIVIOIW VOVIlOVOW 0I0V1V0W0 OIVIVIOOOV OWlIWOOV O9V2 O5V2 0VV2 OCVZ O2V2 II0000V1VI OOOVWVIIV WOOIOVOIV OVIVWWVO IVIVIIIV10 OWOOIVIOO 00V2 O6C2 08C2 OLZZ 09t2 05 £ 2 10V01V01II 00V100011V IOOOVIIOOV I1W0V0WV 0I1VI00V0I VOOOOVOOW 0VC2 0G € 2 O2C2 OIZZ 00 € 2 0622 001V00VIIV 1V000VI0W 00V0V00100 OIOOOVOVIO WOVOVlWO IVOOOVOVII 0822 0 ^ 22 0922 0522 0 * 722 Oi22 iOVWOOIVO WOIIOVII WOOOOVOlV 010VII0V00 OWOOOlVOV WOIOWVOV 0222 0122 0022 0612 0812 0Π2 000I0I1W0 VWWVWOI V0010I0I11 1W0IV0I0I VI1I010V0I 10VI1IW00 0912 0512 0VI2 0C12 0212 0Π2 IVIOWOlVO IIV1V01V11 V000V0I01V OVOVWTVIl 00001WV0I WOOIIOIIO 0012 0602 0802 OiO2 0902 0502 2770 2780 2790 2800 2810 2820 GGGAAACAAA TATTGTTTAT AAAGAGGCAA AAGAATCTGT AGATGCTTTA TTTGTAAACT 2830 2840 2850 2860 2870 2880 CTCAATATGA TAGATTACAA GCGGATACCA ACATCGCGAT GATTCATGCG GCAGATAAAC 2890 2900 2910 2920 2930 29AO GCGTTCATAG CATTCGAGAA GCTTATCTGC CTGAGCTGTC TGTGATTCCG GGTGTCAATG 2950 2960 2970 2980 2990 3000 CGGCTATTTT TGAAGAATTA GAAGGGCGTA TTTTCACTGC ATTCTCCCTA TATGATGCGA 3010 3020 3030 30A0 3050 3060 GAAATGTCAT TAAAAATGGT GATTTTAATA ATGGCTTATC CTGCTGGAAC GTGAAAGGGC 3070 3080 3090 3100 3110 3120 ATGTAGATGT AGAAGAACAA AACMCCACC GTTCGGTCCT TGTTGTTCCG GAATGGGAAG 3130 3140 3150 3160 3170 3180 CAGAAGTGTC ACAAGAAGTT CGTGTCTGTC CGGGTCGTGG CTATATCCTT CGTGTCACAG 3190 3200 3210 3220 3230 3240 CGTACAAGGA GGGATATGGA GAAGGTTGCG TAACCATTCA TGAGATCGAG AACAATACAG 3250 3260 3270 3280 3290 3300 ACGAACTGAA GTTTAGCAAC TGTGTAGAAG AGGAAGTATA TCCAACACAC ACGGTAACGT 3310 3320 3330 3340 3350 3360 GTAATGATTA TACTGCGACT CAAGAAGATE ATGAGGGTAC GTACACTTCT CGTAATCGAG 3370 3380 3390 3400 3410 3420 GATATGACGG AGCCTATGAA AGCAATTCTT CTGTACCAGC TGATTATGCA TCAGCCTATG 3430 3440 3450 3460 3470 3480 AAGAAAAAGC ATATACAGAT GGACGAAGAG ACAATCCTTG TGAATCTAAC AGAGGATATG 3690 3500 3510 3520 3530 35A0 GGGATTACAC ACCACiACCA GCTGGCTATG TGACAAAAGA ATTAGAGTAC TTCCCAGAAA 3550 3560 3570 3580 3590 3600 CCGATAAGGT ATGGATTGAG ATCGGAGAAA CGGAAGGAAC ATTCATCGTG GACAGCGTGG 3610 3620 3630 3640 3650 3660 AATTACTTCT TATGGAGGAA TAATATATGC TTTATAATGT AAGGTGTGCA AATAAAGAATE 3670 3680 3690 3700 3710 3720 GATTACTG \ TTGTATTGAC AGATAAATAA GGAAATTTTT ATATGAATAA AAAACGGGCA 3730 3740 3750 3760 3770 3780 TCACTCTTAA AAGAATGATG TCCGTTTTT GTATGATTTA ACGAGTGATA TTTAAATGTT 3790 3800 3810 3820 3830 3840 TTTTTTGCGA aggctttact taacggggta ccgccacatg cccatcaact taagaatttg 3850 3860 3870 3880 3890 3900 CACTACCCCC AAGTGTCAAA AAACGTTATT CTTTCTAAAA AGCTAGCTAG AAAGGATGAC 3910 3920 3930 3940 3950 3960 ATTTTTTATG AATCTTTCAA TTCAAGATGA ATTACAACTA TTTTCTGAAG AGCTGTATCG 3970 3980 3990 4000 4010 4020 TCATTTAACC CCTTCTCTTT TGGAAGAACT CGCTAAAGAA TTAGGTTTTG TAAAAAGAAA 403Ö 4040 4050 4060 4070 4080 ACGAAAGTTT TCAGGAAATG AATTAGCTAC CATATGTATC TGGGGCAGTC AACGTACAGC 4090 4100 4110 4120 4130 4140 GAGTGATTCT CTCGTTCGAC TATGCAGTCA ATTACACGCC GCCACAGCAC TCTTATGAGT 4150 4160 4170 4180 4190 4200 CCAGAAGGAC TCAATAAACG CTTTGATAAA AAAGCGGTTG AATTTTTGAA ATATATTTTT 4210 4220 4230 4240 4250 4260 ICTGCATTAT GGAAAAGTAA ACTTTGTAAA ACATCAGCCA TTTCAAGTGC AGCACTCACG 4270 4280 4290 4300 4310 4320 TATTTTCAAC GAATCCGTAT TTTAGATGCG ACGATTTTCC AAGTACCGAA ACATTTAGCA 4330 4j40 4350 4360 CATGTATATC CTGGGTCAGG TGGTTGTGCA CAAACTGCAG 48. The method according to claim 33, characterized in that said bifunctional vector is the bifunctional vector pXI 61 (pK61) transformed in B. thuringiensisvar. ku / staki HDI cryB (DSM 4572). 49. The method according to claim 47, characterized in that said bifunctional vector is the bifunktionellon vector pXI 93 (pK93), transformed into B.thuringiensis var. Kurstaki HDI cryB (DSM 4571) and B.cereus 569K (DSM 4573) is. 50. A method for the direct, targeted and reproducible genetic manipulation of B. thuringiensis and / or B.cereus according to claim 1, characterized in that a) genes which are to be cloned in B.thuringiensis and / or B.cereus and optionally expressed, first isolated; b) optionally operably linking the isolated genes to expression sequences operable in Bacillus thuringiensis and / or B. cereus; c) transforming the genetic constructions from section b) into Bacillus thuringiensis and / or B.cereus cells using suitable vectors; and d) optionally expressing a corresponding gene product and, if desired, isolated. 51. The method according to claim 50, characterized in that said gene is a Protoxingen from Bacillus thuringiensis. 52. The method according to claim 50, characterized in that said expression sequences include a sporulation-dependent promoter from Bacillus thuringiensis. 53. The method according to claim 50, characterized in that one uses direct cloning vectors. 54. The method according to claim 50, characterized in that one uses bifunctional ("shuttle") vectors. 55. The method according to claim 50, characterized in that instead of genes, other useful DNA sequences are introduced into the Bacillus cells and cloned there. 56. A method for the direct, targeted and reproducible genetic manipulation of Bacillus thuringiensis and / or B. cereus according to claim 50, characterized in that a) the total DNA of Bacillus thuringiensis digested with the aid of suitable restriction enzymes; b) isolating such suitable size from the resulting restriction fragments; c) splicing said fragments into a suitable vector; d) Bacillus thuringiensis and / or B. cercus cells are transformed with said vector; and e) isolated from the transformants using suitable screening method new DNA sequences. 57. The method according to claim 56, characterized in that said new gene is a protoxin gene. 58. The method according to claim 56, characterized in that one uses direct cloning vectors. 59. The method according to claim 56, characterized in that one uses "shuttle" vectors. 60. The method according to claim 56, characterized in that one uses for detecting new DNA sequences, an immunological screening method. 61. A method of controlling insects, characterized in that insects or their habitat with a) B. thuringiensis or B. cereus cells or treated with a mixture of both transformed with a recombinant DNA molecule containing a structural gene coding for a δ-endotoxin polypeptide naturally present in B. thuringiensis or a polypeptide substantially homologous thereto; or but -n- 203840 b) with cell-free crystal body preparations containing a protoxin produced by besagton-transformed Bacillus cells. 62. The method according to claim 57, characterized in that one uses insecticidal mixtures consisting of transformed, praising or dead B. thi'Hngiensis and / or B. cereus cells according to section a) and from duty-free crystal body preparations containing a protoxin, the produced by said transformed Bacillus cells, according to section b). 63. Method according to one of claims 61 or 62, characterized in that said recombinant DNA molecule is a bifunctional vector gene ^; is one of claims 33 to 47. 64. The method according to any one of claims 61 to 63, characterized in that they are insects Ürc tion Lepidoptera, Diptera or Coleopter «is. 65. The method according to claim 64, characterized in that they are insects of the order Lepidoptera. 66. An insect control agent, characterized in that it contains, besides the commonly used carriers, distributing agents or carriers and distributing agents a) B.thuringiansisorB. cereus cells or a mixture of said Bacillus cells transformed with a recombinant DNA molecule containing a structural gene encoding a δ-F.ndotoxin polypeptide naturally occurring in B. thuriny'ensis or otherwise A polypeptide substantially homologous to it; or but b) cell-free crystal body preparations containing a protoxin produced by said transformed Bacillus cells. 67. An insect control agent according to claim 66, characterized in that it contains insecticidal mixtures consisting of transformed, living or dead B. thuringiensis and / or B. cereus cells in addition to the commonly used carriers, distributing agents or carriers and distributing agents Section a) and cell-free crystal body preparations containing a protoxin produced by said transformed Bacillus cells, according to Section b). 68. A composition according to any one of claims 66 or 67, characterized in that said recombinant DNA molecule is a bifunctional vector according to any one of claims 33 to 47. 69. Use of B. thuringiensis and / or B. cereus, characterized in that they are used as generall host organisms for the cloning and expression of homologous and especially heterologous DNA or a combination of homologous and heterologous DNA.