IE910322A1 - Temperature-stable bacillus thuringiensis toxin - Google Patents

Abstract

The present invention describes an insecticidal toxin, prepared by means of genetic manipulation methods, from Bacillus thuringiensis, which in contrast to its natural counterpart is still stable even at temperatures >25 DEG C, and the DNA encoding said toxin. The invention additionally relates to genetic manipulation methods for preparing a temperature-stable toxin of this type and to the vectors, plasmids and host organisms which contain the toxin-encoding DNA and are employed in this method. Likewise embraced by the present invention are insecticidal agents which contain said temperature-stable B.t. toxin as active component, and the use thereof in a method for controlling insect pests.

Description

OPEN TO PUBLIC HMS^BGTION UHDER SECTION 89 AND R,ULI dNL, HO. OF 3( bF> *>( CIBA-GEIGY AG, a body corporate organised according to the laws of Switzerland, of Klybeckstrasse 141, 4002 Basle, Switzerland. - 1Case GA/5-17948/= Temperature-stable Bacillus thuringiensis toxin The present invention relates to an insecticidal toxin from Bacillus thuringiensis prepared by means of genetic engineering processes, which toxin, in contrast to its natural counterpart, is still stable even at temperatures of > 25°C, and also to the DNA coding for the said toxin.

This invention relates also to genetic engineering processes for the preparation of such a temperature-stable toxin and to the vectors, plasmids and host organisms that are used in that process and contain the DNA coding for the toxin.

The present invention also includes insecticidal compositions that contain said temperature-stable B.t. toxin as active component, and the use thereof in a method of controlling harmful insects.

Most of the Bacillus thuringiensis strains known hitherto produce an insecticidal toxin, the so-called δ-endotoxin, in the course of their sporulation. The insecticidal potential of those toxins was recognised at a very early stage. As early as the late 1920s, B. thuringiensis preparations were being used as bioinsecticides in the control of various cultivated plant diseases caused by insects. Since then, a whole series of B.t. preparations produced from natural isolates have come into existence in this field of indication.

One of the best researched B. thuringiensis strains is without doubt theB.i. strain HD1 subsp. kurstaki. It is known that that strain contains in its genome up to three homologous plasmid-coded genes that code for insecticidally active toxin proteins having a molecular weight of from 130 to 134 kilodaltons. Those genes are classified in the literature in three groups and are referred to in general everyday usage as 6.6 or CrylA(c), 5.3 or CrylA(b) and 4.5 or CrylA(a) genes according to the size of the DNA fragments, obtainable after HindHI digestion, on which the 5’ end of those genes is located. It has in the meantime been possible in all three cases to isolate the corresponding genes, to clone them in E. coli and also to express them there [Adang et al, 1985; Kronstad JW and Whiteley, 1984; -2McLinden et al, 1985]. It was possible to show, with reference to the spectrum of activity, that the various toxin proteins have in some cases very different specificities both from the quantitative and from the qualitative point of view.

With the development of new transformation techniques which, based on electroporation, now permit the cloning of genes in B. thuringiensis sn&jorB. cereus [Schurter et al, 1989], the possibility was for the first time created of cloning toxin genes from B. thuringiensis directly in the natural host and also of expressing them there, which gives numerous advantages.

An interesting phenomenon was observed in the course of those transformation experiments. If, for example, the CrylA(b) gene is cloned in a crystal-body-free mutant of B. thuringiensis and is expressed there at a normal cultivation temperature of from 30°C to 33°C, then, after lysis of the B.t. cells, only very low toxin protein concentrations can be detected in the lysate. If, on the other hand, the transformed B.t. cells are cultivated at 25°C, a normal protein level can be readily obtained. This phenomenon was not observed in the case of the CiylA(a) and CryIA(c) genes. The toxin proteins expressed by those genes can be detected in the lysate in comparable concentrations both at a cultivation temperature of 25°C and at cultivation temperatures of from 30°C to 33°C.

Investigations have shown that the variability in the protein concentration observed in the case of the CrylA(b) genes is demonstrably unconnected with gene transcription or translation, and the anomalies observed can accordingly not be explained at the level of expression. It could accordingly only be assumed that the causes lay with the protein product coded for by the CrylA(b) gene, since that product obviously exhibits certain instabilities at a temperature of > 25 °C.

The problem was accordingly to modify the CrylA(b) toxin gene in such a manner that the modified gene codes for a temperature-stable toxin protein and that protein is expressed to a normal extent. It has surprisingly now been possible to solve this problem within the scope of the present invention.

The present invention accordingly relates especially to modified CrylA(b) genes coding for a gene product that exhibits normal stability at a temperature of > 25°C and is accordingly obtainable in customary yields at the preferred cultivation temperature for#. thuringiensis cells of from 30°C to 33°C, and also to processes for the preparation of said -3modified CrylA(b) genes. Within the scope of this invention, ’customary yields’ are to be understood as being toxin concentrations in the B.t. lysate such as are also achieved under comparable conditions in the case of the expression of the CrylA(a) or CryIA(c) genes.

Also included are mutants and variants of said modified CrylA(b) genes, including partial sequences, that still have the characteristic properties of the starting gene.

This invention relates also to recombinant DNA molecules containing a modified CrylA(b) gene coding for a gene product that exhibits normal stability at a temperature of > 25 °C and is accordingly obtainable in customary yields at the preferred cultivation temperature for B. thuringiensis cells of from 30°C to 33°C.

Also included are vectors and plasmids that contain said modified CrylA(b) gene, as well as the host organisms transformed thereby.

Especially preferred within the scope of this invention are B. thuringiensis or B. cereus cells that have been transformed using a recombinant DNA molecule containing a CrylA(b) structural gene that codes for a modified, temperature-stable δ-endotoxin polypeptide that is stable even at a cultivation temperature of > 25°C, especially at the preferred temperature for the cultivation of the B. thuringiensis cells of from 30°C to 33°C, and is accordingly obtainable in customary yields, or for a polypeptide that is substantially homologous thereto.

The present invention also includes the temperature-stable protoxin molecules that are coded for by said modified CrylA(b) genes and exhibit normal stability at a temperature of > 25 °C and are accordingly obtainable in customary yields at the preferred cultivation temperature for B. thuringiensis cells of from 30°C to 33°C, as well as processes for the preparation of those temperature-stable protoxin molecules.

Also included are mutants and variants of said temperature-stable protoxin molecules, including partial sequences, that still have the characteristic properties of the starting material.

This invention relates also to insecticidal compositions that contain said temperaturestable protoxin and to methods of controlling harmful insects on cultivated plants using -4said insecticidal compositions.

Brief description of the Figures and sequence listings Fig. 1 Construction scheme of plasmid pXI106. a DNA fragments used for the ligase reaction; EZ3 Kpnl/Hindlll fragment of plasmid pXI54 incorporated in the CrylA(b) gene; arrow indicates the reading direction and extent of the CiylA(b) gene or the CryIA(c) gene; A indicates the position of the deletion on the CrylA(b) gene SEQ ID NO; 1 KpnI/Hindin fragment of plasmid pXI36. This fragment comprises a region within the CrylA(b) gene of B. thuringiensis subsp. kurstaki HD1 that extends from position 2329 to 2899 and, in the natural state, has a deletion of 78 bp in comparison with the other known CrylA genes.

SEQ ID NO; 2 Kpnl/HindHI fragment of plasmid pXI54. This fragment comprises a region within the CrylA(c) gene of B. thuringiensis subsp. kurstaki HD73 (see Adang et al, 1985) that extends from position 2564 to 3212.

SEQ Π) NO: 3 shows the differences in the amino acid sequence of the gene products, coded for by plasmids pXI93 and pXI106, in the region 720 to 916 [CrylA(b) toxin] or 719 to 942 [modified CrylA(b) toxin].

SEQ ID NO: 4 shows the complete DNA sequence of the Hpal/Pstl fragment of plasmid pXI106 after eliminating the deletion within the coding region of the CrylA(b) gene of B. thuringiensis subsp. kurstaki HD1.

SEQ ID NO: 5 shows the amino acid sequence of the thermostable gene product that is coded for by the modified CrylA(b) gene of B. thuringiensis subsp. kurstaki HD1.

It is known from comparative investigations of the amino acid sequences of the CrylA genes carried out by Geiser et al (1986) and by Hofte and Whiteley (1989) that the differences between those genes are to be found essentially in the N-terminal region of the -5gene, while the C-terminal half appears to be extremely conservative. It was accordingly assumed that the C-terminal region may possibly have a certain functional significance.

Within the scope of the present invention, however, it has surprisingly now been possible to show that there is apparently a direct link between the C-terminal region of the CrylA genes and the stability of the coded toxin proteins. On the basis of this finding, it has now been possible within the scope of the present invention so to modify the CrylA(b) gene, which codes for a temperature-stable gene product, that the instabilities of the toxin gene product observed at a cultivation temperature of > 25 °C can be eliminated.

The present invention accordingly relates especially to a novel, modified CrylA(b) gene that codes for and expresses a gene product that is stable even at a cultivation temperature of > 25°C, especially at the preferred temperature for the cultivation of the B. thuringiensis cells of from 30°C to 33°C, and is accordingly obtainable in customary yields.

It is known from the literature (Geiser et al, 1986) that the above-considered conservatism of the C-terminal region in the CrylA(b) gene is breached insofar as the CrylA(b) gene has, in comparison with the other two CrylA genes, a short deletion comprising 78 base pairs in the C-terminus. Surprisingly, it has now been found within the scope of this invention that the observed temperature defect of the CrylA(b) gene product can be removed by eliminating that deletion, preferably by means of genetic engineering methods. This can be effected, for example, by replacing a corresponding fragment of the CrylA(b) gene, which contains that deletion, by a completely or partially overlapping fragment of a CrylA(a) or a CrylA(c) gene without a deletion. In this manner, a novel, modified gene is obtained that, in contrast to the known CrylA(b) gene, codes for a toxin protein that exhibits normal stability at a temperature of > 25 °C and is accordingly obtainable in customary yields at the preferred cultivation temperature for B. thuringiensis cells of from 30°C to 33°C.

’Normal’ stability is to be understood in this case as being a stability that is comparable to that of the CrylA(a) and CrylA(c) gene products.

The present invention accordingly relates especially to a novel and modified CrylA(b) gene, wherein the deletion present in the C-terminus has been eliminated by replacing a corresponding fragment of the CrylA(b) gene, which contains that deletion, by a -6completely or partially overlapping fragment of a CrylA(a) or a CryIA(c) gene without a deletion, or by a DNA fragment that is homologous to those CrylA fragments.

Within the scope of this invention, ’homologous DNA fragments’ are to be understood as being fragments that have, essentially, a homology of 60 %, but preferably a homology of 80 % and especially a homology of 90 %.

Likewise included in this invention is the protoxin coded for by said modified CrylA(b) gene, which protoxin exhibits normal stability at a temperature of > 25°C and is accordingly obtainable in customary yields at the preferred cultivation temperature for B. thuringiensis cells of from 30°C to 33°C.

Apart from replacement by completely or partially overlapping DNA fragments of the C-terminus of the CrylA genes, other methods of eliminating the deletion present in the CrylA(b) gene would also be conceivable, such as, for example, replacement of the entire C-terminal region of the CiylA(b) gene.

Preferred within the scope of this invention is the replacement of a fragment of the CrylA(b) gene, which contains that deletion, by a completely or partially overlapping fragment of a CrylA(c) gene without a deletion, or by a DNA fragment that is homologous thereto. This fragment is preferably a fragment, especially a KpnI-Hindlll fragment, that comprises a DNA section extending from position 2769 to 2846 on the CryLA(c) gene (Adang et al, 1985). The size of the fragment is not limiting since processes have in the meantime been developed that permit the preparation of fragments of any size, regardless of restriction sites present, such as, for example, the polymerase chain reaction (PCR) process.

In a special embodiment of the present invention, the deletion present in the CryLA(b) gene is eliminated by replacing a KpnI-Hindlll fragment of the CrylA(b) gene (positions 2329 to 2899) published in EP-A 342 633, which contains the DNA sequence shown in SEQ ID NO: 1, including the above-mentioned deletion, by the corresponding KpnI-Hindlll fragment of the CryIA(c) gene (positions 2564 to 3212) published in Adang et al (1985), the DNA sequence of which is shown in SEQ ID NO: 2.

The novel and modified CrylA (b) gene produced in that manner, the complete DNA sequence of which is shown in SEQ ID NO: 4, codes for a protoxin molecule that, in comparison with the wild-type gene product, has the differences in the amino acid sequence shown in SEQ ID NO: 3 and exhibits normal stability even at a temperature of > °C, especially at the preferred temperature for the cultivation of B. thuringiensis cells of from 30°C to 33°C.

The amino acid sequence of a CrylA(b) gene product preferred within the scope of this invention is shown in ID SEQ NO: 5.

The replacement of a fragment of the CrylA(b) gene which, in comparison with the corresponding CrylA(a) and CrylA(c) genes, has a deletion of 78 base pairs, by a completely or partially overlapping fragment of the corresponding region of a CrylA(a) or CrylA(c) gene, can preferably be carried out using genetic engineering processes.

For example, the replacement of a KpnI-Hindlll fragment of the CrylA(b) gene (positions 2329 to 2899), published in EP-A 342 633, which contains the above-mentioned deletion, by the corresponding KpnI-Hindlll fragment of the CrylA(c) gene (positions 2564 to 3212), published in Adang et al (1985), which replacement is preferred within the scope of the present invention, can be achieved by isolating suitable fragments of different sizes from the said genes and then so joining them together again in a linking reaction known to the person skilled in the art that a complete and operative toxin gene results.

In a preferred embodiment of the present invention, the following fragments are linked to one another in a ligase reaction: (a) the large Pstl-Kpnl fragment of plasmid pXI93 [Schurter et al, 1989] which is obtainable after restriction digestion and essentially comprises the N-terminal portion of the CrylA(b) gene but not the above-mentioned deletion; (b) a 648 bp KpnI-Hindlll fragment of the CrylA(c) gene that covers a DNA region within the C-terminus that is substantially homologous to the homologous region on the CrylA (b) gene, with the exception of the deletion located there; (c) a 1460 bp Hindlll-Pstl fragment of plasmid pXI36 [EP-A 238 441] that contains a major portion of the C-terminus of the CryIA(b) gene, but not the region containing the deletion.

After linking those fragments in a ligase reaction and cloning in a suitable host, such as, for example, a suitable E. coli strain, a complete and modified CrylA(b) gene is obtained which, unlike the wild-type gene, does not contain a deletion in the C-terminus and the gene product of which is accordingly stable at a temperature of > 25°C, especially at the preferred temperature for the cultivation of B. thuringiensis cells of from 30°C to 33°C.

For the transformation and expression of the modified CrylA(b) gene in B. thuringiensis, it is advantageous if at least one of the above-mentioned fragments already contains DNA sequences that are suitable for replication and expression of the modified toxin gene in B. thuringiensis. Said sequence sections are located, for example, on the large Pstl-Kpnl fragment, mentioned under point (a), of plasmid pXI93, a bifunctional plasmid the composition and construction of which are described in Schurter et al (1989) and in EP-A 342 633.

If the starting material required for the preparation of a chimaeric gene is not available in an amount sufficient for the intended genetic manipulation, it can advantageously first be incorporated into a suitable vector and the vector can then be amplified by replication in a heterologous host cell. Bacterial or yeast cells are best suited to the amplification of genes or gene fragments. If a sufficient amount of the required genes or gene fragments is available, those genes or gene fragments can be used for the construction of the chimaeric gene.

Alternatively, the required gene fragments can be obtained, for example, by means of a polymerase chain reaction (PCR). This is a process by means of which up to 105-fold amplification of DNA fragments can be achieved. The polymerase chain reaction process is described in detail in US Patent 4,683,202. Ready-for-use kits for carrying out the PCR process are commercially available [Gene Amp™ DNA Amplification Reagent Kit, Perkin Elmer Cetus, Norwalk].

Before a chimaeric gene can be transformed into B. thuringiensis οτΒ. cereus cells or into another suitable host organism, it is preferably integrated into a suitable vector which may optionally be identical with the vector used for amplification. Either the chimaeric gene can be assembled in the vector molecule or the already completely assembled chimaeric gene is integrated into the vector as a unit. The incorporation of chimaeric genes into bifunctional vectors is especially preferred.

Some examples of bacterial host cells that are suitable for cloning genes or gene fragments include bacteria selected from the genera Escherichia, such as E. coli, Agrobacterium, -9such as A. tumefaciens or A. rhizogenes, Pseudomonas, such as Pseudomonas spp., Bacillus, such as B. megaterium orB. subtilis, etc., and also yeast cells, for example of the genus Saccharomyces (EP-A 0 238 441). In addition, B. thuringiensis andB. cereus themselves can be used as host cells. Processes for cloning heterologous genes in bacteria are described in US Patents 4,237,224 and 4,468,464.

The replication in E. coli of genes that code for the crystalline protein of B. thuringiensis is described by Wong et al. (1983).

Any desired vector can be used for cloning genes or gene fragments provided it has suitable restriction cleavage sites and is capable of replicating in a suitable host cell, such as, for example, a bacterium or a yeast cell. The vector may be derived, for example, from a phage or a plasmid. Examples of vectors that are derived from phages and that can be used within the scope of this invention are vectors derived from M13 and λ phages. Some suitable vectors derived from M13 phages include M13mpl8 and M13mpl9. Some suitable vectors derived from λ phages include Xgtl 1, Xgt7 and XCharon4.

There may be mentioned as examples of the vectors that are derived from plasmids and that are very especially suitable for replication in bacteria: pBR322 (Bolivar et al, 1977), pUC18 and pUC19 (Norrander et al, 1983) and Ti-plasmids (Bevan et al, 1983), but the subject of the invention is not in any way limited to those examples. The preferred vectors for the amplification of genes in bacteria are pBR322, pUC18 and pUC19.

For cloning directly in B. thuringiensis and/or B. cereus, there may be mentioned especially direct cloning vectors, such as, for example, pBD347, pBD348, pBD64 and pUB1664, and also, especially, shuttle vectors, which are described in detail, for example, in Schurter et al (1989) and in EP-A 342 633, but the invention is not limited thereto.

Especially preferred within the scope of this invention are the bifunctional (shuttle) vectors pXI61 andpXI93 [Schurter et al (1989)], which, transformed into 5. thuringiensis var. kurstaki HD1 CryB orB. cereus 569 K, are deposited in accordance with the provisions of the Budapest Treaty at the ’Deutsche Sammlung von Mikroorganismen’ (Brunswick, Federal Republic of Germany), which is recognised as an international depository, under number DSM 4573 (pXI61, transformed into 5. thuringiensis var. kurstaki HD1 CryB) or DSM 4571 (pXI93, transformed into B. thuringiensis var. kurstaki HD1 CryB) and DSM 4573 (pXI93, transformed into B. cereus 569 K). - 10In order to construct a chimaeric gene suitable for replication in bacteria, 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. According to the invention, suitable vectors are those which are capable of replicating themselves in the host cell.

The promoter, the 5’-untranslated region, the coding region and the 3’-untranslated region can optionally first be assembled in one unit outside the vector and then inserted into the vector. Alternatively, however, portions of the chimaeric gene can be inserted individually into the vector.

In the case of the B. thuringiensis andS. cereus cloning vectors, that process step can be omitted since the whole unit isolated from/?, thuringiensis, consisting of a 5’-untranslated region, the coding region and a 3’-untranslated region, can be spliced into the vector.

In addition, the vector also preferably contains a marker gene which confers on the host cell a property by means of which the cells transformed using the vector can be recognised. Marker genes that code for a resistance to antibiotics are preferred. Some examples of suitable antibiotics are ampicillin, chloramphenicol, erythromycin, tetracycline, hygromycin, G418 and kanamycin.

Marker genes that code for enzymes having a chromogenic substrate, such as, for example, X-gal (5-bromo-4-chloro-3-indolyl-B-D-galactoside), are also preferred. The transformed colonies can then be detected very readily by means of a specific colour reaction.

The insertion or the assembly of the gene in the vector is carried out using standard processes, for example by the use of recombinant DNA (Maniatis et al, 1982) in conjunction with homologous recombination (Hinnen etal, 1978).

The processes of recombinant DNA technology are based on the following procedure: first the vector is cleaved and the desired DNA sequence is inserted between the cleaved pieces of the vector; the ends of the desired DNA sequence are then linked to the corresponding ends of the vector. - 11 The vector is preferably cleaved with suitable restriction endonucleases. Suitable restriction endonucleases are, for example, those which form blunt ends, such as Smal, Hpal and EcoRV, and also those which form cohesive ends, such as EcoRI, 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 is in such cases cut out of its original source and, if necessary, so modified that its ends can be joined to those of the cleaved vector. If the ends of the desired DNA sequence and the cleaved vector are blunt ends, they can be joined to one another, for example, with ligases specific to blunt ends, such as the T4 DNA ligase.

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

Cohesive ends are advantageously formed by cleaving the desired DNA sequence and the vector with the same restriction endonuclease. In that case, the desired DNA sequence and the cleaved vector have cohesive ends that are complementary to one another.

Cohesive ends can also be constructed by, with the aid of the terminal deoxynucleotidyl transferase, appending complementary homopolymeric tails to the ends of the desired DNA sequence and the cleaved vector. Alternatively, it is also possible to produce cohesive ends by appending a synthetic oligonucleotide sequence that is recognised by a specific restriction endonuclease and is known as a linker, and cleaving the sequence with the endonuclease (see, for example, Maniatis et al., 1982).

If the vector produced in the manner described above and containing a modified CrylA(b) gene is present in a sufficient amount, it can be used for the transformation of Bacillus thuringiensis or B. cereus cells. This transformation is preferably carried out by means of electroporation, such as described, for example, in Schurter et al, 1989.

In a specific embodiment preferred within the scope of this invention, the B. thuringiensis cells are first incubated in a suitable nutrient medium with sufficient aeration and at a suitable temperature, preferably from 20°C to 35 °C, until an optical density (OD550) of - 12from 0.1 to 1.0 has been reached. The age of the Bacillus cultures provided for the electroporation has a clear influence on the transformation frequency. An optical density of the Bacillus cultures of from 0.1 to 0.3, especially 0.2, is accordingly especially preferred. It should be pointed out, however, that good transformation frequencies can also be obtained with Bacillus cultures from other growth phases, especially with overnight cultures.

Fresh cells or spores are as a rule used as starting material, but deep-frozen cell material can equally well be used, that material preferably being cell suspensions of B. thuringiensis and/or B. cereus cells in suitable liquid media to which a specific amount of an ’antifreezing agent’ is advantageously added.

Suitable antifreezing agents are especially mixtures of osmotically active components and DMSO in water or a suitable buffer solution. Other suitable components for use in antifreezing agent solutions include sugars, polyhydric alcohols, such as, for example, glycerol, sugar alcohols, amino acids and polymers, such as, for example, polyethylene glycol.

If B. thuringiensis spores are used as starting material, they are first inoculated in a suitable medium and incubated overnight at a suitable temperature, preferably from 25 °C to 28°C, and with sufficient aeration. This batch is then diluted and treated further in the manner described above.

There may be used for the induction of sporulation in B. thuringiensis any medium that brings about such sporulation. A GYS medium according to Yousten A A and Rogoff MH, (1969) is preferred within the scope of this invenUon.

The introduction of oxygen into the cultivation medium is generally effected by agitating the cultures, for example on an agitating machine, speeds of from 50 rpm to 300 rpm being preferred.

The culUvadon of B. thuringiensis spores and vegetative microorganism cells within the scope of the present invention is carried out in accordance with known, generally customary methods, liquid nutrient media preferably being used for practical reasons.

The composition of the nutrient media may vary slightly, depending on the strain of B. thuringiensis or B. cereus used. Complex media with loosely defined, readily assimilable -13carbon (C) and nitrogen (N) sources are generally preferred, such as are usually used in the cultivation of aerobic species of Bacillus.

Apart from the LB medium preferably used within the scope of the present invention, any other culture medium suitable for the cultivation of B. thuringiensis and/or B. cereus may be used, such as, for example, Antibiotic Medium 3, SCGY medium, etc.. SporulatedB. thuringiensis cultures are preferably stored on GYS media (slant agar) at a temperature of 4°C. [The exact composition of the mentioned media is given in the section Media and buffer solutions.] When the cell culture has reached the desired cell density, the cells are harvested by means of centrifugation and suspended in a suitable buffer solution which is preferably cooled beforehand with ice. Especially suitable buffer solutions within the scope of this invention are osmotically stabilised phosphate buffers that contain sugars, such as, for example, glucose or saccharose, or sugar alcohols, such as, for example, mannitol, as the stabilising agent, and that have been adjusted to pH values of from 5.0 to 8.0. Very especially preferred are phosphate buffers of the PBS type that have a pH value of from 5.0 to 8.0, preferably from 5.5 to 6.5, and that contain saccharose as the stabilising agent at a concentration of from 0.1M to 1.0M, but preferably from 0.3M to 0.5M.

The period of incubation for the Bacillus cells before and after electroporation is preferably from 0.1 to 30 minutes, but especially 10 minutes. The temperature can be freely selected within a wide range. The preferred temperature range is from 0°C to 35°C, preferably from 2°C to 15°C, 4°C being very especially preferred.

Aliquots of the suspended Bacillus cells are then transferred into cuvettes or any other suitable vessels and incubated, together with the DNA to be transformed, for a suitable period, preferably for a period of from 0.1 to 30 minutes, especially from 5 to 15 minutes, and at a suitable temperature, preferably at a temperature of from 0°C to 35°C, especially at a temperature of from 2°C to 15°C, a temperature of 4°C being very especially preferred.

When operating at low temperatures it is advantageous to use pre-cooled cuvettes or any other suitable pre-cooled vessels.

The DNA concentration preferred forB. thuringiensis orB. cereus transformation is in a IE 91322 - 14range of from 1 ng to 20 gg. A DNA concentration of from 10 ng to 2 gg is especially preferred.

The whole batch containing B. thuringiensis and/or B. cereus cells and also the plasmid DNA to be transformed is then introduced into an electroporation apparatus and subjected to electroporation, that is to say, is exposed briefly to an electrical pulse.

Electroporation apparatuses suitable for use in the process according to the invention are already being offered for sale by various manufacturers, such as, for example, Bio Rad (Richmond, CA, USA; ’Gene Pulser Apparatus’), Biotechnologies and Experimental Research, Inc. (San Diego, CA, USA; ’BTX Transfector 100’), Promega (Madison, WI, USA; ’X-Cell 2000 Electroporation System’), etc..

Any other suitable apparatus can, of course, also be used in the process according to the invention.

The capacity setting at the capacitor is advantageously from 1 gF to 250 gF, especially from 1 gF to 50 gF, 25 gF being very especially preferred. The starting voltage can be freely selected within a wide range. A starting voltage Vo of from 0.2 kV to 50 kV is preferred, especially from 0.2 kV to 2.5 kV and very especially preferably from 1.2 kV to 1.8 kV. The distance between the electrode plates depends, inter alia, on the dimensions of the electroporation apparatus. It is advantageously from 0.1 cm to 1.0 cm, preferably from 0.2 cm to 1.0 cm. A plate distance of 0.4 cm is especially preferred. The distance between the electrode plates and the starting voltage set at the capacitor determine the field strength values that act on the cell suspension. Those values are advantageously in a range of from 100 V/cm to 50,000 V/cm. Field strengths of from 100 V/cm to 10,000 V/cm, especially from 3,000 V/cm to 4,500 V/cm, are especially preferred.

The exponential decay time preferred within the scope of the process according to the invention is from approximately 2 ms to approximately 50 ms, especially from approximately 8 ms to approximately 30 ms. An exponential decay time of from approximately 14 ms to approximately 20 ms is very especially preferred.

The fine-tuning of the freely selectable parameters, such as, for example, capacity, starting voltage, plate distance, etc., depends to a certain extent on the architecture of the devices used and can accordingly vary within certain limits from case to case. It is therefore also IE 91322 -15possible to exceed or fall below the given limit values in certain cases if that should prove necessary to attain optimum field strength values.

The actual electroporation operation can be repeated once or several times until the optimum transformation frequency for the particular system concerned has been reached.

After electroporation, the treated Bacillus cells can advantageously be re-incubated, preferably for a period of from 0.1 to 30 minutes, at a temperature of from 0°C to 35°C, preferably from 2°C to 15°C. The electroporated cells are then diluted with a suitable medium and incubated again for a suitable period, preferably from 2 to 3 hours with sufficient aeration and at a suitable temperature, preferably from 20°C to 35 °C.

After electroporation, the treated Bacillus thuringiensis or 5. cereus cells are transferred onto a selective sporulation medium and are incubated there, until complete sporulation has taken place, at a temperature of from 10°C to 40°C, preferably at a temperature of from 20°C to 35°C and very especially preferably at a temperature of from 30°C to 33°C. The sporulation medium contains as selective substance preferably one of the abovementioned antibiotics, depending on the vector used, and, if appropriate, a suitable solidifying agent, such as, for example, agar, agarose, gelatin, etc..

In the course of sporulation, autolysis of the sporulating cells occurs, which is of great technical advantage for the screening operation which follows since it is unnecessary to break open the cells artificially. In the case of clones that contain the protoxin gene sought and express it under the control of their natural promoter, the crystal proteins formed are present in the medium in readily accessible form. These crystal proteins present in free form in the medium can then be separated out of the culture liquor, for example by means of membrane filters or by other suitable measures, such as, for example, by centrifugation. Suitable membrane filters are, for example, nylon or nitrocellulose membranes. Membranes of that type are readily available on the market.

The crystal proteins isolated in that manner can then be very readily identified and quantified within the framework of a suitable immunoassay.

Preferred within the scope of this invention is an immunoassay based on an ELISA assay using protoxin-specific antibodies, which assay is described in detail, for example, in Antibodies: A Laboratory Manual, eds. Harlow E and Lane D; Cold Spring Harbor -16Laboratories, (1988). The antibodies used in that immunoassay are on the one hand monoclonal antibodies directed specifically against B.t. protoxin and, on the other hand, an anti-immunoglobulin antibody provided with a specific marker, but preferably a peroxidase-labelled anti-mouse rabbit IgG antibody which is obtainable, for example, from DIANAVA GmbH [Hamburg, Federal Republic of Germany; cat.# 315-035-045].

Especially preferred within the scope of the process according to the invention is the use of monoclonal antibodies that recognise a certain portion of the protein molecule very specifically. Those antibodies can be used either individually or in the form of a mixture. In addition, however, it is, of course, also possible to use polyclonal antisera in the immunoassay. Mixtures based on monoclonal and polyclonal antibodies are also possible.

Processes for the preparation of monoclonal antibodies against Bacillus thuringiensis protoxin proteins are known and are described in detail, for example, in Huber-Lukad et al (1986). Those processes can also be used in the present case. In addition, it is, of course, also possible within the scope of this invention to use other suitable immunoassays.

When the above-described procedures are used, a modified B.t. toxin is obtained that exhibits normal stability even at a temperature of > 25 °C and is accordingly obtainable in customary yields at the preferred cultivation temperature forB. thuringiensis cells of from 30°C to 33°C.

Especially preferred within the scope of this invention is a modified B.t. toxin that has the amino acid sequence shown in SEQ ID NO: 5, and also mutants and variants thereof that have undergone structural changes that do not substantially alter the specific properties and the properties according to the invention of the starting molecule.

Bacillus thuringiensis and B. cereus cells that have been transformed by means of the above-described process, and the toxins produced by those transformed Bacillus cells, are extremely suitable for the control of insects, especially for the control of insects of the order Lepidoptera.

The present invention accordingly relates also to a method of controlling insects, wherein the insects or their locus are treated a) with B. thuringiensis or B. cereus cells, or with a mixture of the two, that have been transformed using a recombinant DNA molecule containing a CrylA(b) structural - 17gene that codes for a modified, temperature-stable δ-endotoxin polypeptide that is stable even at a cultivation temperature of > 25 °C, especially at the preferred temperature for the cultivation of the B. thuringiensis cells of from 30°C to 33°C, and is accordingly obtainable in customary yields, or for a polypeptide that is substantially homologous thereto; or b) with cell-free crystal body preparations containing a modified temperature-stable protoxin that is produced by said transformed Bacillus cells.

The present invention also includes insecticidal compositions that contain as active component, together with the carriers, dispersing agents or carrier and dispersing agents customarily used, a) B. thuringiensis οτΒ. cereus cells, or a mixture of the two, that have been transformed using a recombinant DNA molecule containing a CrylA(b) structural gene that codes for a modified, temperature-stable δ-endotoxin polypeptide that is stable even at a cultivation temperature of > 25°C, especially at the preferred temperature for the cultivation of the B. thuringiensis cells of from 30°C to 3 3 °C, and is accordingly obtainable in customary yields, or for a polypeptide that is substantially homologous thereto; or b) cell-free crystal body preparations containing a modified, temperature-stable protoxin that is produced by said transformed Bacillus cells.

For use as insecticides, the transformed microorganisms containing the recombinant B. thuringiensis toxin gene, preferably transformed living or dead B. thuringiensis or B. cereus cells, including mixtures of living and dead B. thuringiensis and£. cereus cells, and also the toxin proteins produced by said transformed cells, are used in unmodified form or preferably together with the adjuvants conventionally employed in the art of formulation, and are formulated in a manner known per se, e.g. into suspension concentrates, coatable pastes, directly sprayable or dilutable solutions, wettable powders, soluble powders, dusts, granulates, and also encapsulations in e.g. polymer substances.

As with the nature of the compositions, the methods of application, such as spraying, atomising, dusting, scattering, coating or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.

It is, of course, also possible to use insecticidal mixtures consisting of transformed living - 18or dead/?. thuringiensis and/or B. cereus cells and also of cell-free crystal body preparations containing a protoxin that is produced by said transformed Bacillus cells.

The formulations, i.e. the compositions or preparations containing the transformed living or dead Bacillus cells or mixtures thereof and also the toxin proteins produced by said transformed Bacillus cells, and, where appropriate, solid or liquid adjuvants, are prepared in known manner, e.g. by homogeneously mixing the transformed cells and/or toxin proteins with solid carriers and, where appropriate, surface- active compounds (surfactants).

The solid carriers used e.g. for dusts and dispersible powders are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are, for example, calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverised plant residues.

Suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good dispersing and wetting properties. The term surfactants will also be understood as comprising mixtures of surfactants.

Both so-called water-soluble soaps and also water-soluble synthetic surface-active compounds are suitable anionic surfactants.

Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C40-C22), e-S· sodium or potassium salts of oleic or stearic acid or of natural fatty acid mixtures which can be obtained e.g. from coconut oil or tallow oil. Fatty acid methyltaurin salts, e.g. the sodium salt of cis-2-(methyl-9-octadecenylamino)-ethanesulfonic acid (content in formulations preferably approximately 3 %), may also be mentioned as surfactants.

More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates or fatty alcohols, e.g. 2,4,7,9-tetramethyl-5-decine-4,7-diol (content in formulations -19IE 91322 preferably approximately 2 %).

The fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and contain a C8-C22alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. the sodium or calcium salt of lignosulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfated and sulfonated fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a condensate of naphthalenesulfonic acid and formaldehyde.

Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

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

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan, e.g. polyoxyethylene sorbitan trioleate, are also suitable.

Cationic surfactants are preferably quaternary ammonium salts which contain, as N-substituent, at least one C8-C22alkyl radical and, as further substituents, unsubstituted or -20halogenated lower alkyl, benzyl or hydroxy-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described, inter alia, in the following publications: 1986 International McCutcheon’s Emulsifiers & Detergents, The Manufacturing Confectioner Publishing Co., Glen Rock, NJ, USA; Helmut Stache Tensid-Taschenbuch Carl Hanser-Verlag Munich/Vienna 1981.

The agrochemical compositions usually contain 0.1 to 99 %, preferably 0.1 to 95 %, of the transformed living or dead Bacillus cells, or mixtures thereof, or of the toxin proteins produced by said transformed Bacillus cells, 99.9 to 1 %, preferably 99.8 to 5 %, of a solid or liquid adjuvant and 0 to 25 %, preferably 0.1 to 25 %, of a surfactant Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations.

Such compositions may also contain further ingredients such as stabilisers, antifoams, viscosity regulators, binders, tackifiers as well as fertilisers or other active ingredients for obtaining special effects.

The transformed living or dead Bacillus cells or mixtures thereof containing the recombinant modified CrylA(b) genes of B. thuringiensis, and the temperature-stable protoxin proteins themselves which are produced by said transformed Bacillus cells, are extremely suitable for the control of harmful insects and are accordingly also included in the present invention.

There may preferably be mentioned in this connection plant-destructive insects of the order Lepidoptera, especially those of the genera Pieris, Heliothis, Spodoptera, Lymanthria and Plutella, such as, for example, Pieris brassicae, Heliothis virescens, Heliothis zea, Spodoptera littoralis, Spodoptera frugiperda, Lymanthria dispar and Plutella xylostella.

The rates at which the Bacillus cells or the toxin proteins produced thereby are used -21 depend on the prevailing circumstances, such as, for example, the weather conditions, the nature of the soil, plant growth and the time of application.

Formulation examples for B. /Zmringiewsis-toxin-containing material In the following formulation examples, the term Bacillus cells is to be understood as meaning those B. thuringiensis and/or B. cereus cells which contain a recombinant CrylA(b) gene of B. thuringiensis according to the invention. (Throughout, percentages are by weight.) Fl. Granulates a) b) Bacillus cells and/or toxin protein produced thereby 5 % 10 % kaolin 94 % highly dispersed silicic acid 1 % attapulgite - 90 % The Bacillus cells and/or toxin protein produced thereby are first suspended in methylene chloride, the suspension is subsequently sprayed onto the carrier and then the suspending agent is evaporated in vacuo.

F2. Dusts a) b) Bacillus cells and/or toxin protein produced thereby 2 % 5 % highly dispersed silicic acid 1 % 5 % talcum 97 % kaolin 90% Ready-for-use dusts are obtained by intimately mixing the carriers with the Bacillus cells and/or the toxin protein produced thereby.

F3. Wettable powders Bacillus cells and/or toxin protein produced thereby sodium lignosulfonate sodium laurylsulfate a) % % b) c) 50% 75% % % -226% 10% sodium diisopropylnaphthalenesulfonate octylphenolpolyethylene glycol ether (7-8 moles of ethylene oxide) highly dispersed silicic acid 5% 10% 10% kaolin 2% % 27 % The Bacillus cells and/or toxin protein produced thereby are carefully mixed with the adjuvants and the mixture obtained is then thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentration.

F4. Extruder granulates Bacillus cells and/or toxin protein produced thereby 10 % sodium lignosulfonate 2 % carboxymethylcellulose 1 % kaolin 87 % The Bacillus cells and/or toxin protein produced thereby are mixed and carefully ground with the adjuvants, and the mixture is moistened with water. The mixture is extruded and then dried in a stream of air.

F5. Coated granulate Bacillus cells and/or toxin protein produced thereby 3 % polyethylene glycol 200 3 % kaolin 94 % The homogeneously mixed Bacillus cells and/or toxin protein produced thereby are uniformly applied, in a mixer, to the kaolin moistened with polyethylene glycol. Nondusty coated granulates are obtained in this manner.

F6. Suspension concentrate Bacillus cells and/or toxin protein produced thereby 40% -2310% tlE ethylene glycol nonylphenol polyethylene glycol (15 moles of ethylene oxide) salt* 3% carboxymethylcellulose silicone oil in the form of a 75 % aqueous emulsion % alkylbenzenesulfonic acid triethanolamine 0.1 % water 39% * Alkyl is preferably linear with from 10 to 14, especially from 12 to 14, carbon atoms, such as, for example, n-dodecylbenzenesulfonic acid triethanolamine salt.

The homogeneously mixed Bacillus cells and/or toxin protein produced thereby are intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desired concentration can be obtained by dilution with water.

General recombinant DNA techniques Since many of the recombinant DNA techniques employed in this invention are a matter of routine for the person skilled in the art, a short description of the generally used techniques is given below so that it is unnecessary to give this general information in the specific embodiment. Except where there is a specific indication to the contrary, all these procedures are described in the Maniatis et al (1982) reference.

A. Cleaving with restriction endonucleases A reaction batch typically contains approximately from gg/ml to 500 pg/ml of DNA, preferably in a buffer solution recommended by the manufacturer. From 2 to 5 units of restriction endonucleases are added for each pg of DNA and the reaction batch is incubated for from one to three hours at the temperature recommended by the manufacturer. The reaction is terminated by heating at 65°C for 10 minutes or by extraction with phenol, followed by precipitation of the DNA with ethanol. This technique is also described on pages 104 to 106 of the Maniatis et al (1982) reference.

B. Treatment of the DNA with polymerase in order to produce blunt ends From 50 pg/ml to 500 pg/ml of DNA fragments are added to a reaction batch, preferably -24in a buffer recommended by the manufacturer. The reaction batch contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. After adding a suitable DNA polymerase, the reaction takes place over a period of 30 minutes at 15 °C and is then terminated by heating at 65°C for 10 minutes.

For fragments obtained by cleaving with restriction endonucleases that produce 5’-projecting ends, such as EcoRI and BamHI, the large fragment, or Klenow fragment, of DNA polymerase is used. For fragments obtained by means of endonucleases that produce 3’-projecting ends, such as Pstl and Sacl, T4 DNA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of the Maniatis et al (1982) reference.

C. Agarose gel electrophoresis and purification of DNA fragments from gel impurities Agarose gel electrophoresis is carried out preferably in a horizontal apparatus, as described on pages 150 to 163 of the Maniatis et al (1982) reference. The buffer used is the Tris-borate or Tris-acetate buffer described therein. The DNA fragments can then be stained, for example, using 0.5 pg/ml of ethidium bromide which is either present in the gel or tank buffer during electrophoresis or, alternatively, is added after termination of electrophoresis. The DNA is made visible by illumination with long-wave ultraviolet light.

If the fragments are to be separated from the gel, an agarose is advantageously used that gels at low temperature and is obtainable, for example, from Sigma Chemical, St. Louis, Missouri. After the electrophoresis the desired fragment is cut out, placed in a plastics test tube, heated at 65 °C for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol. This procedure is slightly different from that described by Maniatis et al (1982) on page 170.

As an alternative, the DNA can be isolated from the agarose gel with the aid of the Geneclean kit (Bio 101 Inc., La Jolla, CA, USA).

D. Removal of 5’-terminal phosphates from DNA fragments During the plasmid cloning steps, treatment of the vector plasmid with phosphatase reduces the recircularisation of the vector [discussed on page 13 of the Maniatis et al -25(1982) reference]. After cleavage of the DNA with the correct restriction endonuclease, one unit of calf intestinal alkaline phosphatase, obtainable, for example, from Boehringer-Mannheim, Mannheim, is added. The DNA is incubated at 37°C for one hour and then extracted twice with phenol and precipitated with ethanol.

E. Linking of the DNA fragments If fragments having complementary cohesive ends are to be linked to one another, approximately 100 ng of each fragment are incubated in a reaction mixture of from 20 gl to 40 μΐ containing approximately 0.2 unit of T4 DNA ligase (for example, from New England Biolabs), preferably in a buffer recommended by the manufacturer. Incubation is carried out for from 1 to 20 hours at 15°C. If DNA fragments having blunt ends are to be linked, they are incubated as described above except that the amount of T4 DNA ligase is in this case increased to from 2 to 4 units.

F. Transformation of DNA into E. coli E. coli strain HB101 is used in most of the experiments. DNA can advantageously be introduced into E. coli using the calcium chloride process, as described by Maniatis et al (1982), pages 250 and 251.

G. Screening of E. coli for plasmids After transformation, the resulting colonies of E. coli are tested for the presence of the desired plasmid by means of a rapid plasmid isolation process. Two customary processes are described on pages 366 to 369 of the Maniatis et al (1982) reference.

H. Large-scale isolation of plasmid DNA Processes for the isolation of plasmids from E. coli on a large scale are described on pages 88 to 94 of the Maniatis et al (1982) reference.

Media and buffer solutions LB medium [g/1] tryptone 10 yeast extract 5 NaCl 5 Antibiotic Medium No. 3 (Difco Laboratories) [g/1] -26bovine meat extract 1.5 yeast extract 1.5 peptone 5 glucose 1 NaCI 3.5 K2HPO4 3.68 KH2PO4 1-32 SCGY medium [g/1] casamino acids 1 yeast extract 0-1 glucose 5 K2HPO4 14 KH2PO4 6 Na3 citrate 1 (NH4)2SO4 2 MgSO4 · 7 H2O 0.2 GYS medium (Yousten & Rogoff, 1969) [g/1] glucose 1 yeast extract 2 (NH4)2SO4 2 K2HPO4 0.5 MgSO4 · 7 H2O 0.2 CaCl2-2H2O 008 MnSO4 ♦ H2O 0.05 adjust pH to 7.3 before autoclaving.

PBS buffer [mM] saccharose 400 MgCl2 1 phosphate buffer, pH 6.0 7 TBST buffer [mM] Tween 20* 0.05 % (w/v) -27Tris/HCl* (pH 8.0) 10 NaCl 150 Buffer High [Maniatis et al (1982); page 104] [mM] NaCl 100 Tris/HCl* (pH 7.5) 50 MgCl2 10 dithiothreitol 1 Nick-translation buffer (lOx) [mM] ' Tris/HCl* (pH 7.2) 500 MgSO4 100 dithiothreitol 1 bovine serum albumin (BSA Pentax fraction V) 500 pg/ml ♦Tween 20 Polyethoxysorbitan laurate ♦Tris/HCl a,tt,tt-Tris(hydroxymethyl)methylaminohydrochloride -28Non-Iimiting Examples Construction of plasmid pXI93 Plasmid pK36: The CrylA(b) gene which is used within the scope of this invention for insertion and expression in B. thuringiensis orB. cereus and which codes for a Kurhdl delta-endotoxin protein originates from plasmid pK36 (=pXI36) which was deposited under deposit number DSM 3668 on 4th March 1986 at the Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany, which is recognised as an international depository, in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

A detailed description of the methods of identifying and isolating the δ-endotoxin genes and the construction of plasmid pK36 is contained in European Patent Application EP-A 0 238 441 and, in the form of a reference, is part of the present invention.

Plasmid pXI61: For the construction of a potent bifunctional vector, first the large EcoRI fragment of pBC16, a naturally occurring plasmid of Bacillus cereus, is spliced into the EcoRI site of plasmid pUC8 with the aid of T4 DNA ligase (28^Vieira J and Messing J, 1982). E. coli cells are then transformed using this construct. A construction recognised as being correct by means of restriction analysis is called pXI62.

The EcoRI cleavage site located in the distal position with respect to the pUC8 polylinker region is then removed. pXI62 is linearised by partial EcoRI digestion. The cohesive EcoRI ends are filled using Klenow polymerase and joined together again with T4 DNA ligase. After transformation into E. coli, a construction recognised as being correct by means of restriction analysis is selected and called pXI61.

That construction can be transformed directly into B. thuringiensis HDlcryB by means of the method of transformation described in Example 7.

Plasmid pXI93: pK36 plasmid DNA is digested completely with the restriction enzymes Pstl and BamHI -29and the 4.3 Kb fragment which contains the Kurhdl δ-endotoxin gene is isolated from an agarose gel. That fragment is then spliced into PXI61 which has previously been digested with Pstl and BamHI and treated with calf intestinal alkaline phosphatase. After the transformation of E. coli HB101, a construction recognised as being correct by means of restriction analysis is isolated and is given the name pXI93. A detailed description of the processes for constructing plasmid pXI93 is contained in European Patent Application EP-A 0 342 633. In addition, plasmids pXI61 and pXI93 are deposited in accordance with the provisions of the Budapest Treaty at the ’Deutsche Sammlung von Mikroorganismen’ (Brunswick, Federal Republic of Germany), which is recognised as an international depository, under number DSM 4572 (pK61 = pXI61, transformed into B. thuringiensis var. kurstaki HD1 CryB) and DSM 4571 (pK93 = pXI93, transformed into B. thuringiensis var. kurstaki HD1 CryB) and DSM 4573 (pK93 = pXI93, transformed into B. cereus 569 K). 2. Digestion of the pXI93 DNA The digestion of 5 pg of pXI93 plasmid DNA with the restriction enzyme Kpnl is carried out in accordance with the manufacturer’s instructions (Boehringer, Mannheim, Federal Republic of Germany). When the DNA has been completely digested, it is precipitated with ethanol at low temperatures (-20°C and below) under the conditions described in Maniatis et al (1982) [see page 461]. The DNA is then resuspended in the buffer suggested by Maniatis et al (1982) for a Pstl digestion or using a commercially available incubation set (’incubation set’; Boehringer, Mannheim; cat# 1082035) in accordance with the manufacturer’s instructions and is there completely digested again. After completion of that second digestion step, the large DNA fragment is separated from the other fragments on an agarose gel and isolated by means of electroelution. 3. Construction of plasmid pXI54 3.1 pHD73: The total DNA obtainable from Bacillus thuringiensis subsp. kurstaki HD73 [the B.t. strain HD73 is described in Adang et al (1985) and is commercially available under number NRRL HD73 from Microbial Property Research, USDA, Northern Reg. Res. Center in Peoria, USA] is digested completely with the restriction enzyme Hindlll and then spliced into the pUC8 vector (New England Biolabs). A correct clone, which is ascertained by hybridisation with a radioactively labelled EcoRI fragment comprising 726 bp (positions 423-1149 of the CrylA(b) gene), is given the name pHD73. A fragment suitable for the above hybridisation reaction can be obtained, for example, from plasmid -30pXI36. It can then be radioactively labelled, for example using a commercially available labelling kit (’oligolabelling kit’; Pharmacia cat# 27-9250-01).

By digesting pHD73 with the restriction enzymes Spel and Hindlll under the conditions recommended by the manufacturer [buffer having a high ionic strength, ’Buffer High’, Maniatis et al (1982), page 104] a corresponding Spel-Hindlll fragment is obtained on which the 5’ end of the CrylA(c) gene [DNA positions 564 to 3212 in the gene described in Adang et al (1985)] is located. The correct fragment, comprising 2648 bp, is then separated by means of gel electrophoresis and isolated from the agarose gel by electroelution. 3.2 pXI36: In plasmid pXI36 (see Example 1) the Hindlll recognition site within the polylinker region is eliminated by first digesting said plasmid with Hindlll and then filling the cohesive ends so formed with dNTP [the reaction batch contains all four deoxynucleotide triphosphates] with the addition of the Klenow fragment of the E. coli polymerase I in a nick-translation buffer [see Maniatis et al (1982), page 394].

The fragments formed, which now have blunt ends, are linked to one another in a ligase reaction, transformed into E. coli HB101 and analysed by means of restriction analysis. A suitable clone, in the case of which the HindHI recognition site within the polylinker region of plasmid pXI36 has been eliminated, is then digested with Spel and HindHI and the large fragment is separated by means of gel electrophoresis and isolated from the agarose gel by electroelution. 3.3 pXI54: The Spel-HindlH fragment (see 3.1) previously isolated from pHD73 is then linked to the large fragment of pXI36 (see 3.2) using T4 DNA ligase and cloned into a suitable E. coli strain (for example E. coli HB101 or DH1) under the conditions indicated in Maniatis et al (1982) (see page 250). A correct clone, which can be ascertained by means of restriction analysis, is given the name pXI54. 4. Isolation of a 1460 bp HindHI-Pstl fragment of pXI36 The 3’ end of the CryIA(b) gene is located on a 1460 bp HindHI-Pstl fragment of plasmid pXI36 [positions 2899 - 4359 of the sequence shown in EP-A 342 633]. 5 gg of pXI36 DNA are digested completely with the restriction enzymes HindHI and Pstl in accordance with the manufacturer’s instructions (Boehringer, Mannheim; ’incubation buffer set’, cat# 1 882 035). The 1460 bp fragment is then separated using an agarose gel. -31 5. Isolation of a 648 bp KpnI-Hindin fragment of pXI54 μg of pXI54 DNA are digested with Kpnl and Hindlll under standard conditions prescribed by the manufacturer (Boehringer, Mannheim, Federal Republic of Germany). The 648 bp fragment [positions 2564 - 3212 in the sequence published in Adang et al (1985)] is then separated using an agarose gel. 6. Linking of the isolated fragments and cloning into B. thuringiensis The large KpnI-Pstl fragment of plasmid pXI93 (see point 2), the 648 bp KpnI-Pstl fragment of plasmid pXI54 containing a section of the CrylA(c) gene (see point 5) and the 1460 bp fragment of pXI36 (see point 4) are linked to one another under standard conditions and cloned into E. coli HB101. After restriction analysis, a correct clone is isolated and is given the name pXI106.

The sequence of the δ-endotoxin gene located on plasmid pXI106 is in principle identical with the known sequence of plasmid pXI93. The only difference concerns the sequence section from positions 2329 (Kpnl cleavage site) to 2899 (Hindlll cleavage site) in the CrylA(b) gene [see SEQ ID NO: 1]. In plasmid pXI106, that fragment has been replaced by a corresponding Kpnl-Hindlll fragment of the CrylA(c) gene which is located there between positions 2564 (Kpnl cleavage site) and 3212 (Hindlll cleavage site) [see SEQ ID NO: 2].

That pXI106 plasmid is then transformed into the B. thuringiensis strain CryB. A corresponding B. thuringiensis strain that has been transformed using plasmid pXI93 acts as a control. 7. Transformation of pXI106 into B. thuringiensis CryB ml of an LB medium (tryptone 10 g/1, yeast extract 5 g/1, NaCl 5 g/1) are inoculated with spores of B. thuringiensis var. kurstaki HDlcryB (Stahly DP etal, 1978), a plasmidfree variant of B. thuringiensis var. kurstaki HD1.

That batch is incubated overnight at a temperature of 27°C on a rotary shaker at 50 rpm. The B. thuringiensis culture is then diluted 100-fold in from 100 ml to 400 ml of LB medium and cultivated further at a temperature of 30°C on a rotary shaker at 250 rpm until an optical density (OD550) of 0.2 has been achieved. -32The cells are harvested by centrifugation and suspended in 1/40 volume of an ice-cooled PBS buffer (400 mM saccharose, 1 mM MgCl2, 7 mM phosphate buffer pH 6.0). The centrifugation and subsequent suspension of the harvested B. thuringiensis cells in PBS buffer is repeated once.

The cells pre-treated in that manner can then be either electroporated directly or, after the addition of glycerol to the buffer solution [20 % (w/v)], stored at from -20°C to -70°C and used at a later time. 800 μΐ aliquots of the ice-cooled cells are then transferred to pre-cooled cuvettes, 0.2 μg of plasmid DNA (20 pg/ml) is then added and the whole batch is incubated for 10 minutes at 4°C.

When deep-frozen cell material is used, first a suitable aliquot of frozen cells is thawed in ice and at room temperature. The further treatment is carried out analogously to the procedure applied when fresh cell material is used.

The cuvette is then introduced into an electroporation apparatus and the B. thuringiensis cells present in the suspension are subjected to electroporation by applying voltages of from 0.1 kV to 2.5 kV by discharging a capacitor once.

The capacitor used in this case has a capacity of 25 pF, the distance between the electrodes of the cuvettes is 0.4 cm, which, when discharge takes place and depending on the setting, results in an exponentially decreasing field strength with initial peak values of from 0.25 kV/cm to 6.25 kV/cm. The exponential decay time is in the range from 10 ms to 12 ms.

The described electroporation experiments can be carried out, for example, with an electroporation apparatus manufactured by Bio Rad (’Gene Pulser Apparatus’, # 165-2075, Bio Rad, 1414 Harbour Way South, Richmond, CA 94804, USA).

Any other suitable apparatus can, of course, also be used in the process according to the invention.

After a further 10 minutes’ incubation at 4°C, the cell suspension is diluted with 1.2 ml of LB medium and incubated for 2 hours at a temperature of 30°C on a rotary shaker at 250 -33rpm.

Suitable dilutions are then plated out onto LB agar (LB medium solidified with agar, 15 g/l) which contains as an additive an antibiotic suitable for the selection of the newly obtained plasmid. In the case of pXI106, the antibiotic is tetracycline which is added to the medium at a concentration of 20 mg/1.

Bacillus cereus cells can be transformed in accordance with the above protocol in the same manner as B. thuringiensis cells. 8. Expression of the modified CrylA(b) toxin (pXI106) and the wild-type toxin (pXI93) in B. thuringiensis CryB at 25°C and at 33°C In order to inoculate the sporulation medium [GYS medium (50 ml) with tetracycline (20 gg/ml)] with the B. thuringiensis strains CryB (pXI106) and CryB (pXI93), 0.5 ml of an overnight culture grown in an LB medium [with 20 gg/ml of tetracycline] is used. The two B. thuringiensis strains CryB (pXI106) and CryB (pXI93) are cultivated in the sporulation medium (GYS medium) at cultivation temperatures of 25°C and 33°C, while being constantly agitated [ca. 250 rpm], until the end of the sporulation phase.

During the course of the growth phase, samples (1.5 ml) are constantly taken and analysed by SDS-PAGE. The samples are also examined under a microscope for the presence of spores or crystals.

After lysis of the cells, the protoxin crystals, together with the cellular constituents, are removed by centrifugation and then resuspended in a suitable buffer. Identical aliquots are then removed from the suspension and separated by means of polyacrylamide gel electrophoresis (7.5 % SDS-PAGE). The protoxin-containing fractions are eluted from the gel and resuspended in PBS buffer. 9. ELISA assay for the quantitative determination of the pXI106 and pXI93 protoxins The detailed protocol for this immunoassay is described in Antibodies: A Laboratory Manual, eds. Harlow E and Lane D; Cold Spring Harbor Laboratories, (1988). The antibodies used in that immunoassay are on the one hand monoclonal antibodies directed specifically against B.t. protoxin and, on the other hand, peroxidase-labelled anti-mouse rabbit IgGs obtainable, for example, from DIANAVA GmbH [Hamburg, Federal Republic -34of Germany; cat.# 315-035-045]. 9.1 Preparation of monoclonal antibodies against B. thuringiensis protoxin The preparation of monoclonal antibodies against δ-endotoxin from Bacillus thuringiensis var. kurstaki HD1 is carried out analogously to the process described in Huber-Lukac et al (1986).

The hybridoma cells used for the preparation of the antibodies are fusion products of Sp2/O-Ag myeloma cells [described in Shulman et al (1978), this cell line is commercially available from the ’American Type Culture Collection’ in Rockville, Maryland, USA] and spleen lymphocytes from BALB/c mice previously immunised with δ-endotoxin and Bacillus thuringiensis var. kurstaki HD1.

Monoclonal antibodies can thus be obtained that are directed specifically against the δ-endotoxin of Bacillus thuringiensis var. kurstaki HD1.

It is, however, quite possible to use other monoclonal or, alternatively, polyclonal antibodies in the immunoassay which follows. 9.2 ELISA assay (1) Aliquots of the partially purified antigen sample in section 8 are bound to microtitre plates by introducing 50 pi of the antigen-containing suspension (in PBS) into each well; (2) there follows 2 hours’ incubation at room temperature in a humid atmosphere; (3) The plates are washed twice with PBS; (4) The remaining free protein binding sites on the plate are saturated by means of a blocking buffer. For that purpose, the wells are filled with a 3 % BSA/PBS buffer containing 0.02 % sodium azide and incubated for 2 hours at room temperature and in a humid atmosphere; (5) The plates are washed twice with PBS; (6) 50 μΐ of an antibody-containing solution (monoclonal antibody against δ-endotoxin -35from Bacillus thuringiensis var. kurstaki HD1) are introduced into each of the wells pre-treated in accordance with the above protocol and the whole batch is incubated for 2 hours at room temperature and in a humid atmosphere; (7) The antibodies that are not bound are removed by washing twice with PBS; (8) 50 μΐ of a second, labelled antibody (peroxidase-labelled anti-mouse rabbit IgGs) are then introduced into each well. The whole batch is incubated for 2 hours at room temperature and in a humid atmosphere. In order to ensure accurate quantitative determination, the second antibody should be present in excess. (9) The antibodies that are not bound are removed by washing 4 times with PBS; (10) After the final washing operation, there are introduced into each of the wells 50 μΐ of the substrate solution [0.1 mg of 3’,3’,5’,5’-tetramethylbenzidine (TMB) dissolved in 0.1 ml of dimethyl sulfoxide. 9.9 ml of 0.1 M sodium acetate (pH 6.0) are added to the solution, the whole is then filtered through a Whatman No. 1 filter or a comparable filter and adjusted with hydrogen peroxide to give a final concentration of 0.01 %]. (11) The incubation time is from 10 to 30 minutes at room temperature. The positives have a pale blue appearance; (12) After adding 50 μΐ of a 1M H2SO4, the positives have a bright yellow appearance; (13) Quantitative determination is effected at 450 nm.

The results are shown in Table 1.

Both the pXI106 and the pXI93 gene products are stable at a cultivation temperature of 25 °C and are accordingly detectable in the nutrient solution.

At a cultivation temperature of 33°C, on the other hand, modified CryLA(b) toxin is found almost exclusively, while the gene product of plasmid pXI93 is present in an approximately 30 times lower concentration.

Table 1: Concentration of the gene products of pXI93 [CryLA(b) gene] and pXI106 IE 91322 -36[modified CrylA(b) gene] in the sporulation culture at 25°C and 33°C. plasmid temperature 25°C 33°C pXI93 [CrylA(b) gene] 14.8* 0.53* pXI106 [modified CrylA(b) gene] 17.1* 17.6* * this information refers to |lg toxin/ml sporulation culture IE 91322 -37Bibliography Antibodies: A Laboratory Manual, eds. Harlow E and Lane D; Cold Spring Harbor Laboratories, (1988) Adang et al, Gene, 36: 289-300 (1985) Bevan et al, Nature, 304: 184-187 (1983) Bolivar et al, Gene, 2: 95-113 (1977) Geiser et al, Gene, 48: 109-118, (1986) Hinnen et al, Proc. Natl. Acad. Sci., USA, 75: 1929-1933 (1978) Hofte and Whiteley, Microbiol. Rew., 53(2): 242-255 (1989) Huber-Lukad et al, Infect. Immunol., 54: 228-232 (1986) Kronstad JW and Whiteley, J. Bacteriol., 160: 95-102 (1984) Maniatis et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, (1982) McLinden et al, Appl. Environ. Microbiol., 50: 623-628 (1985) Norrander et al, Gene, 26: 101-104 (1983) Shulman etal, Nature, 276:269 (1978) Schurter et al. Mol. Gen. Genet., 218: 177-181 (1989) Stahly DP et al, Biochem. Biophys. Res. Comm., 84: 581-588 (1978) Wong et al, J. Biol, Chem., 258(3): 1960 (1983) Young et al, Proc. Natl. Acad. Sci., USA, 80: 1194-1198 (1983) Youston AA and Rogoff MH, J. Bacteriol., 100: 1229-1236,1969 1986 International McCutcheon’s Emulsifiers & Detergents, The Manufacturing Confectioner Publishing Co., Glen Rock, NJ, USA Helmut Stache Tensid-Taschenbuch” Carl Hanser-Verlag Munich/Vienna 1981.

Patent literature EP-A 238 441 EP-A 342 633 US-P 4,237,224 US-P 4,468,464 US-P 4,683,202 -38SEQ ID NO: 1 SEQUENCE TYPE: nucleotide sequence SEQUENCE LENGTH: 570 bases STRANDEDNESS: single MOLECULE TYPE: CrylA(b) gene [positions 2329-2899] ORIGINAL SOURCE ORGANISM: Bacillus thuringiensis subsp. kurstaki HD1 IMMEDIATE EXPERIMENTAL SOURCE ORGANISM: E. coli HB101 (pK36) [DSM 3667] K P n I TGGGTACC TTTGATGAGT GCTATCCAAC GTATTTATAT CAAAAAATAG 2370 ATGAGTCGAA ATTAAAAGCC TATACCCGTT ACCAATTAAG AGGGTATATC 2420 GAAGATAGTC AAGACTTAGA AATCTATTTA ATTCGCTACA ATGCCAAACA 2470 CGAAACAGTA AATGTGCCAG GTACGGGTTC CTTATGGCCG CTTTCAGCCC 2520 CAAGTCCAAT CGGAAAATGT GCCCATCATT CCCATCATTT CTCCTTGGAC 2570 ATTGATGTTG GATGTACAGA CTTAAATGAG GACTTAGGTG TATGGGTGAT 2620 ATTCAAGATT AAGACGCAAG ATGGCCATGC AAGACTAGGA AATCTAGAAT 2670 TTCTCGAAGA GAAACCATTA GTAGGAGAAG CACTAGCTCG TGTGAAAAGA 2720 GCGGAGAAAA AATGGAGAGA CAAACGTGAA AAATTGGAAT GGGAAACAAA 2770 TAT T GT T TAT AAAGAGGCAA AAGAATCTGT AGATGCTTTA TTTGTAAACT 2820 CTCAATATGA TAGATTACAA GCGGATACCA ACATCGCGAT GATTCATGCG 2870 GCAGATAAAC GCGTTCATAG CATTCGAGAA GCTTA 2905 H i n d I I I IE 91322 SEQ ID NO: 2 SEQUENCE TYPE: nucleotide sequence SEQUENCE LENGTH: 648 bases STRANDEDNESS: double MOLECULE TYPE: CrylA(c) gene [positions 2564-3212] ORIGINAL SOURCE ORGANISM: Bacillus thuringiensis subsp. kurstaki HD73 IMMEDIATE EXPERIMENTAL SOURCE ORGANISM: pHD73 K P n I AG TC GTACCTTTGA CATGGAAACT TGAGTGCTAT ACTCACGATA CCAACATATT GGTTGTATAA TGTATCAAAA ACATAGTTTT 2600 AATCGATGAA TTAGCTACTT TCAAAATTAA AGTTTTAATT AAGCCTTTAC TTCGGAAATG CCGTTATCAA GGCAATAGTT TTAAGAGGGT AATTCTCCCA 2650 ATATCGAAGA TATAGCTTCT TAGTCAAGAC ATCAGTTCTG TTAGAAATCT AATCTTTAGA ATTTAATTCG TAAATTAAGC CTACAATGCA GATGTTACGT 2700 AAACATGAAA TTTGTACTTT CAGTAAATGT GTCATTTACA GCCAGGTACG CGGTCCATGC GGTTCCTTAT CCAAGGAATA GGCCGCTTTC CCGGCGAAAG 2750 AGCCCAAAGT TCGGGTTTCA CCAATCGGAA GGTTAGCCTT AGTGTGGAGA TCACACCTCT GCCGAATCGA CGGCTTAGCT TGCGCGCCAC ACGCGCGGTG 2800 ACCTTGAATG TGGAACTTAC GAATCCTGAC CTTAGGACTG TTAGATTGTT AATCTAACAA CGTGTAGGGA GCACATCCCT TGGAGAAAAG ACCTCTTTTC 2850 TGTGCCCATC ACACGGGTAG ATTCGCATCA TAAGCGTAGT TTTCTCCTTA AAAGAGGAAT GACATTGATG CTGTAACTAC TAGGATGTAC ATCCTACATG 2900 AGACTTAAAT TCTGAATTTA GAGGACCTAG CTCCTGGATC GTGTATGGGT CACATACCCA GATCTTTAAG CTAGAAATTC ATTAAGACGC TAATTCTGCG 2950 AAGATGGGCA TTCTACCCGT CGCAAGACTA GCGTTCTGAT GGGAATCTAG CCCTTAGATC AGTTTCTCGA TCAAAGAGCT AGAGAAACCA TCTCTTTGGT 3000 TTAGTAGGAG AATCATCCTC AAGCGCTAGC TTCGCGATCG TCGTGTGAAA AGCACACTTT AGAGCGGAGA TCTCGCCTCT AAAAATGGAG TTTTTACCTC 3050 IE 91322 -40AGACAAACGT GAAAAATTGG AATGGGAAAC AAATATCGTT TATAAAGAGG 3100 TCTGTTTGCA CTTTTTAACC TTACCCTTTG TTTATAGCAA ATATTTCTCC CAAAAGAATC TGTAGATGCT TTATTTGTAA ACTCTCAATA TGATCAATTA 3150 GTTTTCTTAG ACATCTACGA AATAAACATT TGAGAGTTAT ACTAGTTAAT CAAGCGGATA CGAATATTGC CATGATTCAT GCGGCAGATA AACGTGTTCA 3200 GTTCGCCTAT GCTTATAACG GTACTAAGTA CGCCGTCTAT TTGCACAAGT TAGCATTCGA GAAGCTTA 3218 ATCGTAAGCT CTTCGAAT H i n d I I I IE 91322 -41 SEQ ID NO: 3 SEQUENCE TYPE: amino acid sequence [sequence comparison] SEQUENCE LENGTH: sequence (a): 197 amino acids, sequence (b): 223 amino acids MOLECULE TYPE: protein [d-endotoxin] ORIGINAL SOURCE ORGANISM: Bacillus thuringiensis IMMEDIATE EXPERIMENTAL SOURCE ORGANISM: sequence (a) Bacillus thuringiensis CryB (pXI93) sequence (b) Bacillus thuringiensis CryB (pXI106) Val Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr 733 III I I I I I I I I I I Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Thy Pro Thr Tyr 733 Leu Tyr Gin Lys lie Asp Glu Ser Lys Leu Lys Ala Tyr Thr 747 111111111111:1 Leu Tyr Gin Lys lie Asp Glu Ser Lys Leu Lys Ala Phe Thr 747 Arg Tyr Gin Leu Arg Gly Tyr lie Glu Asp Ser Gin Asp Leu 761 I I I I I I I I I I I I I I Arg Tyr Gin Leu Arg Gly Tyr lie Glu Asp Ser Gin Asp Leu 761 Glu lie Tyr Leu lie Arg Tyr Asn Ala Lys His Glu Thr Val 775 I I I I I I I I I I I I I I Glu lie Tyr Leu He Arg Tyr Asn Ala Lys His Glu Thr Val 775 SE 91322 Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Pro I I I I I I I I I I I I I Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gin Ser Pro lie Gly Ser Pro lie Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp ....... Lys Cys Ala His His Ser His His Phe Ser Leu Asp I I I I I I I I I I I I Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp lie Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val I I I I I I I I I I I I I I He Asp Val Gly Cys Thr Asp Leu Asp Glu Asp Leu Gly Val Trp Val lie Phe Lys lie Lys Thr Gin Asp Gly His Ala Arg I I I I III I I I I I I I Trp Val He Phe Lys He Lys Thr Gin Asp Gly His Ala Arg 789 789 793 803 793 817 805 831 819 845 833 859 -43Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly 847 I I I I I I I I I I I I I I Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly 873 Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg 861 I I I I I I I I I I I I I I Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Phe Arg 887 Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn lie Val Tyr 875 I I I I I I I I I I I I I I Asp Lys Arg Glu Lys Leu Glu Phe Glu Thr Asn He Val Tyr 901 Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser 889 I I I I I I I I I I I I I I Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser 915 Gin Tyr Asp Arg Leu Gin Ala Asp Thr Asn He Ala Met He 903 III I I I I I I I I I I Gin Tyr Asp Gin Leu Gin Ala Asp Thr Asn He Ala Met He 929 His Ala Ala Asp Lys Arg Val His Ser He Arg Glu Ala 917 III I I I I I I I I I His Ala Ala Asp Lys Arg Val His Ser He Arg Glu Ala 943 -44SEQ Π) NO: 4 SEQUENCE TYPE: nucleotide sequence SEQUENCE LENGTH: 4438 bases STRANDEDNESS: single MOLECULE TYPE: modified CrylA(b) gene ORIGINAL SOURCE ORGANISM: Bacillus thuringiensis IMMEDIATE EXPERIMENTAL SOURCE ORGANISM: Bacillus thuringiensis CryB (pXI106) 1 GTTAACACCC TGGGTCAAAA ATTGATATTT. AGTAAAATTA GTTGCACTTT 51 GTGCATTTTT TCATAAGATG AGTCATATGT TTT ^AATTGT A.GTAATGAAA 101 AACAGTATTA TATCATAATG AATTGGTATC TTAATAAAAG AGATGGAGGT 151 AACTTATGGA TAACAATCCG AACATCAATG AATGCATTCC TTATAATTGT 201 TTAAGTAACC CTGAAGTAGA AGTATTAGGT GGAGAAAGAA TAGAAACTGG 251 TTACACCCCA ATCGATATTT CCTTGTCGCT AACGCAATTT CTTTTGAGTG 301 AATTTGTTCC CGGTGCTGGA TTTGTGTTAG GACTAGTTGA TATAATATGG 351 GGAATTTTTG GTCCCTCTCA ATGGGACGCA TTTCTTGTAC AAATTGAACA 401 GTTAATTAAC CAAAGAATAG AAGAATTCGC TAGGAACCAA GCCATTTCTA 451 GATTAGAAGG ACTAAGCAAT CTTTATCAAA TTTACGCAGA. ATCTTTTAGA 501 GAGTGGGAAG CAGATCCTAC TAATCCAGCA TTAAGAGAAG AGATGCGTAT 551 TCAATTCAAT GACATGAACA GTGCCCTTAC AACCGCTATT CCTCTTTTTG 601 CAGTTCAAAA TTATCAAGTT CCTCTTTTAT CAGTATATGT TCAAGCTGCA 6?1 AATTTACATT TATCAGTTTT GAGAGATGTT TCAGTGTTTG GACAAAGGTG 701 GGGATTTGAT GCCGCGACTA TCAATAGTCG TTATAATGAT TTAACTAGGC 751 TTATTGGCAA CTATACAC-AT CATGCTGTAC GCTGGTACAA TACGGGATTA 801 GAGCGTGTAT GGGGACCGGA TTCTAGAGAT TGGATAAGAT ATAATCAATT 851 TAC-AAGAGAA TTAACACTAA CTGTATTAGA TATCGTTTCT CTATTTCCGA 901 ACTATGATAG TAGAACGTAT CCAATTCGAA CAGTTTCCCA ATTAACAAGA 951 GAAATTTATA CAAACCCAGT ATTAGAAAAT TTTGATGGTA GTTTTCGAGG 1001 CTCGGCTCAG GGCATAGAAG GAAGTATTAG GAGTCCACAT TTGATGGATA 1051 TACTTAACAG TATAACCATC TATACGGATG CTCATAGAGG. AGAATATTAT 1101 TGGTCAGGGC ATCAAATAAT GGCTTCTCCT GTAGGGTTTT CGGGGCCAGA 1151 ATTCACTTTT CCGCTATATG GAACTATGGG AAATGCAGCT CCACAACAAC 1201 GTATTGTTGC TCAACTAGGT CAGGGCGTGT ATAGAACATT ATCGTCCACT 1251 TTATATAGAA C-ACCTTTTAA TATAGGGATA AATAATCAAC AACTATCTGT 1301 TCTTGACGGG ACAGAATTTG CTTATGGAAC CTCCTCAAAT TTGCCATCCG 1351 CTGTATACAG AAAAAGCGGA ACGGTAGATT CGCTGGATGA AATACCGCCA 1401 CAGAATAACA ACGTGCCACC TAGGCAAGGA TTTAGTCATC GATTAAGCCA 1451 TGTTTCAATG TTTCGTTCAG GCTTTAGTAA TAGTAGTGTA AGTATAATAA 1501 GAGCTCCTAT GTTCTCTTGG ATACATCGTA GTGCTGAATT TAATAATATA 1551 ATTCCTTCAT CACAAATTAC ACAAATACCT TTAACAAAAT CTACTAATCT 1601 TGGCTCTGGA ACTTCTGTCG TTAAAGGACC AGGATTTACA GGAGGAGATA 1651 TTCTTCGAAG AACTTCACCT GGCCAGATTT CAACCTTAAG AGTAAATATT 1701 ACTGCACCAT TATCACAAAG ATATCGGGTA AGAATTCGCT ACGCTTCTAC 1751 CACAAATTTA CAATTCCATA CATCAATTGA CGGAAGACCT ATTAATCAGG 1801 GGAATTTTTC AGCAACTATG AGTAGTGGGA GTAATTTACA GTCCGGAAGC 1851 TTTAGGACTG TAGGTTTTAC TACTCCGTTT AACTTTTCAA ATGGATCAAG 1901 TGTATTTACG TTAAGTGCTC ATGTCTTCAA TTCAGGCAAT GAAGTTTATA 1951 TAGATCGAAT TGAATTTGTT CCGGCAGAAG TAACCTTTGA GGCAGAATAT 2001 GATTTAGAAA GAGCACAAAA GGCGGTGAAT GAGCTGTTTA CTTCTTCCAA 2051 TCAAATCGGG TTAAAAACAG ATGTGACGGA TTATCATATT GATCAAGTAT 2101 CCAATTTAGT TGAGTGTTTA TCTGATGAAT TTTGTCTGGA TGAAAAAAAA -46GAATTGTCCG TTTACTTCAA GCTGGAGAGG aaagagaatt ATATTTGTAT ATCAATTAAG ATTCGCTACA CTTATGGCCG ATCGATGCGC agggatggag TGATGTAGGA TTAAGATTAA CTCGAAGAGA GGAGAAAAAA TCGTTTATAA CAATATGATC AGATAAACGT TGATTCCGGG TTCACTGCAT TTTTAATAAT AAGAACAAAA GAAGTGTCAC TGTCACAGCG AGATCGAGAA GAAGTATATC AGAAGAATAT AGAAAGTCAA GATCCAAACT AAGTACGGA.T acgttacgct CAAAAAATCG AGGGTATATC ATGCAAAACA CTTTCAGCCC GCCACACCTT aaaagtgtgc TGTACAGACT GACGCAAGAT AACCATTAGT TGGAGAGACA AGAGGCAAAA AATTACAAGC GTTCATAGCA TGTCAA7GCG TCTCCCTATA ggcttatcct CAACCACCGT AAGAAGTTCG TACAAGGAGG CAATACAGAC CAAACAACAC GAGGGTACGT ACATGCGAAG TTAGAGGGAT ATTACCATCC ATTGGGTACC ATGAATCAAA GAAGATAGTC TGAAACAC-TA AAAGTCCAAT GAATGGAATC CCATCATTCG TAAATGAGGA GGGCACGCAA AGGAGAAGCG AACGTGAAAA GAATCTGTAG GGATACGAAT TTCGAGAAGC gctatttttg TGATGCGAGA gctggaacgt TCGGTCCTTG TGTCTGTCCG GATATGGAGA GAACTGAAGT GGTAACGTGT ACACTTCTCG CC-ACTTAGTG CAATAGACAA aaggaggcga TTTGATGAGT ATTAAAAGCC aagacttaga AATGTGCCAG CGGAAAGTGT CTGACTTAGA CATCATTTCT CCTAGGTGTA GACTAGGGAA CTAGCTCGTG attggaatgg atgctttatt attgccatga TTATCTGCCT aagaattaga aatgtcatta GAAAGGGCAT TTGTTCCGGA GGTCGTGGCT aggttgcgta TTAGCAACTG AATGATTATA TAATCGAGGA ATGAGCGGAA CTAGACCGTG TGACGTATTC GCTATCCAAC TTTACCCGTT AA.TCTATTTA GTACGGGTTC GGAGAGCCGA TTGTTCGTGT CCTTAGACAT TGGGTGATCT TCTAGAGTTT TGAAAAGAGC GAAACAAATA TGTAAACTCT TTCATGCGGC GAGCTGTCTG agggcgtatt aaaatggtga GTAGATGTAG ATGGGAAGCA atatccttcg accattcatg tgtagaagag CTGCGACTCA tatgacggag 3451 CCTATGAAAG CAATTCTTCT GTACCAGCTG ATTATGCATC AGCCTATGAA 3501 GAAAAAGCAT ATACAGATGG ACGAAGAGAC AATCCTTGTG AATCTAACAG 3551 AGGATATGGG GATTACACAC CACTACCAGC TGGCTATGTG ACAAAAGAAT 3601 TAGAC-TACTT CCCAGAAACC GATAAGGTAT GGATTGAGAT CGGAGAAACG 3651 GAAGGAACAT TCATCGTGGA CAGCGTGGAA TTACTTCTTA TGGAGGAATA 3701 ATATATGCTT TATAATGTAA GG7GTGCAAA TAAAGAATGA TTACTGACTT 3751 GTATTGACAG ATAAATAAGG AAA T T T T T A T ATGAATAAAA AACGGGCATC 3801 ACTCTTAAAA GAATGATGTC CGTTTTTTGT ATGATTTAAC GAGTGATATT 3851 TAAATGTTTT TTTTGCGAAG GCTTTACTTA ACGGGGTACC GCCACATGCC 3901 CATCAACTTA AGAATTTGCA CTACCCCCAA GTGTCAAAAA ACGTTATTCT 3951 TTCTAAAAAG CTAGCTAGAA AGGATGACAT TTT7TATGAA TCTTTC.AATT 4001 CAAGATGAAT TACAACTATT TTCTGAAGAG CTGTATCGTC ATTTAACCCC 4051 rp rprrrrp GAAGAACTCG AGGTTTTGTA AAAAGAAAAC 4101 C-AAAGTTTTC AGGAAATGAA TATGTATCTG GGGCAG7CAA 4151 CGTACAGCGA. GTGATTCTCT CGTTCGACTA TGCAGTCAAT 7ACACGCCGC 4201 CACAGCACTC TTATGAGTCC AGAAGGAC7C AATAAACGCT TTGATAAAAA 4251 AGCGGTTGAA Φττ’ψψζζ AAAT £ ΓΗ *τ< ΓΗ tp ΓΗΓρ m TGCATTATGG AAAAGTAAAC 4301 TTTGTAAAAC ATCAGCCATT TCAAGTGCAG CACTCACGTA TTTTCAACGA 4351 4401 ' ATCCGTATTT TGTATATCCT TAGATGCGAC GGGTCAGGTG GATTTTCCAA GTTGTGCACA GTACCGAAAC AACTGCAG ATTTAGCACA -48IE 91322^ SEQIDNO: 5 SEQUENCE TYPE: amino acid sequence SEQUENCE LENGTH: 1181 amino acids MOLECULE TYPE: protein [d-endotoxin] ORIGINAL SOURCE ORGANISM: Bacillus thuringiensis IMMEDIATE EXPERIMENTAL SOURCE ORGANISM: Bacillus thuringiensis CryB (pXI106) Met Asp Asn Asn Pro Asn He Asn Glu Cys He Pro Tyr Asn 14 Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg 28 lie Glu Thr Gly Tyr Thr Pro He Asp lie Ser Leu Ser Leu 42 Thr Gin Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe 56 Val Leu Gly Leu Val Asp He lie Trp Gly He Phe Gly Pro 70 Ser Gin Trp Asp Ala Phe Leu Val Gin He Glu Gin Leu He 84 Asn Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala He Ser 98 Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gin lie Tyr Ala Glu 112 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu 126 Arg Glu Glu Met Arg He Gin Phe Asn Asp Met Asn Ser Ala 140 91322 Leu Thr Thr Ala He Pro Leu Phe Ala Val Gin Asn Tyr Gin 154 Val Pro Leu Leu Ser Val Tyr Val Gin Ala Ala Asn Leu His 168 Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gin Arg Trp 182 Gly Phe Asp Ala Ala Thr He Asn Ser Arg Tyr Asn Asp Leu 196 Thr Arg Leu He Gly Asn Tyr Thr Asp His Ala Val Arg Trp 210 Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 224 Asp Trp He Arg Tyr Asn Gin Phe Arg Arg Glu Leu Thr Leu 238 Thr Val Leu Asp He Val Ser Leu Phe Pro Asn Tyr Asp Ser 252 Arg Thr Tyr Pro He Arg Thr Val Ser Gin Leu Thr Arg Glu 266 lie Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe 280 Arg Gly Ser Ala Gin Gly He Glu Gly Ser lie Arg Ser Pro 294 His Leu Met Asp He Leu Asn Ser He Thr He Tyr Thr Asp 308 Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gin He Met 322 Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 336 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gin Gin Arg lie 350 Val Ala Gin Leu Gly Gin Gly Val Tyr Arg Thr Leu Ser Ser 364 Thr Leu Tyr Arg Arg Pro Phe Asn He Gly He Asn Asn Gin 378 Gin Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr 392 Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr 406 Val Asp Ser Leu Asp Glu He Pro Pro Gin Asn Asn Asn Val 420 Pro Pro Arg Gin Gly Phe Ser His Arg Leu Ser His Val Ser 434 Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser He lie 448 Arg Ala Pro Met Phe Ser Trp He His Arg Ser Ala Glu Phe 462 Asn Asn lie He Pro Ser Ser Gin He Thr Gin He Pro Leu 476 Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys 490 Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr Ser 504 Pro Gly Gin He Ser Thr Leu Arg Val Asn lie Thr Ala Pro 518 Leu Ser Gin Arg Tyr Arg Val Arg He Arg Tyr Ala Ser Thr 532 Thr Asn Leu Gin Phe His Thr Ser He Asp Gly Arg Pro He 546 Asn Gin Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 560 Leu Gin Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro 574 Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala 588 His Val Phe Asn Ser Gly Asn Glu Val Tyr He Asp Arg lie 602 Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp 616 Leu Glu Arg Ala Gin Lys Ala Val Asn Glu Leu Phe Thr Ser 630 Ser Asn Gin He Gly Leu Lys Thr Asp Val Thr Asp Tyr His 644 lie Asp Gin Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu 658 Phe Cys Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys 672 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp 686 Pro Asn Phe Arg Gly lie Asn Arg Gin Leu Asp Arg Gly Trp 700 Arg Gly Ser Thr Asp He Thr He Gin Gly Gly Asp Asp Val 714 Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp Glu 728 Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser Lys 742 Leu Lys Ala Phe Thr Arg Tyr Gin Leu Arg Gly Tyr lie Glu 756 Asp Ser Gin Asp Leu Glu He Tyr Leu lie Arg Tyr Asn Ala 770 Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 784 Pro Leu Ser Ala Gin Ser Pro He Gly Lys Cys Gly Glu Pro 798 Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp 812 Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His 826 His Phe Ser Leu Asp lie Asp Val Gly Cys Thr Asp Leu Asn 840 Glu Asp Leu Gly Val Trp Val He Phe Lys He Lys Thr Gin 854 Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu 868 Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala 882 Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu 896 Thr Asn He Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala 910 Leu Phe Val Asn Ser Gin Tyr Asp Gin Leu Gin Ala Asp Thr 924 Asn He Ala Met He His Ala Ala Asp Lys Arg Val His Ser 938 lie Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val He Pro Gly 952 Val Asn Ala Ala He Phe Glu Glu Leu Glu Gly Arg He Phe 966 Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val lie Lys Asn 980 Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly 994 His Val Asp Val Glu Glu Gin Asn Asn His Arg Ser Val Leu 1008 Val Val Pro Glu Trp Glu Ala Glu Val Ser Gin Glu Val Arg 1022 Val Cys Pro Gly Arg Gly Tyr He Leu Arg Val Thr Ala Tyr 1036 Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr He His Glu He 1050 Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu 1064 Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr 1078 Thr Ala Thr Gin Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg 1092 Asn Arg Gly Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val 1106 Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr 1120 Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr 1134 Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu 1148 Leu Glu Tyr Phe Pro Glu Thr Asp Gly Glu Thr Glu Gly Thr Phe He Leu Leu Met Glu Glu Lys Val Trp lie Glu He 1162 Val Asp Ser Val Glu Leu 1176

Claims (33)

1. A modified CrylA(b) gene, in which the deletion present in the C-terminus of the wildtype gene has been eliminated and which codes for a gene product that exhibits normal stability at a temperature of > 25°C and is accordingly obtainable in customary yields at the preferred cultivation temperature for B. thuringiensis cells of from 30°C to 33 °C.

2. A modified CryIA(b) gene according to claim 1, wherein said deletion has been eliminated by replacing a corresponding fragment of the wild-type CrylA(b) gene, which contains that deletion, by a completely or partially overlapping fragment of a CrylA(a) or a CrylA(c) gene without a deletion, or by a DNA fragment that is homologous to those CiylA fragments.

3. A modified CrylA(b) gene according to claim 2, wherein the deletion present in the C-terminus of the wild-type gene has been eliminated by replacing a corresponding fragment of the wild-type CrylA(b) gene, which contains that deletion, by a completely or partially overlapping fragment of a CrylA(c) gene without a deletion, or by a DNA fragment that is homologous to that fragment.

4. A modified CrylA(b) gene according to claim 3, wherein said fragment of the CrylA(c) gene is a KpnI-Hindin fragment.

5. A modified CrylA (b) gene according to claim 4, wherein said ΚρηΙ-HindHI fragment comprises a region that extends from position 2564 to 3212 on the CrylA(c) gene.

6. A modified CryIA(b) gene according to claim 5, having the DNA sequence shown in sequence listing 4 [SEQ ID NO: 4], and mutants and variants thereof, including partial sequences, that still have the characteristic properties of the starting gene.

7. A recombinant DNA molecule containing a modified CrylA(b) gene according to any one of claims 1 to 6.

8. A recombinant DNA molecule according to claim 7, which is a vector molecule.

9. A host cell containing a recombinant DNA molecule according to claim 7 or claim 8. -5510. B. thuringiensis or B. cereus cells that have been transformed using a recombinant DNA molecule containing a modified CrylA(b) structural gene that codes for a modified, temperature-stable δ-endotoxin polypeptide that is stable even at a cultivation temperature of > 25°C and is accordingly obtainable in customary yields, or for a polypeptide that is substantially homologous thereto.

10. 11. B. thuringiensis or B. cereus cells according to claim 11, wherein said modified, temperature-stable δ-endotoxin polypeptide is stable at a temperature of from 30°C to 33°C and is accordingly obtainable in customary yields.

11. 12. A modified temperature-stable protoxin that is coded for by a modified CrylA(b) gene according to any one of claims 1 to 6 and exhibits normal stability at a temperature of > 25 °C and is accordingly obtainable in customary yields at the preferred cultivation temperature forB. thuringiensis cells of from 30°C to 33°C.

12. 13. A modified, temperature-stable protoxin according to claim 12, having the amino acid sequence shown in sequence listing 5 [SEQ ID NO: 5], and mutants and variants thereof, including partial sequences, that still have the same characteristic properties as the starting material.

13. 14. A process for the preparation of a modified CrylA(b) gene that codes for a gene product that exhibits normal stability at a temperature of > 25 °C and is accordingly obtainable in customary yields at the preferred cultivation temperature for B. thuringiensis cells of from 30°C to 33°C, which process comprises eliminating the deletion present in the C-terminus of the CryIA(b) gene by replacing a corresponding fragment of the CrylA(b) gene, which contains that deletion, by a completely or partially overlapping fragment of a CrylA(a) or a CrylA(c) gene without a deletion, or by a DNA fragment that is homologous to those CrylA fragments.

14. 15. A process for the preparation of a modified CrylA(b) gene according to claim 14, wherein said overlapping fragment is a fragment of a CrylA(c) gene, or a DNA fragment that is homologous to that fragment.

15. 16. A process for the preparation of a modified CrylA(b) gene according to claim 15, wherein said fragment is a KpnI-Hindlll fragment. IE 91322 -5617. A process for the preparation of a modified CrylA(b) gene according to claim 14, which comprises linking the following fragments to one another in a ligase reaction: (a) the large Pstl-Kpnl fragment of plasmid pXI93 which is obtainable after restriction digestion and essentially comprises the N-terminal portion of the CrylA(b) gene but not the deletion to be eliminated; (b) a 648 bp KpnI-Hindin fragment of the CrylA(c) gene that covers a DNA region within the C-terminus that is substantially homologous to the corresponding region on the CrylA(b) gene, with the exception of the deletion located there; (c) a 1460 bp Hindlll-Pstl fragment of plasmid pXI36 that contains a major portion of the C-terminus of the CrylA(b) gene, but not the region containing the deletion.

16. 18. A process for the preparation of a modified, temperature-stable protoxin, which comprises (a) transforming B. thuringiensis and/or B. cereus cells using a recombinant DNA molecule according to claim 7 or claim 8; (b) cultivating the transformed Bacillus cells in a suitable medium; and, (c) after sporulation and lysis of the Bacillus cells, isolating the protoxin crystals present in the cultivation medium.

17. 19. A method of controlling insects, which comprises treating insects or their locus a) with B. thuringiensis or B. cereus cells, or with a mixture of the two, that have been transformed using a recombinant DNA molecule containing a CrylA(b) structural gene that codes for a modified, temperature-stable δ-endotoxin polypeptide that is stable even at a cultivation temperature of > 25 °C and is accordingly obtainable in customary yields at the preferred temperature for the cultivation of the B. thuringiensis cells of from 30°C to 33°C, or for a polypeptide that is substantially homologous thereto; or b) with cell-free crystal body preparations containing a modified temperature-stable protoxin that is produced by said transformed Bacillus cells.

18. 20. Insecticidal compositions that contain as active component, together with a suitable carrier, dispersing agent or a carrier and a dispersing agent customarily used, IE 91322 -57a) B. thuringiensis or B. cereus cells, or a mixture of the two, that have been transformed using a recombinant DNA molecule containing a CryIA(b) structural gene that codes for a modified, temperature-stable δ-endotoxin polypeptide that is stable even at a cultivation temperature of > 25°C and is accordingly obtainable in customary yields at the preferred temperature for the cultivation of the B. thuringiensis cells of from 30°C to 33°C, or for a polypeptide that is substantially homologous thereto; or b) cell-free crystal body preparations containing a modified, temperature-stable protoxin that is produced by said transformed Bacillus cells.

19. 21. A process for the preparation of B. thuringiensis and/or B. cereus cells containing a modified CrylA(b) gene according to claim 1, which comprises transforming said Bacillus cells using a recombinant DNA molecule according to claim 7 or claim 8.

20. 22. A process for the preparation of insecticidal compositions, which comprises mixing an insecticidally effective amount of a) B. thuringiensis or B. cereus cells, or a mixture of the two, that have been transformed using a recombinant DNA molecule containing a CryIA(b) structural gene that codes for a modified, temperature-stable δ-endotoxin polypeptide that is stable even at a cultivation temperature of > 25°C and is accordingly obtainable in customary yields at the preferred temperature for the cultivation of theB. thuringiensis cells of from 30°C to 33°C, or for a polypeptide that is substantially homologous thereto; or b) cell-free crystal body preparations containing a modified, temperature-stable protoxin that is produced by said transformed Bacillus cells, with a suitable carrier, dispersing agent or a carrier and a dispersing agent customarily used.

21. 23. A modified CrylA (b) gene according to claim 1, substantially as hereinbefore described and exemplified.

22. 24. A recombinant DNA molecule according to claim 7, substantially as hereinbefore described and exemplified.

23. 25. A host cell according to claim 9, substantially as hereinbefore described and exemplified.

24. 26. A B. thuringiensis or a B. cereus cell according to claim 10, substantially as hereinbefore described and exemplified. IE 91322

25. 27. A modified temperature-stable protoxin according to claim 12, substantially as hereinbefore described and exemplified.

26. 28. A process for the preparation of a modified CrylA(b) gene according to claim 1, substantially as hereinbefore described and exemplified.

27. 29. A modified CrylA(b) gene according to claim 1, whenever prepared by a process claimed in any one of claims 14-17 or 28.

28. 30. A process for the preparation of a modified temperature-stable protoxin according to claim 12, substantially as hereinbefore described and exemplified.

29. 31. A modified temperature-stable protoxin according to claim 12, whenever prepared by a process claimed in claim 18 or 30.

30. 32. A method according to claim 19 of controlling insects, substantially as hereinbefore described.

31. 33. An insecticidal composition according to claim 20, substantially as hereinbefore described and exemplified.

32. 34. A process for the preparation of a B. thuringiensis or a B. cereus cell according to claim 10, substantially as hereinbefore described and exemplified.

33. 35. A B. thuringiensis or a B. cereus cell according to claim 10, whenever prepared by a process claimed in claim 21 or 34.