IE891635L - Bacillus thuringiensis transformation - Google Patents

Abstract

The present invention describes a process which makes it possible to carry out direct and targeted genetic manipulation of Bacillus thuringiensis and the closely related B. cereus using recombinant DNA technology. The present invention also relates to the construction of plasmids and shuttle vectors and to the B. thuringiensis strains transformed therewith. Likewise described is a process for the direct cloning, expression and identification of genes in B. thuringiensis and the closely related B. cereus. <IMAGE> [EP0342633A2]

Description

H 9 :: " o jl. v. v.' - ■ Bacillus thuringiensis and Bacillus cereus recombinant transformation The present invention describes a process that: for the first tine renders possible a direct and targeted genetic manipulation of Bacillus shuringiensis and the closely related 3. cereus using recombinant DMA technology„ based on an efficient transformation process for the said Bacillus species.

In particular, the present invention relates to a process for inserting and cloning and, if desired, also expressing genes or other useful DMA sequences in Bacillus thuringiensis and/or Bacillus caraus, but ■j q especially to a process for inserting and expressing pro sent in genes.

The present invention also includes a process for the direct cloning and, if dssired, expression and identification of novel genes or othssr useful DNA sequences in Bacillus thuringiensis and/or Bacillus cereus, as a result of which it is possible for the first time to establish gene banks -] 5 directly in Bacillus thuringiensis and/or Bacillus cereus and to express thaa therein.

The present invention furthermore relates to the use of plasaids and "shuttle" vectors in the process according to the invention and to eh® 3- thuringiensis and/or B. cereus strains that have been transformed 20 therewith.

Bacillus thuringiensis belongs to the large group of gram-positive, aerobic, endospore-forming bacteria. Unlike the very closely related special of Bacillus, B. cereus and 8. anthracis, the majority of the hitherto known B. thuringiensis species produce in the course of their 51 2 5 sporulstion a patasporal inclusion body which, on account of its crystalline structure, is generally referred to also as a crystalline body. This crystalline body is composed of insecticidally active crystalline proeoxin proteins, the so-called £-endotoxin.

Thesa protein crystals are responsible for the toxicity to insects of B. thuringiensis. The ^-endotoxin does not exhibit its insecticidai activity until after oral intake of the crystalline body, when the latter is dissolved in the alkaline intestinal juice of the target insects and the actual toxic component is released from she proeoxin as a result of limited proteolysis caused by the action of proteases from the digestive tract of the insects.

Th« ^-endotoxins of the various B. thuringiensis strains are distinguished by high specificity with respect to certain target insects, especially with respect to various Lepidoptsra, Coleopters and Diptera larvae,, and by their high degree of activity. Further advantages in using e-endotoxins of B. thuringiensis reside in the obvious difficulty that the target insects have in developing resistance so the crystalline protein and in the fact that the toxins are harmless to humans, other mammalse birds. fish and insects, with the exception of the above-mentioned target insects.

The insecticidai potential of 3. thuringiensis protoxins was recognised very early on- Since the end of the twenties B. thuringiensis preparations have been used as bioinsecticides for controlling various diseases caused by insects in cultivated plants. With the discovery of 1) B. thuringiensis var. israelensis by Goldberg and Margalit (1S77) and 2) B. thuringiensis var. tenebrionis by " Krieg et al. (1983) it was possible for the range of use of B. thuringiensis to be extended even to mosquito and beetle larvae.

With the introduction of genetic engineering and the new possibilities resulting from it, the field of B. thuringiensis toxins has received a fresh impetus.

For example„ the cloning of o-endotoxin genes in foreign host organisms, such as, for example, in E. coli, is already routine. The result of this, meanwhile, has been that the DNA sequences of a whole series of 3 3) ^-endotoxin genes ars now known (for example Schnepf H.£. end A) " C\ Whiteley H.R., 1981; Klier A. st al», 1982; G « Hose of zhe B. thuringiensis species contain sevare.1 g©nes that cods for ^ as? insecticidally active protaia. These genes, which ar* expressed only during the sporulation phase, ar« in the majority of cases located on large transferable plasnids (30 - 150 Hd) and can therefor® very easily be interchanged between the TOriows 3. thuringiensis strains ©nd between B. thuringiensis and B. cereus, provided thss® are compatible 7% ( Gonzalez «J«M. as al., 1982)„ The protoxin genes of B. thuringiensis var. kurstaki belong to a family of related genes, various, of which have already been cloned and sequenced. This work has been carried out especially in an £. coli cloning system. 1 5 The cloning of B. thuringiensis genas has thus so far essentially been limited to some few and exclusively heterologous host systems, of which the £. coli system is the best researched and understood.

In the meantime, however, reports have also been published on the successful eloning ©nd expression of protoxin genes in other host 4) systems, such as, for example, iss B. subtilis ( Klier sc al., 1982), g) Pseudomonas fltforescens ( Obukowicz H.G. et al., 1986) , and Saccharoayces cerevisiae (EP 0 238 4*1). The insertion and expression of the S-endotoxin gene in plant host cells has als© been successful (EP 0292 435).

In cloning in E. coli, advantage is taken of the fact that some protoxin genes happen to contain, in addition to gram~positive promoters, also an E. eoli-lika promoter. These promoter-like DMA sequences make it possible for the B. thuringiensis protoxin genes to be expressed also in heterologous host systems, provided these are capable of recognising the . * above-mentioned control sequences. 4 After breaking open the hose calls, tha expressed protoxxn proteins can than be isolated and identified vising known methods.

Is has since been demonstrated, however, that E. coli-like promoters are 9) noc present in all protoxin genes ( Donovan et al.„ 1988), and 5 consequently so far only very specific protoxin genes that meet the above-mentioned prerequisites can be expressed and thus identified in heterologous host systems.

The cloning of genes outside the natural host organism and the us® of these strains as bioinsecticides in practice is thus associated with a 10 nunber of disadvantages, some of which are serious: ®) Expression of B. thuringiensis protoxin genes from the native; expression sequences is possible only in certain cases. b) Generally there is no, or only a slight, secretion of expressed foreign proteins. e) Correct folding of the 6-endotoxins is not always guaranteed in the reducing medium of heterologous host cells, and this could result in an undesirable change in the specific activity or in tha host range of tha toxins. d) If expression occurs at all, the expression rates of the cloned 20 foreign genes among the native expression sequences are mostly only low. 3)s.0)Schnepf and Whitley (1981: 1985) estimate that the B. thuringiensis toxin cloned in E. coli constitutes only 0.5 % to 1 % of tha total cell protein of E. coli, whereas tha crystalline protein in 3. thuringiensis amounts to between 30 % and 40 % of the dry weight of sporulating 25 cultures. These considerable discrepancies between the expression rates ssay possibly be attributed to tha lack of sporulation-specific control signals in the heterologous host systems and to difficulties in tha recognition of the B. thuringiensis promoters and/or to problems in the post-translatxonal modification of the toxin molecule by the foreign 30 host.

•%) Kany of she host strains generally used for expression ars to jcieologically not as harmless as B. thuringiensis and 3. csteus. * £) 3. thuringiensis and B. cereus form a natural major component of #» microbial soil flora™ which is not true of most of she hose strains 5 generally ysed for expression.

Th« problems and difficulties mentioned above could b» overcome if the said B- thuringiensis genes could be cloned directly in fchs homologous host system where it is possible to use the natural gram-positive promoters of the protoxin genes for the expression. 1q As y®s. however, there is no process that would make B. thuringiensis, this very important bacterium from tha commercial point of view, amenable to direct genetic modifications and that would consequently render possible, for example, efficient reinsertion of ® cloned protoxin gene into a B. thuringiensis strain.

The reason for this can be regarded, in particular, as being the fact that the development of an efficient transformation system for B. thuringiensis and the closely related B. cereus that would ensure adequately high transformation ratsis and consequently render possible the .application also to 3. thuringiensis of established rDVA techniques has 20 not as yet been'successful.

The processes used so far to produce new B. thuringiensis strains having novel insecticidai properties are based chiefly on transfer by conjugation of plasmid-encoded protoxin genes.

Successful reinsertion of a cloned B. thuringiensis crystalline protein gens issto 3. thuringiensis has to date been described only in one case 11) ( 4 Klier A. et al., 1983), but in that case too. owing eo the lack of a suitable transformation system for B. thuringiensis, it was necessary to ' ' resort to transfer by conjugation between 3. subtilis .and B. thuringiensis. Furthermore,, in this process described by Klier et al.

E . coli is used as intermediate host. 8 The processes of transfer by conjugation, however, have a whola series of serious disadvantages that makes them appear unsuitable for routine wse for the genetic Modification of B. thuringiensis and/or B. cereus. a) The transfer of plasmid-encoded protoxin genes by conjugation is ^ possible only between B. thuringiensis strains and between B. cereus and B. thuringiensis strains that are compatible with on® another. b) With transfer of plasmids by conjugation between more distant strains, often only a low transfer frequency is achieved. c) There is no possible way of regulating or modifying the expression of 10 the protoxin genes. d) There is no possible way of modifying the gene itself.

®) If several protoxin genes are present in one strain the expression of individual genes may be greatly reduced as a result of the so-called gene-dosage effect. f) Instabilities may arise as a result of a possible homologous recombination of related protoxin genes.

Alternative transformation processes, which have since been used routinely for many gram-positive organisms, have proved unsuitable both for B. thuringiensis and for 8. cereus.

One of the above-mentioned processes is, for example, the direct transformation of bacterial protoplasts by means of polyethylene glycol treatment, which has been used successfully in the case of many 12) Streptomyces strains (* Bibb J.J. et al., 1978) and in the case of 13) B. subtilis ( Chang S. and Cohen S.N. . 1979), B. megaterium 1 ^) ( Brown B.J. and Carlton B.C., 1980), Streptococcus laccis (" "^Kondo J.K. and McKay L.L., 198&)„ S. faecalis C^Wirth R. et al.)» 17) Corynebactsrium glutamicum ( Yoshihama M. et al., 1985) and numerous other grara-posicive bacteria. 7 To use this process, the bacterial calls oust first of all be converted So protoplasts, chat is to say tha cell walls ar-s digested using lytic enzymes. #> Another prerequisite for the success of this direct csaasfosMtion 5 process is the expression of the newly introduced gsaetie Information and the regeneration of tha transformed protoplasts on complex solid media before successful transformation can be detected, for example using a selectable marker.

This transformation process has proved unsuitable for B. thuringiensis 10 the closaly related B. cereus. As a result of the high resistance of B» thuringiensis cells to lysozyme and tha very poor regenerability of tha protoplasts to intact call wall-containing cells, the rates of transformation achievable remain low and difficult to reproduce (18)Alikhanian S.J. «2t al.. 1981; 19>Hartin P.A. «c al., 1981; 1 5 "^Fischer H-M et al., 1984) .

With this process it is possible therefore, at the snost9 for very simple plasmids, which are unsuitable for work with recombinant DNA, to be inserted as a low frequency into B. thuringiensis or 3. cereus cells.

Individual reports on satisfactory ras«s of transformation that it has been possible to achieve using the afore-described process rely on the formulation of very complex optimising programmes, but these programmes are always applicable specifically to one particular 3- thuringiensis strain only and involve high expenditure in terms of si ma and money 21) ( Schall D. , 1986). Such processes are therefor® unsuitable for routine 25 application on an industrial scale.

As the intensive research work in the field of B. thuringiensis genetics demonstrates„ there is substantial interest in developing new processes * £#. that would make B. thuringiensis or the closely related B. cereus amenable to direct genetic modification and would thus* for examples, render possible she cloning of protoxin genes in the natural host system. Despite this research there are still no satisfactory solutions to che existing difficulties and problems.

Suitable transferaseion processes that render possible an efficient and reproducible transformation of B» thuringiensis and/or 3. cereus at a transformation frequency sufficient to overcome the restriction present in the bacterial calls are not available currently, and neither are suitable cloning vectors that permit the application also to B. thuringiensis of the recombinant DNA techniques already established for other bacterial host systems- The same is true for B. cereus.

This object has now surprisingly been achieved within the scope of the present invention by the use of simple process steps, some of which are known.

The novel process according to the present invention is based on recombinant DNA technology, chat for the first time renders possible a direct and reproducible genetic manipulation of B- thuringiensis and of B. cereus by transforming Bacillus thuringiensis and/or Bacillus cereus with high efficiency by means of a simple transformation process using a recombinant DNA that is suitable for the said genetic manipulation of Bacillus thuringiensis and/or Bacillus cereus.

The present invention thus relates to a process for the transformation of B. thuringiensis and/or B. cereus by inserting, cloning and. optionally expressing recombinant DNA. especially plesmid and/or vector DNA, into B. thuringiensis and/or B. cereus cells by means of electroporation.

In particular, the present invention relates to a process for inserting and cloning DNA sequences in gram positive bacteria selected from the group consisting of 8- thuringiensis and B. cereus, which comprises: a) isolating the DNA to be introduced? b) cloning the thus isolated DNA in a cloning vector that is capable of replicating in a bacterial host cell selected from the group consisting of 3- thuringiensis and B. cereus cells in a heterologous cloning system; 9 c) directly introducing the thus cloned vector DNA in'co the said bacterial call via alac troporacion 9 at a transformation ret® sufficient to ov®rcoms tha restriction present in tha said bacterial calls; sand d) cultivating the thus transformed bacterial calls and isolating the thus cloned vector DNA. . s The present invention also provides a process for inserting, cloning and expressing DMA sequences in grass, positive bacteria selected £roa cha gro^ap consisting of Bacillus thuringiensis and Bacillus cereus, comprising: 1 0 s) isolating the DM to be introduced amid optionally ligating the thus isolated DMA with expression sequences that are capable of functioning in bacterial cells selected fro® the group consisting of Bacillus thuringiensis and Bacillus cereus cells: b) cloning the thus isolated DMA in a cloning vector that is capable of 1 5 replicating in a bacterial host cell selected from the group consisting of Bacillus thuringiensis and Bacillus cereus cells in a heterologous cloning system; c) directly introducing the thus cloned vector DNA into the said bacterial call via clectroporetion at a transformation rate sufficient 20 to overcom® the restriction present in eh® ssid bacterial cells; and d) cultivating the thus transformed bacterial colls and isolating the ehus cloned vector DHA and she expressed gen® product.

The DNA to be introduced into the bacterial host cell may b® a recombinant DNA, which is of homologous or heterologous origin or is a 25 combination of homologous or heterologous DMA.

The recombinant DMA preferably may contain one or more structural genes and 3" and 5° flanking regulatory sequences that are capable of functioning in the said bacterial host cells such as, for example, a * sporu1stion-dependent promoter of B. thuringiensis which sequences ®re 30 operably linked to the structural gene(s) and thus ensure she expression • » of rhe said structural gene(s) in said bacterial host cells. ill Preferred as a structural gene to be used in a process sccoring to tne invention are DNA sequences coding lor a 6-endotoxin polypeptide occurring naturally in B. thuringiensis, or tor a polypeptide chat has substantial structural homologies therewith end has still substantially the toxicity properties of the said crystalline fi-endotoacin polypeptide.

Also preferred ars 6-endotoxin-encoding DMA sequences uhich ars substantially homologous vith at least tne part ©r parts* or the natural 6~endotoxin~encoding sequence that is (are-) responsible Sror the insecticidai activity.

Apart from structural genes it is obviously also possible for any other useful DNA sequences to be used in the process according to the inventions, such as, for example,, non-coding DNA sequences that have a regulatory function, such as, for example, "anti-sens® DNA".

The present invention involves the use of bifunetional vectors, so-called "shuttle" vectors, for B. thuringiensis and/or B. cereus in the transformation of B. thuringiensis and/or B. cereus cells.

Preferred are bifunccional vectors that in addition to replicating in B. thuringiensis and/or B. cereus also replicate in one or more other heterologous host systems, but especially in E- coli calls.

The present invention thus specifically relates to a process for inserting, cloning and, optionally expressing DKA sequences wherein the vector used is a bifunctionsl vector that apart from being capable of replicating in bacterial cells selected from the group consisting of 3. thuringiensis and B- cereus is capable of replicating at least in one other heterologous host organism, and that is identifiable in both the homologous and the heterologous host system. 11 Xh«8 said bifunetional vacsors can also be used for tha transformation of 2. thuringiensis and/or 3. eergus cells and the expression ©f the DMA sequences presenc an the said "shuttle"* vectors t especially those DMA sequences that coda for a <5-endotoxin of 3- thuringiensis off least for a proteisa that has substantially the insect-toxic properties of the B- thuringiensis toxins.

Use present invention thus especially relates to a process for inserting,, cloning and expressing DKA sequences using a bifunetional vector wherein the ssid bifunetional vector comprises under the control -of exptsssion sequences that are capable of functioning in bacterial sells selected front the groap consisting of Bseillus thuringiensis and Bacillus cereus eeJlls si structural gene encoding a S-endotoxin polypeptide that occurs naturally in B. thuringiensis,3 or a polypeptide that has substantial structural homologies therewith and h&s still substantially the toxicity properties of the said crystalline o-endotoxin polypeptid«3.

Especially preferred in this regard ars the bifunetional ("shuttle") vectors pXSol (»pK61) and pXl93 («pSC93) which, introduced by trans-fornation into B. thuringiensis var. kurstaki HDlcryB and into 3. cereus 569K, have been deposited at the "Deutsche Sffimmlung wr, Kikroorganistaen" (3raunschw«ig, Federal Republic of Germany), recognised as an International Depository, in accordance with the Budapest Treaty under the number DSM *572 (pXI61, introduced by transformation into B. thuringiensis var. kurstaki HDlcryB) and DSM 4571 (pX193, introduced by transformation into B. thuringiensis var. kurstaki HDlcryB) and DSM 4573 CpXI93„ introduced by transformation ineo 3. cereus SS9K).

The present invention also relates to the use of B. thuringiensis .sod/or B. cereus ss general host organisms for cloning and expressing homologous and especially also heterologous DMA, or a combination of homologous and heterologous DKA and to 8. thuringiensis and B. cereus cells that have 20 been transformed according to the invention with one of the vector molecules as hereinbefore defined. 12 j.he present invention relates especially to novel B. thuringiensis and B. cereus varieties that have been transformed according to the invention with a DNA sequence that codes for a £-endotoxin of B. thuringiensis and chac can be expressed, or transformed with a DKA sequsenc© coding for at least one protein that has 5 substantially the toxic properties of the B. thuringiensis toxins.

Th® transformed B. thuringiensis and B. cereus cells and the toxins produced by them can be used for the preparation of insecticidai compositions, to uhieh the present invention .mlso relates.

The invention also relates to methods of, and to compositions for, 1o controlling insects using the sbov® mors closely characterised transformed B. thuringiensis and/or B. cereus cells or a cell-free crystalline body*-( tf-endoeoxin) preparation containing protoxins produced by the said transformed Bacillus cells. 13 In particular, the present invention relates to a method of controlling insects, or their habitat, a) with a bacterial host cell selected from B. thuringiensis and B. cereus cells, mmsmd by a process: 1) for inserting and dating DMA sequences in gram positive bacteria selected from B. thuringiensis and B. oaaos, comprising: isolating the DNA to be introduced; cloniag the thus isolated DNA in a dotting vector thae is capable of replicating in a bacterial hose cell selected fitom thuringieasis and B. cereas cells in a heterologous cloning system; directly the thus ctoned vector DNA into the said bacterial cell, via electroporation, as a ' transformation rate sufficient to overcome the restriction present in the said bacterial cells; 1 o and cultivating the thus transformed bacterial cells and isolating the thus cloned vector DNA; or' 2) for inserting, doning and expressing DNA sequences in gram positive bacteria selected from B. thuringiensis and B. cereus, comprising: isolating she DMA to be introduced and optionally ligating the thus isolated DNA with expression sequences that are capable of 15 functioning in bacterial cells selected from B. thuringiensis and B. cereus cells; cloning the thus isolated DNA in a doning vector that is capable of replicating in a bacterial host cdl selected from B. ehwringiensis and B. cereus cells in a heterologous doning system; directly introdudng the thus doned vector DNA into the said bacterial cell via elecaoporation at a transformation me sufficient to overcome the restriction present in the 2 0 said bacterial cells; and cultivating the thus transformed bacterial cells and isolating the thus cloned vscior DMA md the expressed gene product; or wish a mixture of these host: cells; or b) with a cell-free ctystal body preparation containing a protoxin which is produced by a bacterial host cdl a). 2 5 The present Invention also provides a composition for controlling insects comprising a host cell a), defined above; and earners and/or dispersing agents.

The bacterial host eel? used in the method or composition of the Invention comprises a recombinant DMA molecule selected from a recombinant DMA which is of homologoos or heterologous origin, or is a combination of homologous and heterologous DNA, preferably 30 a recombinant DNA containing one or more structural genes and 3' and 5' flanking regulatory sequences, preferably sequences comprising a sporulation*dependent promoter of B. thuringiensis, that arc capable of functioning Ins the bacterial host cells, which sequences are operably linked to the structural gcne(s) and thus ensure the expression of the structural gene(s) in the bacterial host cells. 14 The structural gene preferably codes for a 8-endotoxin polypeptide occurring naturally in B. thuringiensis, or for a polypeptide that has substantial structural homologies therewith, and still has substantially the toxicity properties of the crystalline 5-endotoxin polypeptide, especially a polypeptide which is substantially homologous with a 5-endotoxin polypeptide 5 of a suitable sub-species of B. thuringiensis selected from kurstaki, berlimer, alesti, sotto, colworthi, dendiolimus, tenebrionis and israelensis.

Preferably, the S-endocoxin-encodixig DNA sequence is substantially homologous with at least the pan or parts of the natural 5-endotoxin-encoding sequenced) that is (or are) responsible for the insecticidai activity, especially a DNA fragment of B. thuringiensis var. 1 o kurstaki HDl located between nucleotides 156 and 3623 in formula I (as hereinafter defined), or is any shorter DMA fragment that still codes for a polypeptide having insect-toxic properties.

Alternatively, the bacterial, host cell used in the method or composition of the invention comprises a bifunetional vector which, apart from being capable of replicating in bacterial 15 cells selected from B. thuringiensis and B. cereus cells, is capable of replicating at least in one other heterologous host organism, and that is identifiable in both the homologous and rJ>,e heterologous host system.

Preferably, the heterologous host organism is a) prokaryotic organisms selected from the genera Bacillus, Staphylococcus, Streptococus, Serepiomyces, Fscudomonas, Escherichia, 2 o Agrobacterium, Salmonella and Ewinia, especially E. coli; or b) eukaryotic organisms selected from yeast, animal and plant cells.

Preferably, the bifunetional vector comprises, under the control of expression sequences that are capable of functioning in bacterial cells selected from B. thuringiensis and B. cereus cells, a structural gene encoding a S-endotoxin polypeptide that occurs naturally in 25 B. thuringiensis, or for a polypeptide that has substantial structural homologies therewith and still has substantially the toxicity properties of the crystalline S-endotoxin polypeptide. Preferably, the expression sequences comprise a spoliation - dependent promoter of B. thuringiensis.

The subject of she present invention is accordingly a process, based on s pronounced increase in che efficiency of B. thuringiensis/B- cereus transformation compared with knovn processes, that for she first time renders possible a direct genetic modification of che B- churisigiensis 5 and/or 3. cereus genome.

The process ol the invention thus opens op a large number of nw possibilities that are of extraordinary interest from bogh scientific and commercial points of view.

For example, it is now possible for the first tine to obtain information 10 o® a genetic level about the regulation of 6-endotoxin synthesis, especially in respect of sporulacion.

Also* it should now be possible to clarify at which position of the toxin aoi®Gule the regio^(s) responsible tor the toxicity to insccts is (ars) locsted, and to what extent this (these) is (ate) also associated with 1 5 the host specificity.

Knovledge of tha molecular organisation of the various toxin molecules and of the toxin genes coding for these molecules from cbe various species of B. thuringiensis is of ■aastraordinax-y practical interest for a controlled genetic manipulation of those genes, which is now possible for 20 she first time using che process of the invention. 16 In addition to a controlled modification of tha ^-endotoxin genes themselves, tha novel process of the invention permits also the manipulation of the regulatory DKA sequences controlling tha expression of those genes? as a result of uhich the specific properties of the {"endotoxins, such as, for ejcampla. their host specificity, their resorption behaviour inter alia, can be modified and the production rates of the ^-endotoxins can b«a increased, for example by the insertion of stronger and more efficient promoter sequences.

By mutation of selected genes or subgenus in vitro it is thus possible to obtain new B. thuringiensis and/or B. cereus variants.

Another possible way of constructing novel B- thuringiensis and/or B. cereus variants comprises splicing together genes or portions of genes that originate from different B. thuringiensis sources, resulting in B. thuringiensis and/or 3. cereus strains with £ broader spectrum of use. It is also possible for synthetically or sesni-syntheticaily produced toxin genes to be used in this manner for constructing new B. thuringiensis and/or B. cereus varieties.

In addition, the process according to the invention renders possible for the first time, as a result of the pronounced increase in the transformation frequency and the simplicity of tha process, the establishment of gene banks and tha rapid screening of modified and new genes in B. thuringiensis and/or B. cereus.

In particular, the process of the invention now for the first time renders possible direct expression of gene Banks in B. thuringiensis and/or B. cereus and the identification of new protoxin genes in B. thuringiensis using known, preferably immunological or biological processes.

Preferred within the scope of the invention is a process for the identification of new 6-endotoxin encoding genes in Bacillus thuringiensis, which process comprises 1? (a) digesting the tocal DHA of Bacillus thuringiensis using suieabl® restriction enzymes (b) isolating from the resulting restriction fragments those of suitable size; (e) inserting said fragments ineo a suitable vector, preferably a bifunetional vector; (d) const rue ting a genonic DMA library by transforming Bacillus thuringiensis host calls with che said vector using a process according Co the invention; and {©) screening the- thus obtainable DMA library for new 6-endotoxin encoding genes,s preferably by use e£ an issa^nological screening process.

The following is a brief description of the Figures: Figure 1: Transformation of E. coli HB 101 with pBR322 (o) and *3. thuringiensis HDlcryB with p3C16(-) (Anumber of surviving *HDlcryB cells).

Figure t% Influence of tha age of a *B. thuringiensis HDlcryB culture on the transformation frequency.

Figure 3: Influence of the pH value of eha PBS buffer solution on the transformation frequency.

Figure 4: Influence of the saccharose concentration of the PBS buffer solution on the transformation frequency.

Figure 5? Interdependence of the number of transformants and the amount of DNA used per transformation.

Figure a; Simplified restriction map of the "shuttle" vector *pXX61> The shaded region characterises the sequences originating from the gram-positive pBC16„ the remainder originating from the gram-negative plass&id pUC8. 18 Figure 7: Simplified restriction saap of *pXI93. The shaded region characterises the protoxin structural gene (erto«, Kurhdl) and che 5' and 3' non-coding sequences. The remaining unshaded part originates from the "shuttle" ■vector *pXl6i.

Figure 8s SDS (sodium dodecyl sulfate) /polyacrylasnide gel electrophoresis of extracts of speculating cultures of "*B. thuringiensis HDlcryB,, B. cereus 569K and their derivatives. [1° *HDlcryB (pXI93)s 2: *HDlctyB (pXI61), 3: *HDlcryB, 4: HDl, LBG B-&449, 5: *B. cereus 569K (pXI93)5 6: 569K] a) Comassie-dysd, H: molecular weight standard, KW: molecular ua ight (Dalton), arrow; position of tha 130,000 Dalton protoxirs, b) Western blot of the same gel, to which there have bean added polyclonal antibodies to the K-l crystalline protein of B. thuringiensis HDl.

Positive bands were found with the aid of labelled anti-goat antibodies. Arrow: position of the 130,000 Dalton protoxin. Other bands: degradation products of the protoxin.

Figure 9: Transformation of B. subtil is LBG B-&&68 with pBCl 6 plassaid DNA using the alectroporation process optimised for B. thuringiensis. (o: transformants/}jg plassaid DNA: number of living bacteria/ml) * The internal reference pK selected for tha nomenclature of tha plasmids in the priority document has been replaced for che Auslandsfassung (foreign filing text) by the officially recognised designation pXI.

Also, the names for the asporogenic B. thuringiensis HDl mutants used in the Embodiment Examples have been changed from cryS to cryB.

An essential aspect of the present invention concerns a novel transformation process for B. thuringiensis and B. cereus based on the i insertion of plassaid DNA into B. thuringiensis and/or B. cereus cells using electroporation technology„ which is known per se.

All attempts up to tha time of che present invention co apply tha transformation processes already established for other bacterial host systems to B. thuringiensis and the closely related B- cereus having been frustrated, it is now possible «ithin the scop© of this invention Co achieve surprising success using ®l®ctroporatAoii technology and seeosspsnying steps.

This success must elso bo considered surprising and unexpected, especially since ■alectroporation tests with B- thuringiensis protoplasts were 22) carried our. at an earlier date by a Soviet group ( Shivarova M- ®t al., 1983), but the transformation frequencies achieved vera so low that this process was subsequently regarded as unusable for 3. thuringiensis transformation and consequently received no further attention.

Building upon investigations into tha pro.cess parameters critical for an electroporation 'of B. thuringiensis and/or B. cereus cells, it has now surprisingly been possible to develop a transformation process that is ideally adapted to the requirements of 3. thuringiensis and B* cereus snd results in transformation rates ranging from 10£ to 10* cells/yig of plassaid DNA, but especially frosa 10* to 10y cells/pg of plasmid DHA.

Roughly equally high transformation rates with values from 10s to a maximum of 10s transformants/pg of plassaid DMA could hitherto be achieved only with the PEG (poly®thylenc3 glycol) transformation process described 21) by *"* Schall (1986). High transformation rates remained restricted, however, to those 3. thuringiensis strains for which the PEG process was specifically adapted in very time-consuming optimisation studies, which makes this process appear unsuitable for practical use.

Furthermore, the reproducibility of that process in practice is in many cases s?on™eKistant or poor.

Iss contrast, che process of tha present invention is a transformation process that in principle is applicable to all B. thuringiensis and B. cereus strains, and that is less time-consuming, more rational and consequently more efficient than the traditional PEG transformation process.

For examples in the process of the invention it is possible so use. for example, whole intact cells, thus dispensing with ehe time-consuming production of protoplasts critical for B. thuringiensis and 3. cereus and with the subsequent regeneration on complex siut riant saedia* Furthermore, when using the PEG process, carrying out the necessary process steps can take up to a week,, whereas with the transformation process of the invention the transformed cells can be obtained within a feu hours (as a rule overnight).

Another advantage of che process of the invention concerns the number of "10 3. thuringiensis and/or B. cereus cells that can be transformed per unit of time.

Whereas in tha traditional PEG process only small aliquots can be plated out simultaneously in order to avoid inhibition of the regeneration as a result of the growth of the cells being too dense, when using the 15 electroporation technique large amounts of B. thuringiensis and/or B- cereus cells can be placed out simultaneously.

This renders possible the detection of transformants even at very low transformation frequencies, which with the afore-described processes is not possible or is possible only with considerable expenditure.

Furthermore, amounts of DHA in the nanogram range are sufficient to obtain at least some transformants.

This is especially important if a very efficient transformation system is necessary, such as. for example, when using DNA material from E. coli, which on account of a strongly pronounced restriction system in 2 5 B. thuringiensis calls can lead to a reduction of the transformation frequencies by a factor of 103 compared with B. thuringiensis DNA.

The transformation process of the invention, which is based essentially on electroporation technology known per se, is characterised by the following specific process steps: 21 a.) Preparation of a suspension of host calls in an aerated stadium sufficient to allow for the growth of che cells; b) separation of the grown cells from che cell suspension and ^suspension of Eh© grown cells in an inoculation buff®?; e) addition of a DHA sample, comprising the clomid DHA, lis a concen tration suitable for the electroporation, to sha buffer; d) introduction of the batch of step c) into an «l®ctroporation apparatus; «) subjecting the thus introduced bstch to at least on® capacitor 1 0 discharge of a capacitor to produce a high electric fi^ld strength that is sufficient to render'tha bacterial cell wall permeable to the DMA to be introduced, for a period of time sufficient to trustor® the bactorial host cells with the recombinant DHA: f) selection of the thus transformed bacterial host cells.

In * specific embodiment of the process o£ the invention that is preferred vichin the scope of the invention, eh® B. thuringiensis cells are first of all incubated in a suitable nutrient medium with adequate aeration and at a suitable temperature, preferably of from 20*C to 35°C„ until an opticsrl density (ODsjo) of frost 0.1 to 1.0 is achieved. The age 20 of Bacillus cultures provided for the electroporation has a distinct effect on the transformation frequency. An optical density of the Bacillus cultures of from 0.1 to 0.3, but especially of 0.2, is therefore especially preferred. Attention is, however, drawn, to the fact chat it is also possible to achieve good transformation frequencies with Bacillus 25 culture® from other growth phases, especially with ovarnight cultures (see Figure 2). 22 Generally, fresh cells or spores are used as starting material, but it is also squally possible to use deep-frozen cell material■ The call material is preferably cell suspensions of B. thuringiensis and/or 3. cereus cells in suitable liquid stadia co which, advantageously, a certain amount of an "antifreeze solution'5 has been added.

Suitable antifreeze solutions are especially mixtures of osiaotieally active components and DM50 in water or a suitable buffer solution. Other suitable components that can be used in antifreeze 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 of all inoculated in a suitable medium and incubated overnight at a suitable temperature, preferably of from 25°C to 28°C, and with adequate aeration. This batch is then diluted and further treated, in the manner described above.

To induce sporulation in B. thuringiensis it is possible to use any medium that causes such a sporulation. i-Tithin the scope of this invention 23) a GYS medium according to ~ Yousten A.A. and Rogoff M.H., (1969) is preferred.

Oxygen is usually introduced into the culture medium by moving che culture, for example using a shaker, speeds of rotation of from 50 revs/min to 300 revs/sain being preferred.

B. thuringiensis spores and vegetative microorganism cells are cultured within the scope of the present invention according to known generally customary processes, liquid nutrient media preferably being used for reasons of practicability. 23 The composition of the nutrient saedi®. may -vary slightly depending on the strain of B. thuringiensis oc B. cereus used. Generally, complex media with loosely defined, readily assimilable carbon (C-) ®>nd nitrogen (H-) sources are preferred« like those customarily used for culturing aerobic 5 Bacillus species. la addition, vitamins and essential ssetal ions are necessary, bus Chess are usually contained in an adequate concentration as constituents or ... 1U lapurisi.es in che complex nucrienc asdia used.

If desired, the said constituents, such as, for example, essential + + 2+ 2* 2+ 3* © 2~ f q vitaains and also la , K , Cu , Ga , Kg , Fe , NB* , P0%~ , SO* , — 2~ Gl , COa" ions and the trace elements cobalt and manganese, sine* In addition to yeast extracts, yeast hydrolysates, yeast autolysates and y«ast cells, especially suitable nitrogen sources are in particular soya 15 s»al, asise seal, oatsisal9 edamine (enzymatically digested lactalbunin) , peptone, casein hydrolysate, corn steep liquors and »eat extracts,* without tha subject of the invention being in any way limited by this list of 'examples.

The preferred concentration of the mentioned N-sources is from 20 1-0 K/l to 20 g/1.

Suitable C-sources are especially glucose, lactose, sucrose, dextrose, maltose, starch, cerelose, cellulose and malt extract- The preferred concentration range is from 1.0 g/1 to 20 g/1.

Apart from complex nutrient media it is obviously also possible to use 25 semi- or fully-synthetic media that contain the above-described nutrients in a suitable concentration.

Apart frosa the LB medium preferably used within the scope of the present invention it is also possible to use any other culture medium suitable for calcuring B- thuringiensis end/or B. cereus, such as, for example.

Antibiotic Medium 3, SCGY medium, etc.. Sporuiated 3. thuringiensis cultures are preferably stored on GYS media (inclined agar) et a temperature of 4°C.

After the cell culture has reached tha desired cell density, the cells are harvested by means of centrifugation and suspended in a suitable buffer solution that has preferably been cooled beforehand wich ice.

In the course of the investigations, the temperature proved not to b>s critical and is therefore freely selectable within a broad range. A temperature range of from 0°C to 35°C„ preferably from 2°C to 15°C and mors especially a temperature of 4°CS is preferred. The incubation period of the Bacillus cells before and after alectroporation has only a slight ■affect on che transformation frequency attainable (see Table 1). Only an excessively long incubation results in a decrease in the transformation frequency. An incubation period of from 0.1 to 30 minutes, especially of 10 minutes, is preferred. In the course of the investigations, the temperature proved not to be critical and is therefore freely selectable within a broad range. A temperature range of from 0°C to 35°C, preferably from 2°C to 15°C and more especially a temperature of 4°C, is preferred. This operation can be repeated one or snore times. Buffer solutions that ®.re especially suitable within the scope of this invention are osmotieally stabilised phosphate buffers that contain as stabilising agent sugars such as, for example, glucose or saccharoseor sugar alcohols, such as, for example, snannitol, and have pfi values set to from 5.0 to 8.0. More especially preferred are phosphate buffers of the PBS type having a pK value of from 5.0 to 8.0, preferably of from 5-5 to 6«5„ that contain saccharose as stabilising agent in a concentration of from 0.1M to 1.0M, but preferably of from 0.3M to 0.5H (sea Figures 3 and 4).

Aliquots of che suspended Bacillus cells are then transferred into cuvettes or any other suitable vessels and incubated together with a DNA sample for a suitable period, preferably for a period of from 0.1 to 30 minutes, buc especially of from 5 to 15 minutes, and at a suitable temperature, preferably at a temperature of from 0°C to 35°CS but especially at a temperature of from 2°C to 15°C and more especially at a temperature of £°C.

Whesra operating ©c low temperatures it is advantageous to u$<® cuvettes that have «lready been precooled* or any other suitable preeooled vessels* ©ver a vide range there is a linear relationship between the nisssbsr ®£ 5 trsoMsforwed c«lls and the DHA concentration used for the electroporation, ■she ao»b«r» of tratisfonxutd cell® increasing as che DMA concentration increases (see Figure 5). The DHA concentras ion preferred ■within eha scope of ehis invention is in a range of from 1 -ng to 20 |ig. A DHA concentration of froan 10 ng to 2 pg is especially preferred.

Subsequently the entire batch containing B. thuringiensis mnd/oz 3. cereus e*lls and plassaid DMA or another suitable DNA sample is introduced into an electroporation apparatus and subjected to •electroporation, that is to say is briefly exposed to an electric puis®.

Sleceroporation apparatus suitable for tas«$ in the process of the 15 invention is already available from a of manufacturers, such as, for example, froa Bio Had (Richaond. CA, USA; "Gene Pulser Apparatus"), Biotechnologies and Experimental Research Inc. (San Diego. CA, USA; "BTX Transfector 100"), Pranega (KM) (Madison, WI USA; "X-Cell 2000 Electro-poration System"). etc..

It is obviously also possible so yse amy other suitable apparatus in the process of the invention.

Various pulse forms can be used, for example rectangular pulses or •alternatively exponentially decaying pulsus.

The latter are preferred within the scope of this invention. They are 25 produced by the discharging of a capacitor and are characterised by an initially very rapid increase in voltage and by a subsequent exponential decaying phase as a function of resistance and capacitance. The tisia constant EC provides a measure of the length of ehs exponential decay slaws. It corresponds to tb® tiiae nec&ssary for tha voltage to decay to 30 3^ * of the initial voltage (v0)« 26 One parameter decisive in influencing the bacterial call concerns the strength of the electric field acting on tha cells, «hich is calculated from tha ratio of tha voltage applied to eh® distance between the electrode pieces.

Also of great importance in this connection is the exponential decay time, which depends on the configuration of the apparatus used (for example the capacitance of the capacitor) and on other parameters, such as9 for example, the composition of the buffer solution or the -volume of cell suspension provided for tha alectroporation.

In the course of the investigations it has been demonstrated, for example, that reducing by half the volume of the cell suspension provided for the electroporstio.n results in an increase in the transformation frequency by a factor of 10.

A prolongation of che exponential decay time by way of an optimisation of the buffer solution used also results in a distinct increase in the transformation frequency.

All measures that result in a prolongation of the exponential decay time and consequently in an increase in the transformation frequency are therefore preferred within the scope of this invention.

The decay time preferred within the scope of tha process of the invention is from approximately 2 ms to approximately 50 sns, but especially from approximately 8 ms to approximately 20 ms. Most especially preferred is an exponential decay time of from approximately 10 ms to approximately 12 ms.

Within the scope of the present invention, the bacterial cells are acted upon for short periods by very high electric field strengths by means of brief discharge(s) of a capacitor across the DMA-c on t a ining cell suspension; as a result of this, the permeability of the B. thuringiensis cells is briefly and revsrsibly increased. The ®lectroporation parameters 27 &sre so coordinated wish each other in the eourise of she process of the invention that optimum absorption ins© the Bacillus calls of the DNA located is, the electroporation buffer is ensured.

Use capacitance setting of she capacitor within che scope of this invention is advantageoosly from 1 jiF to 250 jiF, bat especially from 1 j*T to 50 'pF arad saors especially is 25 pF» Tho choice of the initial voltage is not critical, and is therefore freely selectable, wiehin wide ranges. Asii initial voltage V of fro® 0.2 kv to 50 kv, bwt especially of fro® 0.2 k¥ te 2.3 kv a nd snore «@p®ciislly of fro® 1.2 kv to i.8 kVg, is preferred. The distance between the electrode plates depends, inter alia, the size of the electroporation apparatus. It is advantageously fro® 0.X ss co 1.0 cm. preferably frosa 0.2 cm to 1-0 csa9 and aor© especially is 0.4 ca. The field strength values that ®cc on che cell suspension result frosa the distance between the electrode plates and the initial voltage set in the capacitor. These values are advantageously in a range of fro» 100 V/cm to 50,000 V/cm= Field strengths of from 100 V/cm to 20„®OQ V/ca, bwt particularly of froa 3,000 V/cas Co A„ 500 V/c®» are especially preferred.

The fine coordination of the freely selectable parameters, such as, for example, capacitance, initial voltage, distance between plates etc., depends to a certain esssssat on eha architecture of the apparatus used and can therefore vary from case to case within certain limits. In certain cases, therefore, it is possible to exceed or fall below the limiting values indicated, should this be necessary in order to achieve optimum field strengths.

The actual electroporation operation can be repeated one or more times until the optimum transformation frequency for the system in question has beea achieved.

Tollovisisg the electroporation. tha treated Bacillus calls can advantageously be reincubated, preferably for a period of from 0=1 so 30 minutes, as a temperature of froa 0°C to 35°C. preferably from 2°C to 15°C. The electroporated cells are then diluted with a suitable medium and in- 2£ > cubated again for a suitable period, preferably from 2 to 3 hours, wieh adequate aeration and at a suitable temperature, preferably of fro® 20°C co 35°C.

The B. thuringiensis cells are then plated osst onto solid saedi® that contain as an additive at* agent suitable tot selecting the new DNA sequences introduced into che bacterial cell. Depending on the nature of the DMA «as@d„ the said agent nay be, for example, an antibiotically active compound or a dye, inter alia. Antibiotics selected fros the group consisting of tetracycline, kanamycin, chloramphenicol sad erythromycin are especially preferred within the scops of this invention for ths selection of Bacillus thuringiensis end/or B. csraus cells.

Also preferred are ehromogenic substrates, such as, for example, X-gal (5-brosno-^~ehloro-3~indoiyl-£-D-galactoside), which can be detected by way of a specific colour reaction.

Other phenotypic markers are known to ths skilled parson and can also be •osed within the scope of this invention.

It is possible to use any nutrient medium suitable for culturing B. thuringiensis cells, to which one of the conventionally employed solidifying media, such as, for example, agar, agarose, gelatin, etc., is added.

The process parameters described hereinbefore in detail for B- thuringiensis are applicable in the same manner to B. cereus cells.

Unlike the processes hitherto available in the prior arc, the process of the invention for the transformation of B. thuringiensis and 3. cereus described hereinbefore is not limited to the use of specific natural plasmids occurring in B. thuringiensis and/or B. cereus but is applicable to all types of DNA. 2S Iz is accordingly now possible for eh® first cisae to transform B- thuringiensis and/or B. cereus in a controlled aannar, is being passible to use apart from homologous plasaid DMA, that is so s®.y plasmid DHA occurring naturally in 3. thuringiensis ox sis® closely related 33. cereus, also plasaid DMA of heterologous origin.

This may be either plasaid DHA that occurs ssaturally in &P. organism other than B. thuringiensis or che closely related B- cereus, such ess, for example, plasmids pUBllO and pCl94 £r©ai Staphylococcus aureus (^""^Horinouchi S. end Weisblura B., 1982; "^Polak J- ©nd Hovick R.P., 26) 1982) and plasmid pIMl3 from B. subtilis ( M*hler J. and Slalvorson H.O., 1980), which are capable of r«plicsting in B. thuringiensis and/or B. cereus, or hybrid plasmid DMA constructed by recombinant DHA technology from homologous plasmid DHA or trow, heterologous pl&smid DKA or alternatively from a combination of homologous and heterologous plasmid DNA. The last-mentioned hybrid plasmid DM is better suited for work with recombinant DHA than the natural isolates.

There way be Mentioned by way of example here, without the subject of the present application in any way being limited, the plasmids pBD6& 27} C Gyyczan T. et al., 1980)» pBD347„ p3D348 and pUB1664.

The cloning vectors already established for B. subtilisv such as, for example, pBD66, may be of particular importance for carrying out the cloning experiments in various B. thuringiensis ®nd 3. cereus strains.

Apart from plasmid DHA, it is now possible within the scope of the present invention to introduce any other DMA into B. thuringiensis and 3. cereus by transformation. The transformed DHA can replicate either autonomously or integrated in the chromosome. It may be, for example, a vector DKA derived not from a plasmid but from a phage.

Especially preferred within the scop® of this invention is the use of bifunetional (hybrid) plasmid vectors, so-called "shuttle" vectors, that arts capable of replicating in on© or in several heterologous host organisms apart from in 3, thuringiensis or the closely related B. cereus, and that are identifiable both in homologous and in heterologous host systems.

Heterologous host organisms to be ssnderstood vichin the scop® of this invention ss all those organisms that do not belong to tha B. thuringiensis/8. cereus group and that at re capable of maintaining in a stable condition a self-replicating DNA.

According to the above definition it is therefore possible for both prokaryotic and eukaryotic organisms to function as heterologous host organisms. At this point there saay be mentioned by way of example, as representatives from the prokaryotic host organism group, individual examples from the genera Bacillus, such as, for example, B. subtilis or B. megaterium, Staphylococcus, such as, for example, S. aureus. Streptococcus, such as, for example. Streptococcus faeealis, Streptomyces, such as, for example Streptomyces spp., Pseudoaonas. such as, for example, Pseudomonas spp., Escherichia» such as, for example„ £■ coli, Agrobacteriusra, such as, for example. A. tumefaciens or A. rhisogenes, Salmonella,, Etwinia, etc- From the eukaryotic host group there may be mentioned especially yeasts and animal and plane ceils. This list of examples is not final and is not intended to limit the subject of the present invention in any way. Other suitable representatives from che prokaryotic and eukaryotic host organism groups are known to tha skilled person.

Especially preferred within the scope of this invention are B. subtilis or B- megaterium, Pseudomonas spp., and especially E. coli from the group of prokaryotic hosts as well as yeasts and animal or plant cells from the group of eukaryotic hosts.

Hore especially preferred are bifunetional vectors that are capable of replicating in both B. thuringiensis and/or B. cereus cells as well as in E. coli.

The present invention involves the use of the said bifunetional vectors for the transformation of B. thuringiensis and B. cereus. 31 "Shuttle88 vectors ars constructed sasing recombinant DMA technology, plasmid DM of homologous (B. thuringiensis, B- carees) or heterologous origin initially being cleaved using suitable restriction enzymes ©ad cluen those DMA fragments containing the functions essential for replication in the respective desired host system boing joined to one another again la she presence of saifcsbla enzyxaes.

The afore-mentioned heterologous host: organisms can ace as a source of plasmid DM of heterologous origin.

The joining of the various DMA fragments must be effected in such a manner that the functions essential for replication in the different host systems are retained.

Ira addition9 obviously also plasaid DMA of purely heterologous origin can be used for the construction of "shuttle" vectors, but at least on® of the heterologous fusion partners must contain regions of DMA that render possible a replication in homologous 3. thuringiensis/B. cereus host systems.

As a source of plasmid DNA of heterologous origin thas is nevertheless capable of replicating in a B. thuringiensis/3. cereus host system there nay be mentioned at this point, by way of example, a feu representatives from ths group "of gram-positive bacteria, selected from eh® group consisting of the genera Staphylococcus, such as, for example, Staphylococcus aureus, Streptococcus, such as, for example, Streptococcus faeealis. Bacillus, such as, for example, Bacillus sueg^tariuia or 3. subtilis, Streptomyces, such as, for example, Streptomyces spp., etc. In addition to the representatives from the group of gram-positive bacteria listed here by way of example« there is a whole series of other organisms known to the skilled person that can be used in the process of che invention.

The production of bifunetional vectors that are suitable for transforming 3. thuringiensis and/or 2. cereus can be achieved by 32 a) first of all breaking down plasmid DNA of homologous or heterologous origin into fragments using suitable restriction enzymes and b) than joining to one another again, in the presence of suitable enzymes, those fragments containing che functions essential for replication ssid selection in she respective desired host system, this being effected in such a manner that the functions essential for replication and selection in che various host systems ars retained.

Ira this manner bifunetional plasmids are obtained that contain, in addition to the functions necessary for replication in B. thuringiensis or 3, cereus, further DNA sequences that ensure replication in at least one other heterologous host system.

To ensure rapid ©nd efficient selection of the bifunetional vectors in both homologous and heterologous host systera(s) it is advantageous to provide tha said vectors with specific selectable markers that can be aased in B. thuringiensis and/or B. cereus as well as in heterologous host system(s), that is to say that render possible a rapid and uncomplicated selection. Especially preferred within the scope of this invention is the ase of DNA sequences coding for antibiotic resistances, especially DNA sequences that code for resistance to antibiotics selected from the group consisting of kanaraycin, tetracycline, chloramphenicol, erythromycin «stc..

Also preferred are genes that code for enzymes with a chroraogenic substrate„ such as for example, X~gal ( 5-bromo-&-ehloro~3-indolyl-B~D~galaetoside) • The transformed colonies can then be detected very easily by way of a specific colour reaction.

Other phenotypic marker genes are known to the skilled person and can also be used within the scope of' this invention. 33 .Ms© preferred are "shuttle"' vectors that r^pliesiee an the osn® hand either ia" B. thuringiensis or B. eer««« or is. both, ©nd on the ochor hand Is eukaryotic nose systems selected xxom che gronap consisting of yeast, stniaal ssd plant cells, ate..

More especially preferred is the use of "shuttle"" vectors that, in addition to DHA sequences that are necessary for replication of the said vectors, in 3. thuringiensis or B. cereus or in both systems, also eoascsia DMA sequences that render possible replication of the said "shuttle" vectors in £. coli.

Examples of such starting plasmids for the construction of "" shut tie'0 vectors for the B. thuringiensis~B. cereus/E. coli system, which nust not, however, be regarded as in say way limiting, are the B. cereus plasmid pBCifi, and the plasmid pUC8 derived from the E. coli plasmid p3R322 (^®^vieira J. and Hessing J., 1982)» ^ The present invention also relates to tha «se of bifunetional ("shuttie1") actors chat. in addition co the functions essential for replication and selection in homologous and heterologous host systems, also contain one or more genes in expressible form or other useful DKA sequences. These vectors can be prepared by inserting the said genes or other useful DHA 2Q sequences into these bifunetional vectors with the aid of suitable enzymes.

Using the "shuctle" vectors in the afore-described transformation process it is thus now possible for the first time to introduce into B. thuringiensis and/or 3. cereus cells by transformation, wish a high degree of efficiency, DNA sequences that have been cloned outside B. thuringiensis cells in a foreign host system.

H Accordingly it is now possible for the first time for genes or other useful DNA sequences, especially also those having a regulatory function, to be introduced in a stable manner into B. thuringiensis and 3. cereus cells and, if desired, expressed therein, as a result of which B. thuringiensis and B. cereus cells with novel and desirable properties are obtained.

Both homologous end heterologous gena(s) or DMA snd synthetic gasie(s) or DNA according to the definition given within the scop® of the present invention, as well as combinations of the said DMAs* can be used as genes in the process of the invention.

The coding DNA sequence can be constructed exclusively from genomic DNA, from cDNA or from synthetic DNA. Another possibility is the construction of a hybrid DNA sequence consisting of both cDNA and of genomic DNA and/or synthetic DNA, or alternatively a combination of those DNAs.

In that case, the cDNA may originate from the same gene as the genomic DNA, or alternatively both the cDNA and the genomic DNA may originate from different genes. In any case, however, both the genomic DNA and/or the cDNA may each be prepared individually from the same or from different genes.

If the DNA sequence contains pares of mors than one gene, these genes may originate from one and the same organism, from several organisms that belong to different strains, or to varieties of the same kind or different species of the same genus, or from organisms that belong to more than one genus of the same or of another taxonomic unit.

In order to ensure the expression of the said structural genes in the bacterial cell, the coding gene sequences must first of all be operably joined to expression sequences capable of functioning in 3. thuringiensis and/or B. cereus cells.

The hybrid gene constructs of che present invention thus contain, in addition to che structural gen-aCs),, oxprassioa signals ch«c include both promoter asrad terminator sequences as well as other regulatory sequences o£ 3*1 ssid 5" untranslated regions.

Especially preferred within the seope of this invention are the natural expression signals of 3» thuringiensis and/or 3. cereus themselvos aad sutamts aad variant:© thereof that are substantially ihoaologons with the natss«l sequence. Within the scop® of this invention, one DKA sequence is substantially homologous with a second DKA sequence uhen at least 70 %, preferably at least 80 %, but especially at least 90 %, of the active regions of the DHA sequence are homologous. According to the present definition of the expression "substantially homologous", two different nucleotides in a DHA sequence of a coding region are regarded as hoaaolegoMs if tha exchange of tha one nucleotide for the other is a silent mutation.

Host especially preferred is eh® use of sporulatiors~dependent promoters of B« thuringiensis that ensure expression ®s a function of the sporulation.

Especially praf^rr©d for the transformation of B. thuringiensis or B. cereus within the scope of this invention is the use of DKA sequences that codc for a {-endotoxin.

The coding region of the chiaaaric gene of the invention preferably contains a nucleotide sequence coding for a polypeptide that occurs aatssraliy in B. thuringiensis or, alternatively, for a polypeptide that is substantially homologous therewith, that is to say that at laasc has substantially the toxicity properties of a crystalline fi-sndotoxin protein of B. thuringiensis- U-ithin the scope of the present invention, by defissitio^ a polypeptide has substantially the toxicity properties of the crystalline 5-endotoxin protein of B. thuringiensis if it has an insecticidai activity against a similar spectrum of insect larvae to that of the crystalline protein of a sub»species of B. thuringiensis. Soma suitable sub-species are, for example, those selected from the group 31 consisting of kurstaki s berliner, alesti, tol«orthi, sotto, dendrolimus, eenebrionis and israelansis. The preferred subspecies for Lapidoptera larvae is kurstaki and, especially, kurstaki HDl.

The coding region may thus be a region that occurs naturally in B. thuringiensis. Altenatively, tha coding region ean if desired also contain & sequence that is different from the sequence in B- thuringiensis but that is equivalent to it on account of tha degeneration in the genetic code.

The coding region of the chimaeric gene can also code for a polypeptide that is different from a naturally occurring crystalline 6-endotoxin protein but that still has substantially the insect-tojcicity properties of the crystalline protein. Such a coding sequence will normally be a variant of s natural coding region. A "variant" of a natural DNA sequence within the scope of this invention should, by definition, be understood as a modified form of a natural sequence that, however, still fulfils the same function. The variant way be a mutant or a synthetic DNA sequence and is substantially homologous with tha corresponding natural sequence. Within the scope of this invention a DNA sequence is substantially homologous with a second DNA sequence when at least 70 %, preferably at least 80 %, but especially at least 90 %, of the active regions of the DKA sequence are homologous. According to the present definition of the expression "substantially homologous", two different nucleotides in a DKA sequence of a Coding region are regarded as homologous if the exchange of one nucleotide for the other is a silent mutation.

Within the scope of the present invention, it is accordingly possible to use any chimaeric gene coding for an amino acid sequence that has the insecticidai properties of a 3. thuringiensis ^-endotoxin and that meets the disclosed and claimed requirements. Especially preferred is the use of a nucleotide sequence that is substantially homologous at least with the part or the parts of the natural sequence that is (are) responsible for the insecticidai activity and/or the hose specificity of the 3. thuringiensis toxin. 37 The polypeptide expressed by tha chimaeric gene as a rule also has at least soa© immunological properties in common wish a natural crystalline protein, because it has at least som® of the same antigenic determinants.

Accordingly, the polypeptide that is encoded by th« said chimaeric gene is preferably structurally related s® the 5-endotoxin of the crystalline protein produced by B« thuringiensis. B. thuringiensis produces a crystallia® protein with a subunit shac corresponds to a protoxin having a aolocular weight (HW) of approximately from 130,000 to 140,000. This subunit ean cleaved by proteases or by alkali into insecticidai fragments having a HW of 70,000 and possibly even less.

For th« construction of chimaeric genes in which eh© coding region includes such fragments of the protoxin or even smaller parts, fragmenting the coding region can be continued for as long as the fragments or parts of those fragments still have the necessary insecticidai activity. The protoxin, insecticidai fragments of the procsssin and insecticidai parts of those fragments can be joined to other molecules, such as polypeptides and proteins.

Coding regions suitable for use within the scope o£ the process of the invention can b Formal I 20 30 40 50 60 GTTAACACCC TGGGTCAAAA ATTGATATTT AGTAAAATTA GTTGCACTTT GTGCATTTTT 70 80 90 100 U0 120 TCATAAGATG AGTCATAIGT TTTAAATTGT AGTAATGAAA AACAGTATTA TATCATAATG 3.8 130 140 150 160 170 180 AATTGGTATC XXAAXAAA&G AGAXGGAGGT AACXXAIGGA XAACAAXCCG AACAXCAAIG 190 200 210 220 230 240 AATGCATTCC XTATAATTGT TIAAGTAACC CXGAAGXAGA AGTAITAGGT GGAGAAAGAA 250 2 60 2 70 280 2 9 0 300 TAGAAACTGG XTACACCCCA AICGATAITT CCXTGTCGCT MCGCAATXX CITIXGAOXG 310 320 330 340 350 360 AAITXGXXCC CGGTGCXGGA TTXGTGXTAG GACXAGXXGA XATAAXAXGG GGAAXXTTTG 370 380 390 400 410 420 GTCCCXCTCA AXGGGACGCA TTTCTXGTAC AAATTGAACA GTTAATXAAC CAAAGAATAG 430 440 450 460 470 480 AAGAATXCGC XAGGAACCAA GCCAXXXCXA GAXXAGAAGG ACXAAGCAAX CXXXAXCAAA 490 500 510 520 530 540 TTTACGCAGA AXCXXTTAGA GAGTGGGAAG CAGAXCCXAC TAAXCCAGCA XTAAGAGAAG 550 560 570 580 590 600 AGATGCGXAT XCAATTCAAT GACAXGAACA GXGCCCXTAC AACCGCXAXX CCXCXXXTXG 610 620 630 640 650 660 CAGXXCAAAA TTAXCAAGXX CCXCXXXXAX CAGXATATGT XCAAGCTGCA AAITXACAXT 670 680 690 700 710 720 XAXCAGXXXT GAGAGAXGXX TCAGXGTXXG GACAAAGGXG GGGAXXXGAX GCCGCGACXA 730 740 750 760 7 70 780 XCAAXAGXCG XTAXAAXGAX TXAACXAGGC XXAXXGGCAA CXAXACAGAT CAXGCXGXAC 790 800 810 820 830 840 GCXGGXACAA TACGGGATIA GAGCGTGXAX GGGGACCGGA XICXAGAGAX XGGAXAAGAT 39 350 860 870 880 890 900 ATAATCAATT TAGAAGAGAA XIAACACIAA CXGXAXXAGA TAXCGTTTCT CIAXXTCCGA 910 920 930 940 950 960 ACXAXGAXAG XAGAACGXAX CCAATICGAA CAGTTTCCCA AXXAACAAGA GAAAXXXAXA 970 980 990 1000 1010 1020 CAAAGGCAGT ATTAGAAAAT XTXGAXGGXA GTTTTCGACSG CXCGGCTCAG SGGAXAGAAG 1030 1040 1050 1060 1070 1080 GAAGXAXTAG GACXCGACAT XXGAXGGAXA XACXXAACAG XAXAACCAXC XATACGGAXG 1090 1100 1110 1120 1130 1140 CXCATAGAGG AGAATAXXAX TGGTCAGGGC AXCAAAXAAX GGCXXCXCCT GXAGGGXXXX 1150 1160 1170 1180 1190 1200 CGGGGCCAGA ATTCACTTXT CCGCTATAXG GAACXAXGGG AAAXGCAGCX CCACAACAAC 1210 1220 1230 1240 1230 1260 GXAXXGXTGC TCAACTAGGT CAGGGCGIGT ATAGAACAXX AICGXCCACX XXAIATAGAA 1270 1280 1290 1300 1310 1320 GACCTXTXAA XATAGGGATA AATAAXCAAC AACTAXCXGX TCXXGACGGG ACAGAATXTG 1330 • 1340 1350 1360 1370 1380 CTTATGGAAC CICCXCAAAX TTGCCATCCG CTGXAIACAG AAAAAGCGGA ACGGIAGAIX 1390 1400 1410 1420 1430 1440 CGCTGGATGA AATACCGCCA CAGAATAACA ACGTGCCACC XAGGCAAGGA XXTAGTCATC 1450 1460 1470 1480 1490 1500 GATTAAGCCA TGTTICAATG TTTCGTTCAG GCXXXAGXAA TAGXAGTGXA AGXAIAATAA 1510 1520 1530 1540 1550 1560 GAGCTCCXAI GITCTCTTGG ATACAXCGXA GIGCIGAAXX XAAXAATAXA ATICCTTCAT 40 1570 1580 1590 1600 1610 1620 CACAAATTAC ACAAATACCT TXAACAAAAT CTACTAAXCX TGGCICTGGA ACTTCTGTCG 1630 1640 1650 1660 1670 1680 TTAAAGGACC AGGATTTACA GGAGGAGAXA ITCXTCGAAG AACTTCACCT GGCCAGATTT 1690 1700 1710 1720 1730 1740 CAACCTTAAG AGTAAATAXT ACTGCACCAT TAICACAAAG ATATCGGGTA AGAATTCGCT 1750 1760 1770 1780 1790 1800 ACGCTXCTAC CACAAAXTTA CAATTCCAXA CATCAATTGA CGGAAGACCT AXXAATCAGG 1810 1820 1830 1840 1850 1860 GGAATTTXTC AGCAACTATG AGTAGTGGGA GXAATTTACA GTCCGGAAGC TITAGGACTG 1870 1880 1890 1900 1910 1920 TAGGTXTXAC XACTCCGXXX AACTTTICAA ATGGATCAAG TGTAXTXACG TXAAGXGCTC 1930 1940 1950 1960 1970 1980 ATGTCTXCAA XXCAGGCAAT GAAGTXTAXA XAGAXCGAAX TGAAXTTGXT CCGGCAGAAG 1990 2000 2010 2020 2030 2040 XAACCTTXGA GGCAGAAXAT GATTXAGAAA GAGCACAAAA GGCGGTGAAT GAGCIGXXXA 2050 • 2060 2070 2080 2090 2100 CTTCXTCCAA XCAAAXCGGG TXAAAAACAG AXGTGACGGA TTATCAXATT GATCAAGXAT 2110 2120 2130 2140 2150 2160 CCAATTTAGT TGAGTGTTTA TCTGATGAAT TXTGTCTGGA TGAAAAAAAA GAATTGTCCG 2170 2180 2190 2200 2210 2220 AGAAAGTCAA ACATGCGAAG CGACTTAGTG ATGAGCGGAA TTTACTTCAA GATCCAAACT 2230 2240 2250 2260 2270 2280 TTAGAGGGAT CAATAGACAA CTAGACCGTG GCTGGAGAGG AAGTACGGAT ATTACCATCC 41 2290 2300 2310 2320 2330 2340 AAGGAGGCGA TGACGTATTC AAAGAGAATX ACGTTACGCT ATTGGGTACC XXXGATGAGT 2350 2360 2370 2380 2390 2*00 GCXAXCCAAC 6XATXIAXAX CAAAAAATA6 ATGAGTCGAA AXtAAAAGCC TAIACCCGXX 2410 2420 2430 2440 2450 2460 ACCAATXAAG AGGGIAXATC GAAGATAGTC AAGACTTAGA AAXCXAXTTA AXXCGCXACA 2470 2480 2490 2500 2510 2520 ATGCCAAACA CGAAACAGXA AATGXGCCAG GTACGGGXTC CTTATGGCCG CTXTCAGCGC 2530 2540 2550 2560 2570 2580 CAAGTCCAAT CGGAAAATGT GCCCATGATT CCCATCAXXX CXGCTTGGAC AXTGAXGTXG 2590 2600 2610 2620 2630 2640 GATGTACAGA CXXAAATGAG GACXTAGGTG TATGGGXGAT ATXCAAGATT AAGACGGAAG 2650 2660 2670 2680 2690 2700 ATGGCCATGC AAGACXAGGA AAXCTAGAAX XTCTCGAAGA GAAACCATTA GTAGGAGAAG 2710 2720 2730 2740 2750 2760 CACTAGCXCG XGTGAAAAGA GCGGAGAAAA AAXGGAGAGA CAAACGXGAA AAATTGGAAX 2770 • 2780 2790 2800 2810 2820 GGGAAACAAA TAXTGTTTAT AAAGAGGCAA AAGAAXCXGX AGAXGCTTTA TTTGTAAACT 2830 2840 2850 2860 2870 2880 CTCAATATGA XAGAXXACAA GCGGATACCA ACATCGCGAX GATTCATGCG GCAGATAAAC 2890 2900 2910 2920 2930 2940 GCGTTCATAG CATTCGAGAA GCTTATCTGC CTGAGCXGXC TGTGATTCCG GGTGTCAATG 2950 2960 2970 2980 2990 3000 CGGCTATTXX XGAAGAAXXA GAAGGGCGTA XTXXCACXGC ATTCTCCCTA XATGATGCGA it 3010 3020 3030 3040 3050 3060 GAAATGICAI XAAAAAXGGT GAXXXTAAXA ATGGCTTAXC CTGCTGGAAC GTGAAAGGGC 3070 3080 3090 3100 3110 3120 AXGXAGAXGX AGAAGAACAA AACAACCACC GTTCGGTCCT TGXTGTTGCG GAATGGGAAG 3130 3140 3150 3160 3170 3180 CAGAAGTGTC ACAAGAAGTT CGTGTCTGTC CGGGTCGTGG CXAXAXCCXX CGTGTCACAG 3190 3200 3210 3220 3230 3240 CGXACAAGGA GGGAXAXGGA GAAGGXXGCG XAACCATXCA TGAGAXCGAG AACAAXACAG 3250 3260 3270 3280 3290 3300 ACGAACTGAA GXIIAGCAAC TGXGTAGAAG AGGAAGTAXA XCCAAACAAC ACGGTAACGX 3310 3320 3330 3340 3350 3360 GXAAXGAXXA XACXGCGACT CAAGAAGAAT ATGAGGGTAC GXACACITCI CGXAATCGAG 3370 3380 3390 3&00 3410 3420 GAXAXGACGG AGCCXAXGAA AGCAAIXCXX CXGXACCAGC XGAXXAXGCA XCAGCCXAXG 3430 3440 3450 3460 3470 3480 AAGAAAAAGC AT AT AC AG AT GGACGAAGAG ACAAXCCTXG XGAAXCXAAC AGAGGAXAXG 3490 • 3500 3510 3520 3530 3540 GGGAIXACAC ACCACXACCA GCXGGCXATG TGACAAAAGA ATXAGAGXAC TXCCCAGAAA 3550 3560 3570 3580 3590 3600 CCGAXAAGGX ATGGAXTGAG AICGGAGAAA CGGAAGGAAC AIICATCGIG GACAGCGXGG 3610 3620 3630 3640 . 3650 3660 AATTACTTCX XAXGGAGGAA IAATAIATGC TTTAXAATGT AAGGXGTGCA AAXAAAGAAT 3670 3680 3690 3700 3710 3720 GAXXACIGAC ITGXAXXGAC AGAXAAATAA GGAAAXTTTX AXAXGAAXAA AAAACGGGCA 43 3730 3740 3750 3760 3770 3780 TCACTCTXAA AAGAATGATG XCCGXXXXXT GTATGATTTA ACGAGTGATA TTTAMTGTT 3790 3800 3810 3820 3830 3840 TTTXTTGCGA AGGCTTXACT XAACGGGGTA CCGCCAGATG CCCATCAACT TAAGAATTTG 3850 3860 3870 3880 3890 3900 CACXACCCCC AAGTGTCAAA AAACGTTATT CTTTCTAAAA AGCTAGCTAG AAAGGATGAC 3910 3920 3930 394Q 3950 3960 ATTTTTTATG AAXCXXTGAA TTCAAGATGA ATTACAACTA XXTICTGAAG AGCXGXAXCG 3970 3980 3990 4000 4010 4020 ICATTTAACC CCTXCICXXX TGGAAGAACX CGCXAAAGAA XXAGGTXTXG TAAAAAGAAA 4030 4040 4050 4060 40/0 4080 ACGAAAGTXT TCAGGAAATG AAIIAGCIAC CATAXGXATC TGGGGCAGIC AACGTACAGC &090 &100 4110 4120 4130 4140 GAGTGAXTCX CTCGTTCGAC XAXGCAGXCA ATXACACGCC GCCAGAGGAC XCXXAXGAGI 4150 4160 4170 &180 &190 A200 CCAGAA.GGAC XCAAXAAACG CXXTGAXAAA AAAGCGGTTG AAXTTTTGAA AlAXAXXTTX 4210 • 4220 4230 4240 4250 4260 XC1GCATTA! GGAAAAGXAA ACXXXGXAAA ACAXCAGCCA XXICAAGXGC AGCACTCACG 4270 4280 4290 4300 4310 4320 XAXTTXCAAC GAAXCCGXAX XXXAGAXGCG ACGAXXTXCC AAGXACCGAA ACAXXXAGCA 4330 4340 4350 4360 CAXGIAXATC CTGGGXCAGG XGGXXGXGCA CAAACIGCAG The coding region defined by nucleotides 156 to 3623 of foEsrcala I cod-as for « polypeptide of formula II. tl Formal II Ma £ Asp Asn Asn Pro Asn Ila Asn Glu ■ o CI He Pro Tyr Asn Cys Lau Sa& Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg 11® Glu Gly Tyr Thr Pro He Asp H© Ser Lets 40 Sar Leu Thr Gin Phe Lau Leu S2 r Gin Lau Thr Arg Glu Ila Tvr A j 6.

Thr Asn 270 Pro Val Lau Glu Asn Phe Asp Gly Ser Phe 280 Arg Gly Sar Ala Gin Gly Ila Glu Gly Sar 290 Ila Arg Sar Pro His Leu Met Asp lie Lau 300 Asn Ser Ila Thr Ila Tyr Thr Asp Ala His 310 Arg Gly Glu Tyr Tyr Trp Ser Gly His Gin 320 lis Met Ala Sar Pro Val Gly Phe Sar Gly 330 Pro Glu Phe Thr Phe Pro Leu Tyr Gly ihx 340 Ms Z Gly Asn Ala Ala Pro Gin Gin Arg Tig 350 45 Val Al® Gin Lau Gly Gin Gly Val «yr Arg 360 j!1b 3C Ser Ser Thr Lau Tyr Arg Arg Pro 370 Phe »1 <&> Gly lis Asn Asn Glu Glsi Leu 380 Ser Val Levi Asp Gly Thr Glu Phe Ala Tyr 390 Slv Thr S er Ser Pro Sar Al® Val 400 "" Arg Lys S»sr Gly Thr Val Asp Ser Leu 410 Asp 61b Tl*© Pro Pr© Gin Asn Asn Val 420 Pro Fro Arg @l*i Gly Ph« Ser" lis Arg 2»e« &3Q S«@r Hxs Val S»sr Mae Phe Arg Ser Glv Phe 440 Scsr Asia S Xl^iT Thr Pro Ph® Asa Ph^j Ser Asn Gly 580 S^&T Ser Val Ph»a Thr L@u Sar A 1 <*) His Val 590 Phs a?l S'sr Gly Asn Glu Val «■» rf •» Xl^s Asp $00 Asg 'i. 1« Glu phs Val Pro Als Glu Val Thr 610 Phe Glu Ala Glu Tyr Asp L®u Glu Arg Al A £e« examples of bacterial host cells that are suitable for replication of the chimaeric gene include bacteria selected from the genera Escherichia. such as E. coli. Agrobccterium, such as A. tumefaciens or A. rhisogenes, Pseudomonas, such as Pseudomonas spp.. Bacillus, such as 3. megaterium or B. subtilis, etc.. As a result of the transformation 25 process of the invention it is no*-' possible for the first time, vithin the scope of this invention, also to use B. thuringiensis and B. cereus themselves as host cells. Processes for cloning heterologous genes in bacterid ar® described in US Patents 4 237 224 ©nd 4 ASS 464.

The replication of genes in £. coli gh®£ code for the crystalline protein '9) of B. thuringiensis is described by "* Wong et al. (1983). .48 Some examples of yeast boss: calls chat ara suitable for che replication of tha genes of che invention include those selected from the gen us Saccharorayces (European Patent Application EP 0 238 4&1)„ Any vector into which the chimaeric gen® can be inserted .-and which is 5 replicated in a suitable host call, such as in bacteria or yeast, can be used for the amplification of che genes of the invention. The vector may b® derived, for example, from a ph*ge or from a plasaid. Examples of vectors fchat are derived fro® phages and that can be used «ichin the scop® of this invention are vectors derived from Ml 3- ©nd from X-phages.

Some suicable vectors derived from Mi 3 phages include Ml3apl8 and ml3mpl9. Some suitable vectors derived from X-phages include Xgcll, Xgt7 and XCharon^.

Of the vectors chat are derived from plasmids and are especially suitable for replication in bacteria, there may be saentiened here by way of 30) 1 5 example pBR322 ( Bolivar et al., 1977), pUC18 and pUCl9 (^'^Norrander «t al. , 1983) and Ti-plasmids (Sevan et al., 1983), without the subject of the invention being in any way limited thereby.

Preferred vectors for the amplification of genes in bacteria are pBR322, pUC18 and pUC19. 2Q Without any limitation being implied, especially direct cloning vectots, such as, for example, pBD3&7„ pBD3^8, pBD64 and pUBl664„ nay be mentioned for cloning directly in B. thuringiensis and/or B„ cereus.

Amongst the bifunetional vectors especially preferred within the scope of this invention are pXI61 (=pK61) and pXI93 (=pK93) which vectors, introduced by transformation into B. thuringiensis var. kurstaki HDlcryB and B. cereus 569K, have been deposited at the "Deutsche Samsnlung von Kikroorganismen" (Braunschweig, Federal Republic of Germany), recognised as an International Depository, in accordance with the requirements ot the Budapest Treaty under the number DSM. 4572 (pXIol , introduced by transformation into B. thuringiensis var. kurstaki HDlcryB) and DSM 4571 49 (pXl93. introduced by transformation into B. thuringiensis var. kurstaki HDlcryB) and DSM 4573 (pX!93, introduced by transforation into 3* cereus 569iD.

Ia order to construct a chimaaric gene suitable for replication in 5 bacteria, a promoter sequence, a 5" untranslated sequence, a coding sequence and a 3' untranslated sequence are inserted into a vector or are assembled in the correct sequence in one ®£ che afore-described teeters. Suitable vectors according to the invention are those that as® capable of being replicated in the host cell.

The proooter, the 5° untranslated region, the coding region and the 3* untranslated region can* if desired, first of all be conbined in on® unit outside the vector and than inserted into the vector. Alternatively, part® of the chimaerie gene can also be inserted into the vector individually.

In the ess© of B. thuringiensis and B. cereus cloning w©etors this process step c®n be omitted sine® the entire unit isolated from 3. thuringiensis, consisting of a 5" untranslated region, the coding region and a 3* untranslated region. can be inserted into che vector.

The vector furthermore preferably also contains a marker gene which 20 confess on the host cell a property by which it is possible so recognise the cells transformed vith the vector. Marker genes that code for an antibiotic resistance are preferred. Some examples of suitable antibiotics are ampicillin, chloramphenicol, erythromycin, tetracycline, hygroaiycin, G 418 and kanaraycin.

Al so preferred are marker genes that code for enzymes having a chro»ogenic substrate, such as, for example, X-gal (5-brosao~4-chloro"3-indolyl-S-D-galactoside). The transformed colonies can then be detected vary easily by way of a specific colour reaction. 58 The insertion of the gene into, or the assembly of the gene in, the vector is carried out by way of standard processes, for example using 33) recombinant MA ( Maniatis at al.. 1982) and using homologous 34) recombination ( Hinnen *t al., 1978).

The srscosabinsnt DHA technology processes are based on the vector first of all being cleaved snd the desired DMA sequence being inserted between the cleaved portions of the vectors the ends of the desired DNA sequence ar The vector is preferably cleaved with suitable restriction endonucleases. Suitable restriction endonucleases era. for example, those that form blunt ends, such as Ssaa I, Hpa I and Zco rv, as well as those that form cohesive ends, such as Eco RI, Sac I and Bam HI.

The desired DNA sequence normally exists as a region of a larger DNA molecule, such as a chromosome, a plasmid, a transposon or a phage. The desired DNA sequence is in these cases excised from its original source and, if desired, so modified that its ends can be joined to those of the cleaved vector. If the ends of the desired DNA sequence and of the cleaved vector are blunt ends, then they can, for example, be joined to one another with ligasas specific for blunt ends, such as T& 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 for cohesive ends, which may also be T4 DNA ligase, is used. Another suitable ligase specific for 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 which case tha desired DNA sequence and the cleaved vector have cohesive ends that ara complementary to each other.

The cohesive ends can also be constructed by adding complementary homopolymer tails to tha ands of the desired DNA sequence and of the cleaved vector with the aid of terminal deoxynucleotidyl transferase. 51 Alternatively, cohesive ends can be produced by adding a synthetic oligonucleotide sequence that is recognised by a particular restriction endonudease and is known as a linker,, and cleaving the sequence with tha 33) endonuclease (see, for example, Maniatis et al- • 1982). ^ le Is thus now possible for sh The possibility of modifying the ©-endotoxin genes and tha control sequences seguilating the expression of those genes is of particular interest hero.

Apart from ehisnaeric genes, it is obx'iously also possible for any other 2q cSsimasrie genetic construct to be inserted into Bacillus thuringiensis and/or Bacillus'cereus cells using tha process of eha invention.

It is thus, for example, conceivable, using che process of the invention, to insert non-coding "anti-sense" DNA into tha genome of a Bacillus thuringiensis and/or Bacillus cereus cell, so that in the course of the 25 expression of the said "anti-sense" DKA & raRHA is transcribed that inhibits the expression of the corresponding "sense" DNA. In this manner it is possible to inhibit in © specifically controlled manner the expression in Bacillus thuringiensis and/or Bacillus cereus of certain undesired genes. 52 Furthermore, apart from the preparation of improved, well-defined B. thuringiensis strains for tha preparation of improved bioinsecticides, it is now also possible to us® B. thuringiensis as a general host for cloning .and. if desired, expressing heterologous and/or homologous genes.

Ia a specific and preferred embodiment of ths process of che invention it is furthermore now possible for tha first time to clone new genes, and especially new protoxin genes, diraetly in the natural hose, that is, to say in 3. thuringiensis or B. cereus.

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

In a first process step, the total DNA of a protoxin-producing B. thuringiensis strain is isolated by processes that are known par se and then broken down into individual fragments. The B. thuringiensis DNA can be fragmented either mechanically., for example by the action of shearing forces, or. preferably, by digestion with suitable restriction enzymes. Digestion of the DNA sample is partial or complete, depending on the choice of enzymes. W'ithin the scope of this invention, the use of restriction enzymes that contain quaternary recognition sites and/or result in a partial digestion of th® B. thuringiensis DNA ars especially preferred, such as„ for example, the restriction enzyme Sau IIIA, but this preference does not imply any limitation. Obviously, it is also possible to use any other suitable restriction enzyme in che process of the invention.

The restriction fragments obtained in the afore-described manner are then separated according to size by processes known per se. Size-dependent separation of DNA fragments is usually effected by cent rifuging processes, such as, for example, saccharose gradient centrifugation, or by electrophoretic processes, such as agarose gel electrophoresis, or by a combination of those processes.

Tho se fractions containing fragments of the correct size, that is to say-fragments that on account of their size are capable of coding for a protoxin, are pooled and used for the next process steps. 53 The previously isolated frrngmmtnts ars first of all inserted ineo suitable elonissg vectors using standard processes® sad tliea inserted directly ineo Bacillus thuringiensis or 3» cereus* but preferably iaea prososis-free strains of Bacillus thuringiensis, using che transformation process of eh® lavencioa- The vectors used are che "shuttle" vectors described ia detail hereinbefore. Th® shuttle vector pXJ200, which is described in detail hereinafter (see Example 9.1), is especially pe®reread wifchia che seope of this invention. Suitable vectors contain DKA sequences that 1 0 ensure easy identification of tha transformed vector-containing clones fros among the immense number of untransformed clones. Especially preferred are DHA sequences coding for a specific marker that on expression results in an easily selectable feature, such as, for example ass antibiotic resistance. There may be mentioned by way of example here a 15 res.isis.snee so ampicillin, chloramphenicol, erythromycin, tetracycline., hygr«mycin. 64IS or kanamycin.

Also preferred are marker genes that sods for ensymes; having a chromogenie substrate, such as, for example. X-gal CS-bromo-A-chloro-3-indolyl-6-D-g,alactoside). The transformed colonies 20 can then be detected very easily by «ay of a specific colour reaction.

After electroporation che traae«sd Bacillus thuringiensis or B- cereus cells are transferred to a selective sporulation medium ®nd ate incubated until sporulation is complete at a tesnparacur© of from 10°C to 40°C. preferably from 20°C to 35°C, and more especially at a temperature of 25 from 29°C to 31 °C.' Tha sporulation medium contains as selective substance preferably one of the ebova-mentioned antibiotics, depending on th© veccor used,, and a suitable solidifying agent, such as, for example, agar, agarose, gelatin etc..

In the course of sporulation, autolysis of the sporulating cells occurs, 30 which is advantageous in industrial seals processing for the subsequent screening since breaking open the cells artificially is dispensed with. 54 In clones that contain eha desired protoxin gena and ara expressed under eha control of their natural promoter» che crystalline proteins formed srs freely accessible in tha medium. These crystalline protains which exist freely in eha medium can than be immobilised s for example with the aid of membrane filters or by other suitable measures.. Suitable membrane fHears ®r®9 for example, nylon or ssierocellulosa msmbrejnss. Membranes of this kind ars freely available on che market.

The crystalline protains immobilised ±n this manner can than be located and identified very simply in a suitable screening process.

Immunological screening using protoxin-specifie antibodies is preferred within the scope of this invention. Immunological screening processes are ) known and are described in detail, for example* in Young et al.. 1983. The use of monoclonal antibodies that recognise quite specifically a particular region of the protein molecule is especially preferred within the scope of the process of the invention- These antibodies can be used either on their own or in the form of a mixture, le is, of course, also possible, however, to use polyclonal antissra for the immunological screening. Mixtures based on monoclonal and polyclonal antibodies are also possible.

Processes for the production of monoclonal antibodies to Bacillus thuringiensis protoxin proteins are known and are described in detail, for example, in' ^^Huber-Lukac (198^) and in Huber-Lukac et al, (1986). These processes can also be used in the present case.

The immunological screening process based on antibodies is part of the present invention.

It is obviously also possible within the scope of this invention to use other suitable screening processes for locating novel DNA sequences in B. thuringiensis and/or B. cereus. 55 Baeill^s churingiensis and 3. cereus cells chat have been transforaed using the afore-described process, and tbe toxins produced by these xramsfarsMsd Bacillus sells, ars oxcalleatly suitable for controlling isas-ects,, but especially for controlling insects ©£ eh® orders Lepidoptera, Diptera and Coleoptera* For use as insecticides, the transformed living or dead B« thuringiensis or B„ cereus cells, containing the recombinant B. thuringiensis toxin gene„ including mixtures of living 2nd dead 3. thuringiensis and 3. cereus cells, as vail as she toxin proteins produced by the ssid transformed cells, are used in unmodified farm or, preferably, together with adjuvants customarily .employed ia the art of formulation, and are formulated in a manner known per se. for exanple into suspension concentrates, coatable pastes, directly sprayable or dilutable solutions, uettable powders, soluble powders, dusts, granulates, and also encapsulations in, for example, polymer substances. 56 As with the nature of the compositions, the methods of application, such as spraying, eternising, dusting, scattering, costing or pouring, ars chosen in accordance with th® intended objectives and eha prevailing circumstances.

Furthermore it is obviously also possible to use insecticidai mixtures consisting of transformed living or deed B- thuringiensis end/or B. cereus cells and cell-free crystalline body preparations containing a protoxin produced by the said transformed Bacillus cells.

The formulations, that is to say che compositions or preparations containing the transformed living or dead Bacillus cells or mixtures thereof and slso the toxin proteins produced by the said transformed Bacillus cells and, where appropriate, solid or liquid adjuvants, are prepared in known manner, for example by intimately 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 sttapulgite. 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 o bentonite: and "suitable nonsorbent carriers are. for example, calcite or sand. In addition, a great number of pregranulated materials of inorgani 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. 57 Suitable soaps are che alkali metal sales, alkaline earth rascal salts or tmsubstituted or substituted ammonium salts of higher fatty acids (Ci#-Cjj), e.g. the sodium or potassium salts of oleic or stearic acid or of natural facey acid mixtures which can be obtained e.g. from coconut oil or sallow oil. Mention may ®lso b® made of facey .acid saothylt«,isria sales, sisch as, for example, the sedissa salt of cis-2~(sa®ehyl-9"©eta-deeenylandho)-e£hanesulfonie acid (cogent ia formulations preferably approximately 3 %).

Hore frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkyl®rylsulfonat'8s or fatty alcohols, such as, for example, 2,4,7,9-tetr«methyl-5-decyne-4,7~diol (eontant in formulations preferably approximately 2 %).

The fatty sulfonates or sulfates are usually in che form of alkali metal salts, alkaline earth metal salts or unsubsticuced or substituted asmnoniura salts ©nd contain a Cg-Cg^alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. che sodium or calcium salt of lignosulfonic acid, of dodecylsulfate or of a saixeues of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise che sales of sulfated and sulfonated fatty a1cohoI/e thylene oxide adducts. The sulfonated b«nziaida:sole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 3 to 22 carbon'acorns. Examples of alkylarylsulfonates are the sodium, e®,lciusa or triethanolamine salts of dodecylbensenesulfonic acid, di~ lau t y 1 naphthalenesulfonic 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 adduce of p-nonylphenol with 4 to !« moles of ethylene oa:ide. 58 Non-ionic surfactants ars preferably polyglycol ather derivatives of aliphatic or cycloaliphatic alcohols,, saturated or unsaturated fatty acids and alkylphenoIs9 said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in tha (.aliphatic) hydrocarbon moiety and S Co 18 carbon atoms in the alkyl moiety of the alkylphanols.

Farther suitable non-ionic surfactants are tha water-soluble adduces of polyethylene oxide with polypropylene glycol, ethylenediaminopoly-p-ropylane glycol and alkylpolyproaylana glycol containing 1 to 10 carbon scorns in the alkyl chain, which adduces 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 par propylene glycol unit.

Examples of non-ionic surfactants are nonylphenolpolyethoxyachanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adduets, tributylphenoxypolyethoxyethanol, polyethylene glycol and oetylphenoxy-polyethoxyethanol. Fatty acid esters of polyoKyethylene sorbitan, e.g. polyoxysthylene sotbitan trioleate, are also suitable non-ionic surfactants .

Cationic surfactants are preferably quaternary ammonium salts which contain, as N-substituent, at least one Cg-Cszalkyl radical and, as further substituents, unsubstitutad or halogenated lower alkyl. benzyl or hydroxy-lower alkyl radicals. The salts are preferably in tha form of halides, mechyl'sulfates or ethylsulfates, e.g. stearyltrimethylammoniusi chloride or ben2yldi( 2-chloroethyl) The surfactants customarily employed in tha art of formulation are described, inter alia, in the following publications: 1986 International HcCutcheon's Emulsifiers & Detergents, The Manufacturing Confectioner Publishing Co.. Glen Rock, NJ, USA; Helmut Stache "Tensid-Taschenbuch" Carl Hanser-Verlag Munich/Vienna 1981. 59 The ©groch®aic&l compositions usually contain 0.1 to 99 %, preferably 0.1 to 95 %, of che transformed living or dead Bacillus cells or mixtures thereof or of che toxin protcaiinss produced by the said transformed Bacillus cells. 99.9 so 1 %, preferably 99.8 to 5 S, of a solid as liquid adjuvant, and 0 eo 25 %, preferably 0.1 to 25 %, of Vh&reas commercial products will preferably be formulated as concentrates, the end uses will nosaally employ dilute forssulaeio^s - The compositions may also contain further auxiliaries such ®s stabilisers. antifoams, viscosity regulators, binders, tackifiers as ucll as fertilisers or other active ingredients for obtaining special effects.

The transformed living or dead Bacillus cells or mixtures thereof containing tha recombinant B. thuringiensis toxin genes, as ■well as the toxin proteins produced by the said transformed Bacillus cells, are excellently suitable for controlling insect pests. Plant-destructive insects of the order Lepidoptera should preferably bs mentioned her®, especially those of tha genera Pieris. Heliothis, Spodoptera and Plutella, such as, for example, Pieris brassicae, Heliothis virescens, Heliothis zes, Spodoptera littoralis and Plutella xylostella.

Other insect pests that can be controlled by the afore-described insecticidai preparations are, for example, beetles of che order Coleoptera, especially choss of she Chrysomelidae family, such as, for example„ Diabrotie® undecimpunctata. D. longicornis, D. virgifera, D. undecimpunctata howardi, Agelastica alni, Leptinotarsa decemlineata etc. • as «ell as insects of the order Diptera, such as, for example. Anopheles sergentii, Uranatenia unguiculata, Culex upoivittatus, Aedes aegyptio Culex pipiens, etc..

The amounts in which the Bacillus cells or she toxin proteins produced by then ar© used depends on the respective conditions, such as, for example, eh® weather conditions, the soil conditions, the plant growth and che time of application. 60 Formulation Examples for material containing B. ehurineiansis toxin In tha following Formulation Examples the t®ra "Bacillus cells" is used to mean those B> thuringiensis and/or B> cereus calls containing a recombinant: B. thuringiensis gen® of the invention. (Tha figures given ere percentages by weight throughout). r1. Granulates Bacillus calls and/or toxin protein produced by these calls kaolin highly dispersed silicic acid attapulgite ••a) % 94 % 1 % b) % 90 % The Bacillus cells and/or toxin protein produced by these calls are first of all suspended in methylene chloride, then the suspension is sprayed onto the carrier, and the suspending agent is subsequently evaporated off in vacuo.

F2. Dusts Bacillus cells and/or toxin protein produced by these cells highly dispersed silicic acid caleum kaolin a) 2 % 1 % 97 % b) % 5 % 90 % Eeady-for-usa dusts ate obtained by intimately mixing che carriers with the Bacillus cells and/or with toxin protein produced by these cells.

F3. Weccable powders 25 Bacillus cells and/or toxin protein produced by these cells sodium lignosulfonate sodium laurylsulfate sodium diisoptopylnsphthalsns-30 sulfonate a) % % 3 % b) 50 % % 6 % c) 75 % % % fit octylphenol polyethylene glycol ether (7-8 moles of ethylene oxide) - 2 % - highly dispersed silicic acid 5 % 10% 10% kaolin 62 % 27 % - The Bacillus cells and/or toxin protein produced by these cells .are carefully nixed with the adjuvants and the resulting aixture is then thoroughly ground in a suitable mill, affording uettable powders* which can be diluted with uater to giv© suspensions of the desired concentration. % 2 % I % 87 % Ths Bacillus calls and/or toxin protein produced by these cells are mixed with the adjuvants, carefully ground, and tha mixture is subsequently jsoistsnsd with «aiar. The mixture is extruded and then dried in a stream <®f air. 3 % 3 % 94 % The homogeneously mixed Bacillus cells snd/or toxin protein produced by these calls are uniformly appli-sd„ in a mixer, to tha kaolin moistened with the polyethylene glycol. Mon-dusty coated granulates ars obtained in this manner.

FA. Extruder granulates Bacillus calls and/or toscin protsis", produced by these cells sodins lignosulfonate carboxymethylcellulose kaolin F5- Coated granulate Bacillus cells and/or toxin protein produced by these cells polyethylene glycol 200 kaolin F6. Suspension concentrate Bacillus cells and/or tosjin protein produced by these cells &0 % 62 ethylene glycol 10 % nonylphenol polyethylene glycol (15 saoles of ethylene oxide) 5 % s»lkylbenzenesuIfonic acid triethanolaaiins salt* 3 % carboxymethylcellulose 1 % silicon® oil ia the form of a 75 % aqueous aiaulsion 0.1 % water 39 % *Alkyl is preferably linear alkyl having fros 10 to especially from 12 to 14, carbon atoms, such ass for example, n-dodecylbenzenesulfonic acid triethanolamine salt.

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

Examples General recombinant DNA techniques Since many of the recombinant DNA techniques used in this invention are routine for the- skilled person, a brief description of tha techniques generally used is given in che following so that these general details need not be given in the Embodiment Examples themselves. Unless expressly indicated otherwise, all of these methods are described in the reference 33) work by Maniacis et al., 1982.

A. Cleaving with restriction endonucleases The reaction mixture uill typically contain about 50 pg/ml to 500 pg/ml DNA in the buffer solution recommended by the manufacturer. New England Biolabs* Beverly, HA.. From 2 to 5 units of restriction endonuclease are added for every pg of DNA and the reaction mixture is incubated at the temperature recommended by the manufacturer for from one to three hours. 83 The reaction is stopped by heating at: 6S°C for 10 minutes or by extraction wish phenol, followed by precipitation of eh® DMA with athanol. This 33) technique is also described on pages 104 ep 106 of tha Maniacis ec al. reference work.

B. Treatment of tha DMA with polymerases to produco blunt «nds SO pgiwl to 500 pg/sal DHA frsgsasnts are added, to ® reaction saixturg ia tchei buffer recommended by the manufacturer. Mew England Biolabs. The reaction mixture contains all four deoxynucleotide triphosphates in concentrations of 0.2 mH. An appropriate DMA polymerase is added and ch<8 reaction is carried out for 30 minutes at 15°C and is then stopped by heating for 10 minutes at 65°C. For fragments obtained by cleaving with restriction endonucleases that produce 5* cohesive ends, such as Eco RX and Bara HI, the large fragment, ©r Klcnow fragment, of DNA polymerase is used. For fragments obtained using endonucleases that produce 3° cohesive ends, such as Pst I and Sac I, DMA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of the 33) Kaniatis et al. reference work.

C. Agarose gel electrophoresis and cleaning DNA fragments to remove gel contaminants Agarose gel electrophoresis is carried ©us in a horizontal apparatus as 33) described on pages 150 to 163 of she Manietis ee al. reference work. The buffer used corresponds to the Tris-borate buffer or Tris-acetate described therein. The DNA fragments are stained with 0.5 pg/sl ethidium bromide which either is present in tha gel or tank buffer during electrophoresis or is not added until after slectrophorasis, as desired. This DNA is made visible by illumination with long-^av© ultra-violat light. If the fragments are to be separated from the g«l. an agarose that g©ls at low temperature, obtainable from Sigma Chemical. St. Louis, Missouri, is ssec!. After electrophoresis, the desired fragment is excised, placed in a ssaall plastics tube, heated at 65°C for about.IS minutes, extracted three times «ith phenol and precipitated twice uith ethanol. This method has been changed slightly compared with the method described by 33) Maniatis et al. on page 170. 64 Alternatively, eha DNA can be isolated fro© the agarose gel with tha aid of the 'Gensclean Kit' (Bio 101 Inc., La .lolls, CA, USA).

D. Removal of 5° terminal phosphates fro® DHA fragments During che plasmid cloning seeps, treatment of the plasmid vector with phosphatase reduces the recircularisacion of the vector (discussed on 33) page 13 of the Maniatis et al. reference work). After cleaving the DNA with the appropriate restriction andonucleasa«, one unit of calf intestinal alkaline phosphatase, which can be obtained from Boehringer-Mannheim, Mannheim, is added. The DNA is incubated for one hour at 37°C and then extracted twice with phenol and precipitated with ethanol.

E. Joining of DNA fragments If fragments having complementary cohesive ends are to be joined to one another, about 100 ng of each fragment are incubated in a reaction mixture of from 20 yl to 4Q yl with about 0.2 unit of DNA ligase from Mew England Biolabs in the buffer recommended by tha manufacturer. The incubation is carried owe for from 1 to 20 hours at 35°C. If DNA fragments having blunt ends are to be joined, thay are incubated as described above except that the amount of T4 DNA ligase is increased to from 2 to A units.

F. Transformation of DNA in E. coli E. coli strain HB101 is used for most experiments. DNA is introduced into 33) E- coli using the calcium chloride process described by Maniatis et al. pages 250 to 251.

G. Screening of £. coli for plasmids After transformation, the resulting colonies o£ E. coli sr«? examined for th« presene® of che desired plasmid by a rapid plassaid isolation process.

Tfc?o commonly asssd processes aar® described on pages 366 to 369 of the 3*0 "* Haniatis et al. reference ^ork* H- Large-scale isolation of plasmid DKA Processes for the large-scale isolaeion of plasmids frosa E. coli avm 33) described on p&gms 88 to 94 of the Maniatis et al. reference work. Madia and Buffer Solutions LB medium [g/1] tryptone 10 yeast extract 5 NaCl 5 Antibiotic medium Mo. 3 (Difco Laboratories) tg/U bovine meat extract 1.S yeast extract 1.5 peptone " 5 glucose 1 H«C1 3.5 K2HP0«, 3.68 KHzTO* 1.32 SCOT madiMsn [g/l] csszmino acids 1 yeast extract 0.1 glucose 5 XzHPOk 14 KH2P0«, 6 II Naa-citrate 1 (NHOaSOi, 2 MgSQk • 7 HgO 0.2 22) GYS wedium ( Tonsten & Rogo£f, 1969) le/il glsscos® 1 yeast extract: 2 (MiHUhSOfc 2 KgHPOfe 0.3 HgSOt, - / HgO 0.2 CaCl2 • 2 H20 0.08 MnSOt, • H20 0.05 pH adjusted so 7.3 before autoclaving.

PBS buffer saccharose 400 MgCl 2 1 phosphate buffer, pH 5.0 7 TBST buffer [mM] Jw-aan 20* 0.05 % (Wv) Tris/HCl* (pH 8.0) 10 NaCl 150 *Tween (RIM) 20: polyet±ioxysorbitan laurate *7ris/HCl: a,a ,c-Tr is (hydroxyme thy l)sne thy laminohydrochlo ride Xh© internal reference pK chosen for designating the plasmids in the 25 Priority Document has been replaced in the Ausiandsfassung (foreign filing text) by the officially recognised reference pXI.

S7 Also, the designation for tha asporogsn.ic B. thuringiensis HDl muesnes used in the Eabodirasnt Zxanples ha® been changed from cryfi so cryB.

Exaapie 1 ; Transformation of 3- thurinisiensis using electroporation Example 1.1; 10 ml of am LB medium (trypton* 10 g/1, yeast extract S g/1, MaC! 5 g/1) are inoculated ^ixh spores of 3- thuringiensis ■war. kurstaki HDlcryB (^'^Stahly D.P. et al.» 1978), a plasmid-free variant of B. thuringiensis var. kurstaki HDl.

This batch is incubated overnight at a temperature of 27°C using a rotary shaker at 50 revs/min. Subsequently che 3. thuringiensis: culture is diluted 100-fold in from 200 ml to 400 sal of LB Biadiusi-, and further cultured at a temperature of 30°C using a rotary shaker at 250 revs/min until an optical density (OD350) of 0.2 is reached.

The cells are harvested by centrifugation and suspended in 1/40 volume of an ice-cooled PBS buffet (400 mM saccharoses, 1 mM HgCla. ^ mM phosphate 15 buffer pH S.0). Centrifugation and subsequent suspension of the harvested B- thuringiensis cells in PBS buffer is repeated once snors.

The cells pretreated in this snannstr can b® eleetroporated eith®r directly. or alternatively after the addition of glycerin to the buffer solution [20 % (w/v)], and are stored ac from -20°C to -70°C, and used at 20 a later point in time. 800 wl aliquots of the ice-cooled cells are then transferred into 40) precooled cuvettes, 0.2 |ig pBClo plasmid DNA ( Bernhard K. et al. „ 1978) (20 tig/ml) is subsequently added, and the encire batch is incubated at 4°C for 10 minutes.

If deep~frosen call material is used, a suitable aliquot of frozen calls 25 is first thawed in ice or at room temperature. The further treacment is analogous to the procedure used for fresh cell macerial. 68 The cuvette is then introduced into an elsetroporation apparatus and the B. thuringiensis cells present in tha suspension are eleetroporated by the action of voltages of from 0.1 kv to 2.5 kv from a single discharge of a capacitor.

Tha capacitor used has a capacitance of 25 |iP and tha distance between she electrodes in the cuvette is 0.& can, which, when discharge occurs results,, depending on the setting, in an exponentially decreasing field strength with initial peak values of from 0.25 kW/csa to 6.25 kV/cai. Tha exponential decay tine lies in the range of from 10 ms to 12 ms.

An electroporation apparatus from the firm Bio Red ("Gene Pulser Apparatus", #165-2075, Bio Rad, 1M& Harbour Way South, Richmond, CA 9^80^, USA), for example, can be used for the described electroporation experiments.

It is obviously also possible to use any other suitable apparatus, in the process of the invention.

After a further 10 minutes' incubation at &°C, the cell suspension is diluted with 1.2 ml of LB medium, and incubated for 2 hours at a temperature of 30°C using a rotary shaker at 250 revs/min.

Suitable dilutions are then plated out onto LB agar (L3 medium solidified with agar, 15 g/1), which contains as an additive an antibiotic suitable for the selection of the newly obtained plasmid. in the case of pBCl6 this is tetracycline, which is added to the medium in a concentration of 20 mg/1.

The transformation frequencies achieved for B. thuringiensis HDlcryB and pBCJ6 as a function of the initial voltage applied for a given distance between plates are reproduced in Figure 1. 68 ihs expression of eha inserted DMA can be detected by way of the tetracycline resistance that occurs. As soon as 2 hours after eh® introduction by transformation of p3Cl6 ineo 3. thwringieasis a complete phenotypic expression of che newly introduced tetracycline resistance occurs (sec 5 Table 2).

Example 1.2; The transformation of B. thuringiensis cells is carried out 1st exactly the same manner as that described in Example 1.1, except that the volume of the cell suspension provided for the electroporation is in this CBSQ 400 i*l* The transformation frequency can be increased by a factor of 10 by this measure.

Example 2: Transformation of B.'thuringiensis HDlcryB with a number of different plasmids Host of tha tests ars carried out with plasmid pBClS, a naturally 15 occurring plasmid of B. cereus. In addition, however, other naturally occurring plasmids can also be successfully inserted into ) B. thuringiensis cells, sssch as, for example. pUBllO ( Polack J. and 24} Novik R.P., 1982), pC19& ( Horinouchi S. and Weisblum B. „ 1932) and pIHl3 ("^Mahler I. and Halvorson 1.0. 1980) (see Table 3).

Also, variants of these plasmids that are better suited the natural isolates for work with recombinant DHA can be introduced by transformation into tha B. thuringiensis strain HDlcryB using the process of the invention, such as, for example, the 3. subtilis cloning vector pBD64 ">D C Gsrycsan T. et al., 1980) and plasmids pBD347, pBD3~8 and pUBI554 (sag 25 Table 3; plasmids pBD3»/ „ pBD3&8 ®nd pUB1654 can ba obtained from Dr. Wo Schurter, CIBA-GEIGY AG, Basle).

The transformation results in Table 3 show clearly that using tha transformation process of the invention, transformation frequencies are achieved that, with one exemption* arc all in the range of from 30 10s t© 10' , irrespective of the plasmid DMA issed- 70 Example 3: Construction of a "shuttle" vector for Bacillus thuringiensis Existing bifuaccional vectors for E. coli and B» subtilis, such .as? for ■example, pHV33 (^Primrose S-B. and Ehrlich S.D., Plasraid, 6: 193-201. 1981) are not suitable for B. thuringiensis HDlcryB (sea Table 3) • For the construction of a potent bifianctional vector, first of all th® large Eco RI fragment of pBClo is inserted wish the aid of DNA ligase 9g) into the Eco RI site of plasaid pUC8 (~ Vieira J. and Messing J. 1982). E. coli calls are then transformed with this construct- A construct recognised as correct by restriction analysis is designated pXI62.

The removal of the Eco RI cleavage sits situated distally from the pUC8 polylinker region than follows. pXl62 is linearised by a partial Eco RI digestion. The cohesive Eco RI ends are made up with Klenow polymerase and joined together again with T4 DHA ligase. After introduction into E. coli by transformation, a construct.recognised as correct by restriction analysis is selected and designated pXIol- A map of pXIol with the cleavage sites of restriction enzymes that cleave pXIol only once, is shown in Figure 6.

This construct can be introduced directly into B. thuringiensis HDlcryB losing the transformation process described in Example 1.

On account of the strong restriction barriers in 3. thuringiensis strains, the transformation rates are lower when using pXI 61 DKA originating from E. coli than when using plasmid DNA originating from B. thuringiensis HDlcryB (see Table 3). Nevertheless pXI61 proves to be very suitable for carrying out cloning experiments in B. thuringiensis.

Example Insertion of the Kurhdl delta-endotoxin gene into strains of B. thuringiensis and B. cereus The DNA sequence coding for a Kurhdl delta-endotoxin protein used within the scope of this invention for insertion and expression in B. thuringiensis and B. cereus originates from plasmid pK36. which was deposited on ^th March 1986 under the Deposit Number DSM 3668 in accordance with the requirements of the Budapest Treaty for the International ?!• Beeognition of the Deposit of Microorganisms for tha Purposes of Patenting, at the Deutsche Samml^ng ^oa Mikroorganismen, Federal Republic of G«sr®8^ys which is recognised && an International Depository.

A detailed description of the process for identifying and isolating the ^-endotoxin genes and for the construction of plasaid p:<36 ia contained ia European Patent Application EP 0 238 441 ssid is a part of che present invention is the forsa of a reference. pK36 plassaid DMA is completely digested uich the restriction enzymes Pst 1 and Sasa HI and the 4.3 Kb fragment, which contains the Kurhdl delta-endotoxin gene (cf. formula I)a is isolated from an agarose gel. This fragment is then inserted into pXX61, Which has previously been digested with Pst I and Bam HI and treated with alkaline phosphatase from calf's stosaach. After the transformation of E. coli H3101, a construct recognised as correct by restriction analysis is Isolated and designated pXX93. A restriction sap of pXI93 is reproduced in Figure 7. pXl93 can be introduced into 3. thuringiensis HDlcryB in 2 different ways . a) 3. thuringiensis cells are transformed directly with a pXI93 isolate of E. coli using the transformation process of tha invention described in Example 1. b) pXI93 is first of all introduced into B- subtilis cells by transformation. as described by Chang and Cohen, 1979. The complete and intact pXI§3 plasmid DMA contained in a transformant is isolated and then introduced into B. thuringiensis HDlcryB by transformation using the electroporation process described in Example 1.

Both methods result in transformants that contain the intact pXl93 plasmid, which can be demonstrated by restriction analysis. 72 Example 5: Evidence of tha expression of the dalta-endotoxin gene in B. thuringiensis Sporulating cultures of B. thuringiensis HDlcryB, HDlcryB (pXl61)„ HDlcryB (pXl93) and HDl are compared under a phase contrast microscope at 5 a magnification of &00. The typical bipyriiuidal protein crystals can ba detected only in the strain containing pXl93 and in HDl. Extracts fro© the same cultures sr@ separated electrophoretically on an SDS poly-scrylanide gel. A protein band of 130,000 Dalton, which corresponds to the Xurhdl gene product, could be detected on the gel only for che strain 10 containing plasmid pXX93 and in HDl (Figure 8a).

Ia a Western blot analysis (Figure 8b), this 130,000 Dalton protein and its degradation products react specifically with polyclonal antibodies that have bean prepared previously against crystalline protein of 3. thuringiensis var. kurstaki HDl in accordance with the process 42) described by Huber-LukacH.« 1982. A detailed description of this process can be found in European Patent Application EP 238 441, which is a pare of this invention in tha form of a reference. Located on plasmid pXl93, upstream of the toxin-encoding region, is a 156 Bp DNA region, which contains the afora-described sporulation-dependent tandem promoter 29) { Wong H.C. et al-, 1983). This sequence is adequate for a high expression of the delte-endotoxin gene in B« thuringiensis HDlcryB and 3. cereus 569K.

Example 6: Evidence of the toxicity of recombinant B. thuringiensis HDlcrvB (pX!93) 3- thuringiensis HDlcryB and HDlcryB (pX!93) era cultured at 25°C in sporulation medium (GYS medium). When sporulation is complete, which is checked using a phase contrast microscope, spores and (if present) protoxin crystals are harvested by centrifugation and spray-dried. The resulting powder is admixed in various concentrations with the food of 30 L-l larvae of Heliothis virescens (tobacco budworm). The mortality of the larvae is ascertained after six days. 73 As expected, tha protoxin gsns~fr®e strain HDlcryB is non-toxic to Heliothis virescens, whilst tha strain transformed with plassaid pXX93 causes a dosage-dependent mortality of H. vircscens (Table 4). This demonstrates chat recombinant strains produced by the electroporation 5 process can actually be used as bioinseceicides.

Example 7: Electroporation of various B. thmringiensls and 3. soec. strains The transformation protocol for 3. thuringiensis HDlcryB described under Example 1 can also be applied to other strains. «5-li tested strains of B. thuringiensis var. kurstaki can be very siaply and efficiently transformed by this process (Table 5).

Excellent transformation frequencies can also be achieved with ® laboratory strain of B. cereus. The same applies also to other tested 8. thuringiensis varieties (war. israelensis, var. kurstaki). By 15 contrast, transformation of B. subtilis by tha electroporation process is very poor.

Using the protoplast-dependent PEG method far 3. subtilis. on the other hand* transformation rates of 4 x 10s/ng plasaid DMA war® achieved.

Tha low transformation rates of B. subtilis obtained rising the electro-20 poration technique are not associated uieh incorrectly selected parameters, such as, for example, an unsuitable voltage* or with a high mortality rat® caused by electric pulses, as can be ssss from Figure 9. 74 Example 8: Transformation of 3. thuringiensis HDlcryB wish tha 8-galacto-sidasa gang 8.1. Insertion of a Bam HI restriction cleavage sits directly before tha first AUG codon of the B. tburingiensjg proserin gaaa Befors Eh® 8~gal®ctosidase gene frosa the plassaid pitfiTh5 (obtainable frosa Dr. M. Gaiser. CIBA-GEIGY AG, Basle, Switzerland) can be joined to tha promoter of the Kurhdl o-andoto&in gene of B. thuringiensis. the DHA sequence of the protoxin gene located in the region of the AUG start codon must first be modified.

This modification is carried out by oligonucleotide-diractad mutagenesis, using the single-stranded phage Ml3rap8« which contains tha 1.8 kB Hinc II-Hind III fragment, of tha 6-endotoxin gene containing the 5' region of that gene.

First of all 3 pg of plasmid pK3S (cf. Example 4) sra digastad wish the restriction enzymes Hind III and Hinc II. Tha resulting 1.8 kb fragment is purified by agarose gal electrophoresis and than isolated from the gel.

In parallel with this, 100 ng of Hi3mp8 RF phage DNA (Bio lab, Tozer Road, Beverly MA, 01915, USA or any other manufacturer) are digested with the restriction enzymes Sma I and Hind III, treated with phenol, and precipitated by the addition of ethanol. The phage DNA treated in this manner is then mixed wieh 200 ng of tha previously isolated protoxin fragment and joined thereto by tha addition of T4 DNA ligase.

After the cransfection of E. coli JM103, 6 white plaques are selected and analysed by restriction mapping.

An isolate in which tha join between the B-galactosidase gene and the promoter of the Kurhdl ^-endotoxin gene of B. thuringiensis has been carried out correctly is selected and designated Ml3mp8/Hinc-Hind.

An oligonucleotide with tha following sequence is synthesized using a DNA synthesizing apparatus ("APPLIED BIOSYSTEM DNA SYNTHESIZER"): 75 (5°) GTTCGGATTGGGATCCATAAG (3s) This synthetic oligonucleotide is complementary to the MI 3ap8/Hinc-Kind 1JHA in a region that extends from position 153 to position 173 of the fcrdhl ©-endotoxin gesje (cf. formula I). The oligonucleotide sequencer reproduced above has a "Mismatch" in positions 162 and 163, however, comps.t Buffer, nucleotide triphosphates, ATP. T& DMA ligase ©nd tha large fragment of DMA polymerase are then added ©nd the batch is incubated b, 3) overnight at 15°C in the manner described £ J.H. Zoller and H. Smith). After agarose gel electrophoresis. circular double-stranded DHA is purified and inserted into E. coli strain JM103 by transfection. As an alternative, the E. coli strain JM107 can be used.

Tha resulting plaques ate examined for sequences that hybridize wish 8* P-labelled oligonucleotides the phages are examined by DHA restriction endonuclease analysis.

A phag® that contains a correct construct in which a Bam HI cleavage site is located directly before tha first AUG codon of tha protoxin gene is designated HI 3mp8/Hinc-Hind/Bsm. 8.2. Joining tha B-galactosidase gene to tha g-andoeoxin promoter 8.2.1s The Tha Q-galae cos Idas© gena is isolated frosa plasmid piWilhS. piWiThS DNA is first of all cleaved at the single Hind III cleavage site. Tha 3" recessed ends ssre made xap using the Klenou fragment of DMA polymerase (ef.

Maniatis at al.t 1983, page 113-114) and the modified DNA is than digested with the restriction snzyiaa Sal I. The DMA fragment containing tha fi-galactosidase gena is isolated by agarose gel electrophoresis.

The vector pXIol (cf. Example 3) is digested with the restriction enzymes Eco EI and Sal I and the two previously isolated fragments are inserted into the vector pXl61.

After transformation of this ligation mixture in the E. coli strain HB101 or JM107, the correctly joined clones are salectad by restriction analysis and by thair 6-galactosidase activity ^ith respect to the ehromogenic substrata X-gal (5-bromo-fe-ehloro-3-indolyl-B-D-galactoside). A clone containing a correct genetic construct is designated pXl80. 8.2.2: In an alternative embodiment, the 162 Bp Eco RI/Bam HI fragment containing the ^-endotoxin promoter is isolated by cleavage of Ml 3mp8/Hinc~Kind/Bam with Eco RI and 3am HI, followed by separation by gel electrophoresis.

The Q-galactosidase gana is isolated from plasmid piWiTh5 in this instance too (cf. Example 8.1.). In this case, the plasmid DNA is digested with the restriction enzymes Bam HI and Bgl II and the large fragment is eluted from the agarose gel after gel electrophoresis.

Tha vector pHY300 PLK. (0PHY-OO1; Toyobo Co., Ltd.. 2-8 Dojima Hama 2-Chome, Kita-ku, Osaka, 530 Japan), which can be obtained commercially (cf. Example 9.1), is digested with the restriction enzymes Eco RI and Bgl II. The two previously isolated fragments are then inserted into the vector pH¥300 PLK. n The entire ligation mixture is than introduced by transformation into tha E. coli strain JM107 (Bethesda Research Laboratories (BRL) 3 411 H, Stonestreet Avenue, Rockville, HD 20850, USA). A clone having a 6-g®Xac-tosidase activity is further analysed by restriction digestions. A clon« eoncaining a correct genetic construct is designated pXIlOl. 8.3. Introduction by transformation into B. subtilis and B. thuriajiansis of plasmid pXISO or pXIIOI pXXSO or pXIlOl plasaid DMA is first of all introduced into 3. subtilis protoplasts by transformation according to a known test protocol dess- 13) esibad by Chang and Cohen ( Chan® ©nd Cohan, 1579).

A correct clone is selected, tha DMA to b@ transformed is isolated by standard processes and introduced by transformation into B. thuringiensis HDlcryB cells by way of electroporation (cf. Example 1).

The transformed B. thuringiensis cells are plated out onto GYS agar (sporulation madiua), which contains X-gal as an additive.

Correctly transformed clones turn blu« whan sporulation commences.

A 3. thuringiensis HDlcryB strain transformed by the pXI61 vector, on the other hand, remains whit® under the sasa«3 conditions• Restriction analysis shoe's that with correctly transformed clones, an intact pX!80 or pXIlOl plasmid is present in the B. thuringiensis cells. 3.4. Q-galactosidase gene under the control of a sporulation-dependent promoter 3. thuringiensis HDlcryB cells containing plasmid pXiSO or pXIlOl are cultured on GYS medium in the manner described hereinbefore. At intervals during the growth phas# (both during the vegetative growth phase and daring the sporulation phase) a Q-galactosidase assay is carried out; in 44) accordance with she test protocol described by J.H. Miller ("Experiments in Molecular Genetics"", Cold Spring Harbor Laboratory, 1972, Experiment 48 and 49).

The individual differences from the above-mentioned test protocol concern the use of X-gal as chromogenic substrate and the measurement of tha coloured hydrolysis product, ■which is formed by the cells after approximately 1 hour.

The cells are than removed by centrifugation, and the optical density of the supernatant is ascertained at a wavelength of 650 nm (ODg 5 e) •> An increase in the optical density as a function of sporulation is observed. The non-transformed B. thuringiensis cells, on the other hand, cannot hydrolyse the chromogenic substrate X-gal.

Example 9: Creation of gene banks in Bacillus thuringiensis 9.1. Construction of pXl200 Plasmid pXl200 is a derivative of plasmid pHY300 PLK, uhich can be obtained commercially from Toyobo Co., Ltd. (tfPHY-001; Xoyobo Co., Led. , 2-8 Dojima Hams 2-Chome, Kita-ku, Osaka, 530 Japan). Plasmid pHY300, the construction of uhich is described in European Patent Application R R EP 162 725, contains both an ampicillin (amp ) and a tetracycline (tetr° ) resistance gene.

Plasmid pHY300 PLK is completely digested vith Bgl I and Pvu X. The resulting restriction fragments are then separated by agarose gel electrophoresis. The .& Kb fragment is isolated from the agarose gel, purified and then religated with 14 DNA ligase.

The whole ligation batch is introduced by transformation into E. coli HB101. After incubation of the transformed E. coli HB101 cells at 37°C on a selective L-agar containing 20 pg/ml tetracycline, the tetracycline-resistant (Tcr) transformants are selected. It is then possible to isolate from an ampicillin-sensitive (Aps) clone (100 fig/ml ampicillin) a plasmid that has lost the Pst I cleavage site in the Apr gene together with the 0.3 Kb Pvu I /Bgl I fragment. This plasmid is designated pXI200. 19 9.2 Cloning protoxin gangs of Bacillus thuringiensis var. kurstaki HDl in Bacillus thuringiensis HDlcryB The total DHA (50 |ig) of Bacillus thuringiensis vsr kurstaki HDl is coapletely digested by incubation with the restriction enzynes Pst 1 and Spa 2* The restriction fragment® so obtained are transferred to s coaaeisaaows saccharose gradient {5 % (w/v) - 23 % (w/v)] where they are separated according to sis® by density gradient centrifugation as?,d collected is 500 jil fractions. Th« centrifugation is carried out ia a TST 41-rotor (Kontron (R.ToM„) Ausschwingrotor) at a tercperature of 15°C at was 2»& x 10s % for a period of 16 hours. Subsequently* in order to determine She £ragsesst size aliquots, ®®ch of SO |il, axe transferred to axx agarose gel [0.3 % (w/v) agarose ia Xris acetate EDXA or Tris borate EDTA; see Maniatis et al.. 1982]. Those fractions containing fragments between 3 Kb and S Kb are pooled and concentrated to s volume of 10 yl by etharcol precipitation.

Tjsg of the "shuttle"' ^®ctor pX!200 described in Example 9.1 are digested wish the restriction enzymes Pst 1 and Sna 1. The 5" phosphate groups of she resulting restriction fragments are then removed by treatment with calf intestinal alkaline phosphatase. 0.2 pg to 0.3 yg of the previously isolated HDl DNA is then mixed with 0.5 \ig of the pXJ20Q vector DHA and incubated overnight at 14°C with the addition of 0.1 U of T» DNA ligase (so-called ""Weiss Units": one unit of T4 DNA ligase corresponds to an -enzymatic activity sufficient to convert 1 nH [32P] from pyrophosphate at a temperature of 37°C and within a period of 20 minutes into & Korit-absorbable material). Tha entire ligation batch is then introduced by eransforaaeiotn! directly into Bacillus thuringiensis HDlcryB cells by aeans of electroporation (cf. Example 1). The electroporated B. thuringiensis cells are than plated out onto a selective sporulation agar containing 20 yg/snl of tetracycline as selecting agent, and incubated at a temperature of 25°C until sporulation is complete- 9.3. Manufacture of monoclonal antibodies to B. thuringiensis protoxin grotain Th© aanufaccure of monoclonal antibodies to S-endocoKin of Bacillus thuringiensis var. kurstaki HDl is carried out analogously to the description iss ^^Huber-Luk®c (1984) sand in ^^Huber-Lukacet al., (1986).

The hybridosaa calls used for the antibody manufacture ars fusion produces ^5) of Sp2/0~Ag Eayslosas cells (described in Shulman cc al., 1978? can be obtained at the "American Type Culture Collection" in Rockville, Maryland, USA) and splenocytes of Balb/c mice that nave previously been immunised with 6-endotojcira of B. thuringiensis var. kurstaki HDl.

In this manner it is possible to obtain monoclonal antibodies that are directed specifically against the 6-endotoxin of B. thuringiensis. Especially preferred are monoclonal antibodies that either bind specifically to an epitope in the N-terminal half of the protoxin protein (for example antibody 5&.1 of the Huber-Lukac et al. , 1986 reference)» or recognise an epitope in the part of the protein constant in Lepidoptera-®etive protoxins, the C-terminal half (for example antibody 83.16 of the Huber-Lukac et al., 1986 reference).

It is. however, also entirely possible for other monoclonal or also polyclonal antibodies to be used for che subsequent immunological screening (cf. Example 9.4). 9.4. Immunological Screening The monoclonal antibodies produced in accordance with Example 9.3, or other suitable monoclonal antibodies, are used for the immunological screening.

First of all. the crystalline proteins present in free form after the sporulation of the B. thuringiensis cells are bound by means of transfer ■ membranes (for example Pall Biodyne (KIM) transfer membrane; Pall Ultrafine Filtration Corporation, Glen Cove, N.Y.) by applying che filter membranes to the plates for a period of approximately 5 minutes. The filters are subsequently washed for 5 minutes with TBST buffer (0.05 % (w/v) Tween 209 10 mM Tris/HCl (pE 8.0), 150 mM Had in bidist. H?,0] and than, in order to block non-specific binding, incubated in a mixture of XBSX buffer and 1 % (w/v) skimmed milk for from 15 to 30 minutes.

The filters prepared ia this nanner are then incubated overnight with the protoxin-speeific antibodies (antibody mixture of 5^«1 sad 83.16 37) C H»ber*l«lta8 et al., (1986)]. lbs unbound antibodies arc removed by washing the filter three times with T3ST buffer for from 5 to 10 minutes each Cine. To detect eha encibody-bound protoxin the filters sr® in-cubated with a further antibody. The secondary antibody used is an anti-mouse antibody labelled with alkaline phosphatase, which can b® obtained commercially,, for example, fro® Bio-Ead [Katalog #170-6520, goat's anti-mouse XgG(H+L)-alkaline phosphatase conjugate]. After an incubation period of 30 minutes the unbound secondary antibodies are removed in the manner described above by washing the filters with TEST buffer three times (for from 5 to 10 minutes each time). The filters sr© than incubated with a substrate mixture consisting of NBT [*p-nitro blu® tetrazolium chloride; nitro-blue tetrazoliu® chloride] and. BCIP [5-brono-A-chloro~3~indolylphosphatQ-p-coluidine salt]. Tha enzymatic reaction is carried out in accordance with the manufacturer's instructions [Bio-Rad; 1414 Harbour Way South, Richmond CA, 948Q«. USA].

Positive. that is to say pro toxin-containing clones, can be recognised •wexy easily by their violac colouring. This occurs as a result of tha enzymatic reaction of the alkaline phosphatase with tha afore-mentioned substrate mixture. Between 800 and 1000 transformants result from the transformation described in Example 9.2 with the ligation batch indicated in that Example. Of these transformants 2 colonies ■exhibit clearly positive signals in the above-described enzyme reaction.

Plasmid DNA is isolated frosa positive clones in which expression of ths protoxin gene could b® detected by way of the described enzyme reaction. The cloned protoxin genes can be further characterised ®nd ultimately identified by restriction analysis and comparison with known restriction 82 Bosh clones contain a recombinant plasmid with an insert of ^•3 Kb. Tha subsequent restriction digestions with Hind III, Pvvj II, Eco RI and Xba 1 permit identification of the gens on tha ins®re by comparison with the known restriction maps of th This gens, cloned directly in 3. thuringiensis and identified by immunological screening, furthermore hybridises with a 18&7 Bp Bam Hi/Hind III ) 1q fragment of the 5.3 Kb gene in plasmid pK36 ( Geiser at al., 1986). In the SDS/PAGE, both clones exhibit a band of 130,000 Dalton typical of the protoxin, which in a Western blot ( ^Towbin et al., 1979) react specifically with the afore-described (see Example 9.4) monoclonal antibodies.

Tables ■^5 Table 1: Influence of the incubation time at ^°C, before and after electroporation„ on the transformation frequency. B. thuringiensis HDlcryB was transformed using the electroporation process with 0.2 VsS pBClo per batch. 83 Exanple 1 2 3 A 6 1 8 preincubation * (aifflnatas) 0 subsequent incaba-tion ** Csaisisases) 0 Transformation £sed cells are selected by plating out onto LB agar containing 20 jig/®! tetracycline.

Tisa® taken to express tetracycline resistance (hours) Transformation frequency (Transformants /pgDNA) Number of living cells 0.5 0 A x 108 1 1,6 x 10s 10s . 2 8.8 x 10* 1.4 x 10® 3 8 x 10® 1.6 x 10s 84 Table 3: Plasmid Transformation of eha B. thuringiensis strain HDlcryB wish various plasmids 1 Origin resistance marker gram negative I grass positive Transformation frequancy naturally occuring plasmids pBCIS B. cereus — Tc 1.9 x 10s pUBHO Staphylococcus Km, Ble 3.3 x 10s* aureus - pem S. aureus - Csa 6 x 10s* pIM13 B. subtilis — Eia 1.8 x 10s modified plassaids/cloning vectors pBD64 pUBHO replicon - Km, Cm x 10& pBD347 pIHl3 repli con , - Cm 2.9 x 10s pBD348 pIH13 repli con.

Em. Cm 1.1 x 10s pU3166& pUBHO repli con, — Cm, Em 3.5 x 10fc "shuttle" vectors pHV33 pBR322/pC19*.

Amp, Tc Cm < 50* pK61 pUC8/ pBCl6, Amp Tc 2.8 x 10" 1: Tc: tetracycline; Km: kanamycin; Ble: bleomycin; Cms chloramphenicol; Em: erythromycin 2: All plasmid DKA originates from B. thuringiensis HDlcryB with the exception of. isolated from B. subtilis LBG&A68.

Table Biotest of B. thuringiensis HDlcryB and HDlcryB (pXI93) agains'c Heliothis virsscens.

Spray-dried sporulacad cultures (spores and (if present) protoxin crystals) are admixed, in the amounts indicated, with the food of L-l larvae of Heliothis virescens. 85 Concentration of spores and protoxin crystals (pg/g tood) Mortality (%) of caused by: K. virescens HDl cryB HDl cryB (pXI93) 200 0 57 100 0 43 so 3 27 0 12.5 0 0 Table 5; Transformability of strains of B. thuringiensis, B. careus and 3. subtilis. All strains transformed «ith plassaid pBC16 in accordance with tha «§lacereparation process described undar Example 1 Strain Transformation^ frequency 3. thuringiensis var. kurstaki HDlcryB 1 HDl dipcl 0.25 HDl-9 0.9 HD 73 0-1 HD 191 0.5 B. thuringiensis; var. thuringiensis HD2HD6-4 13. S B. thuringiensis var. israelensis LBG B-6444 2.6 B. cegows 569 X 7.5 3. subtilis LBG 8-&4S8 0.0002 ^relasive values bas«d on the transformation frequency, defined as 1. achieved with B. thuringiensis var. kurstaki HDlcryB.

Deoosit of Microorganisms A culture of ®®ch of tha microorganisms liscad in eh® following that are used within tha scope of the present invention has been deposited at tha "Deatsch# Saamlung von Mikroorganismen", recognised as an International Depository, in Braunschweig, Federal Republic of Germany, in accordance with the requirements of tha Budapest Treaty for ths International 86 Recognition of che Deposit of Microorganisms for che Purposes of Patenting. A declaration concerning tha viability of the deposited samples has been issued by tha said International Depository.

Deposit of Mlcoorganissas Microorganisms Deposit Date Deposit Number Date of tha viability certificate HB 101 (pK36) (E. coli K3101 1q transformed with pK36 plassaid DHA) 4. March 1986 DSM 3668 1. March 1986 *HD1 cry8 (Bacillus thu-15 ringiensis var. kurstaki HDl cryB 4. Hay 1988 DSM A 57A 4. May 1988 *HD1 cryB (*pK 61) (B. thuringiensis HDl cryB crans-20 formed with *pK61 plassnid DNA) 4. Hay 1988 DSM 4572 4. May 1988 *HD1 cryB (*pK 93) (B. thuringiensis HDl cryB trans-25 formed with *pK93 plasmid DNA) 4. May 1988 DSM 4571 4. May 1988 569 K (Bacillus cereus 569 K) 4. Hay 1988 DSM 4575 4. May 1988 569 K (*pK 93) (B. cereus 569 K transformed with *pK93 plasmid DNA) 4. Hay 1988 DSM 4573 4. May 1988 The internal reference pK selected for the designation of tha plassaids in tha Priority Document has bean replaced for the Auslandsfassung (foreign filing text) by the officially recognised designation pXI.

Also, the designation for the asporogenic B. thuringiensis HDl mutants used in the Embodiment Examples has been changed from cryfl to cryB.

Literature refersness 1. Goldberg L. and Msrgalit J.. Mosquico News, 37s 355-358, 1977 2. Xrieg A. or al-, 2. Ang. Ens., 96: 500-508, 1983 3. Schnepf, H.E. and Whiceley H.R., Proc. Had. Acad. Sci., USA. 5 2893-2897, 1981 4. Xlier A. et al., The EM30 J., is 791-799, 1982 5- Geixer K. ©c al., Gene, 48: 109-118, 1986 6. Haider H.Z. oklonal©n Antikorpern •and Lipiden" (on the interaction of tha delts-endotoxin of Bacillus thuringiensis with jaonoclonel antibodies and lipids), ETH Zurich, 1984 37. Huber-Lucac M. et al., Infect. Immunol., 54* 228-232, 1986 38. McCutcheon's, 1986 Intarnational McCuteheon's Emulsifiars &, Detergents. The Manufacturing Confections Publishing Co., Glen Rock, NJ, USA. 39. Stahly D.P. et al.« Biochero. Biophys. Res. Comm., 84: 581-588, 1978 40. Bernhard K. et al., J. Bacteriol., 133: 897-903, 1978 41. Prisirose S.B., Ehrlich S.D., Plasmid 6; 193-201, 1981 42. Huber-LukacH., Dissertation, Eidgenossisehe Technische Hochschule, Zurich, S^itserland, No. 7050, 1982 43. 2oller J.M. and Smith M., Nucl. Acids Res., 10; 6487, 1982 4&. Miller J.H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972 45. Shulman et'al., Nature, 276: 269, 1978 46. Towbin H. et al., Proc. Natl. Acad. Sci., USA. 76: 4350-4354, 1979 Patent Literature EP 162 725 EP 238 441 MO 86/01536 US-P 4 &48 885 US„p 4 Uhi 036 US-P 4 237 224 US-? 4 468 464

Claims (9)

1. 89 What is dsdmed is: 1. A process for kscmng and cloning DMA sequences in grass positive bacteria selected from fee group consisting o£ Bacillus thuringiensis and Bacillus cereus, comprising: (a) isolating fee DMA i© be introduced; Co) dotting the thus isolated DNA in a dotting vector that is capable of replicating in a bacterial host cell selected from me group consisting of Bacillus thuringiensis and Bacillus cereus ecHs in a heterologoos cloning system; (c) directly introducing the thus cloned vector DNA into fee said bacterial cell via decttoporation at a transformation rate suffirient to overcome the restriction present in the said bacterial cells; and (d) cultivating die thus transformed baessria! cells and isolating the thus cloned vector DNA. 2. A process for inserting, dotting and expressing DNA sequences in gram positive bacteria selected from the group consisting o£ Bacillus thuringiensis and Bacillus cereus, comprising: (a) isolating the DNA to be introduced and optionally ligating fee feus isolated DNA with expression sequences feat are capable of functioning in bacterial cells selected from fee group consisting of Bacillus thuringiensis and Bacillus cereus cells; (b) cloning the thus isolated DNA in a cloning vector that is capable of replicating in a bacterial host ccll selected from the group consisting of Bacillus thuringiensis and Bacillus cereus cells in a heterologous cloning system; (c) directly introducing fee feus cloned vector DNA into fee said bacterial cell via dectrqporation at a transformation rate sufficient to overcome the restriction present in the said bacterial cells; and (d) cultivating the thus transformed bacterial cells and isolating the thus cloned vector DNA and the expressed gene product 3. A process according to claim 2, wherein said transforming comprises a) preparing a suspension of host cells in an aerated medium sufficient to allow for growth of fee cells; b) separating the grown cells from she cell suspension and xssuspending the grown cells in an inoculation buffer; c) adding a DNA sample comprising the cloned DMA in a concentration suitable for the decttoporation to die huffcx; 5 d) Introducing the batch of step c) into an electroporation apparatus; c) subjecting the thus introduced batch to at least one capacitor discharge to produce a high electric field strength that is sufficient to render the bacterial cell wall permeable to the DMA m be introduced, for a period of time sufficient to transform the bacterial host cells with the recombinant DNA; 10 f) selecting the thus transformed bacterial host cclls. 4. A process according to claim 3, which comprises using 3. thuringiensis spores as starting material for the preparation of the cell suspension of step (a). 5. A process according to claim 3, which comprises using thawed bacterial cells, which cells have previously been deep-frozen, as starting material for the preparation of the cell 15 suspension. of step (a). 6. A process according to claim 3, wherein 'the culture medium of step (a) comprises a) complex nutrient media with readily assixxiilablc carbon and nitrogen sources that are conventionally employed for cuiruring aerobic Bacillus species; or b) fully synthetic or semi-synthetic nutrient media thai contain 20 bi) a complex or alternatively a defined readily assimilable carbon and nitrogen source or a combination of the two and also t bj) essential vitamins and metal ions. 7. A process according to claim 3, wherein in step a) the said Bacillus cells are grown until an optical density [OD550] of frosa 0.1 to 1.0 is achieved. 25 8. A process according to claim 3, wherein the inoculation buffer of step b) is a phosphate buffer that has been osmotically stabilized by addition of an osmotic stabilizing agent. 9. A process according to claim 8S wherein the said phosphate buffer contains sugars or sugar alcohols as an osmotic stabilizing agent 91 2©. A process according id claim 9, wherein die said stabilizing agent is saccharose, which Is pjsseai ia £ concentration of from 0.

2. M so 1.0 M, 1L A process according to claim 8, wherein die said phosphate buffer has £ pH value of from pH 5.0 to pH 8.0. 12. A process according to claim 3, wherein the incubation of she bacterial cells is earned out as a temperature of fits 0°C to 35°C before, daring and after electroporation. 1

3. A process accotding to claim 12, wherein the incubation of the bacterial cells Is carried out at a temperature of from 2°C to 15°C before, during and after electroporation. 1

4. A process according to claim 3, wherein die concentration of the added DNA sample is from i ag to 20 |xg. 1

5. A process according to claim 3, wherein the field strength axe from 3000 V/cm to 4500 V/cm. 26, A process according so claim 3, wherein die exponential decay time of die pulse acting on die bacterial cdl suspension lies within a range of ftom 2 ms to 50 ms. 17. A process according to claim 3, wherein selection of die transformed bacterial host cdls comprises plating out the dectropotatcd cells, after a subsequent incubation phase, onm solid media .containing an additive suitable for the selection of the -transformed bacterial cdls. 18. A process according to claim 17, wherein the said additive is an antibiotic suitable for the selection B. thuringiensis or B. cereus or both, sdected from the group consisting of tetracycline, kanamycin, chloramphenicol, erythromycin. 19. A process according to claim 18, wherein die said additive is a chxomogenic substrate suitable for die selection of B. thuringiensis or B. cereus or both. 20. A process according to anyone of claims 1 or 2, wherein the DMA to be introduced into the said bacterial host cell is a recombinant DNA which is of homologous or heterologous origin or is a combination of homologous and heterologous DNA. 21. A process according to claim 20, wherein the said recombinant DNA contains one or snore structural genes and 3* and 5® flanking regulatory sequences that are capable of functioning in the said bacterial host cells, which sequences are operably linked to the structural gcae(s) and thus ensure the expression of the said structural gene(s) in said bacterial host cells.;22. A process according so claim 21, wherein the said 3" and 5° flanking regulatory sequences comprise a sporulation-dependent promoter of B. thuringiensis.;23. A process according to claim 21, wherein the said structural gene codes for a S-endotoxin polypeptide occurring naturally in .8. thuringiensis, or for a polypeptide thai feas substantial structural homologies therewith and has still substantially the toxicity properties of the said crystalline S-endotoxin polypeptide.;24. A process according to claim 23, wherein the said S-eadotoxin-encoding DNA sequence is substantially homologous with at least the pan or parts of the natural S-endotoxin-encoding sequence that is (are) responsible for the insecticidai activity.;25. A process according to claim 23, wherein the said polypeptide is substantially homologous with a S-cnaotoxin polypeptide of a suitable sub-species of B. thuringiensis, selected from the group consisting of kurstaki, beriiner, alesti, sotto, tolworthi, dendrolimus, tenebrionis and israelensis.;2

6. A process according to claim 23, wherein the said 5-endotoxin-encoding DNA sequence is a DNA fragment of B. thuringiensis var. kurstaki HDl located between nucleotides 156 and 3623 in formula I, or is any shorter DNA fragment that still codes for a polypeptide having insect-toxic properties:;Formula 1;S3;10 20 30 40 50;GTTAACACCC TGGGTCAAAA ATTGATATTT AGTAkAATTA GTTGCACTTT;60 70 80 90 100;GTGCATTTTT TCATAAGATG AGTCATATGT TTTAAATTGT AGTAATGAAA;110 120 130 140 150;AACAGTATTA TATCATAATG AATTGGTATC TTAATAAAAG AGATGGAGGT;160 170 180 190 200;I-ACTTATGGA TAACAATCCG AACATCAATG AATGCATTCC TTATAATTGT;210 220 230 240 250;TTAAGTAACC CTGAAGTAGA AGTATTAGGT GGAGAAAGAA TAGAAACTGG;260 270 280 290 300;TTACACCCCA ATCGATATTT CCTTGTCGCT AACGCAATTT CTTTTGAGTG;310 320 330 340 350;AATTTGTTCC CGGTGCTGGA TTTGTGTTAG GACTAGTTGA TATA&TATGG;360 370 380 390 400;GGAATTTTTG GTCCCTCTCA ATGGGACGCA TTTCTTGTAC AAATTGAACA;410 420 430 440 450;GTTAATTAAC CAAfeGAATAG AAGAATTCGC TAGGAACCAA GCCATTTGTA;460 470 480 490 500;GATTAGAAGG ACTAAGCAAT CTTTATCAAA TTTACGCAGA ATCTTTTAGA;510 520 530 540 550;GAGTGGGAAG CAGATCCTAC TAATCCAGCA TTAAGAGAAG AGATGCGTA?;81;560 570 580 590 600;TCAATTCAAT GACATGAACA GTGCCCTTAC AACCGCTATT CCTCTTTTTG;610 620 630 640 650;CAGTTCAAAA TTATCAAGTT CCTCTTTTAT CAGTATATGT TCAAGCTGCA;660 670 680 690 700;5;AATTTACATT TATCAGTTTT GAGAGATGTT TCAGTGTTTG GACAAAGGTG;710 720 730 740 750;GGGATTTGAT GCCGCGACTA TCAATAGTCG TTATAATGA? TTAACTAGGC;760 770 780 790 800;10 TTATTGGCAA CTATACAGAT CATGCTGTAC GCTGGTACAA TACGGGATTA;810 820 830 840 850;GAGGGTGTAT GGGGACCGGA TTCTAGAGAT TGGATAAGAT ATAATCAATT;860 870 880 890 900;TAGAAGAG2VA TTAACACTAA CTGTATTAGA TATCGTTTCT CTATTTCCGA;q5 910 920 930 94 0 950;ACTATGATAG TAGAACGTAT CCAATTCGAA CAGTTTCCCA ATTAACAAGA;960 970 980 990 1000;GAAATTTATA CAAACCCAGT ATTAGAAAAT TTTGATGGTA GTTTTCGAGG;1010 1020 1030 1040 1050;20 CTCGGCTCAG GGCATAGAAG GAAGTATTAG GAGTCCACAT TTGATGGATA;1060 1070 1080 1090 1100;TACTTAACAG TATAACCATC TATACGGATG CTCATAGAGG AGAATATTAT;1110 1120 1130 1140 1150;TGGTCAGGGC ATCAAATAAT GGCTTCTCCT GTAGGGTTTT CGGGGCCAGA;95;1X60 1170 1180 1190 1200;attcactttt ccgctatatg GAACTATGGG AAATGCAGCT ccacaacaac;1210 1220 1230 1240 1250;gtattgttgc tcaactaggt cagggcgtgt atagaacatt atcgtccact;1260 1270 1280 1290 1300;ttatatagaa gaccttttaa tatagggata aataatcaac aactatctgt;1310 1320 1330 1340 1350;tcttgacggg acagaatttg cttatggaac CTCCTCAAAT ttgccatccg;1360 1370 1380 1390 1400;ctgtatacag aaaaagcgga acggtagatt cgctggatga aataccgcca;1410 1420 1430 1440 1450;cagaataaca acgtgccacc taggcaagga tttagtcatc gattaagcca;1460 1470 1480 . 1490 1500;TGTTTCAATG TTTCGTTCAG gctttagtaa tagtagtgta agtataataa;1510 1520 1530 1540 1550;gagctcctat gttctcttgg atacatcgta gtgctgaatt taataatata;1560 1570 1580 1590 1600;attccttcat cacaaattac acaaatacct ttaacaaaat ctactaatct;1610 1620 1630 1640 1650;tggctctgga acttctgtcg ttaaaggacc aggatttaca ggaggagata;1660 1670 1680 1690 1700;ttcttcgaag aacttcacct ggccagattt caaccttaag agtaaatatt;1710 1720 1730 1740 1750;actgcaccat tatcacaaag ATATCGGGTA agaattcgct acgcttctac;36;1760 1770 1780 1790 1800;CACAAATTTA CAATTCCATA CATCAATTGA CGGAAGACCT ATTAATCAGG;1810 1820 1830 1840 1850;GGAATTTTTC AGCAACTATG AGTAGTGGGA GTAATTTACA GTCCGGAAGC;* 5 1860 1870 1880 1890 1900 TTTAGGACTG TAGGTTTTAC TACTCCGTTT AACTTTTCAA ATGGATCAAG 1910 1920 1930 1940 1950 TGTATTTACG TTAAGTGCTC ATGTCTTCAA TTCAGGCAAT GAAGTTTATA 1960 1970 1980 1990 2000 10 TAGATCGAAT TGAATTTGTT CCGGCAGAAG TAACCTTTGA GGCAGAATAT 2010 2020 2030 2040 2050 GATTTAGAAA GAGCACAAAA GGCGGTGAAT GAGCTGTTTA CTTCTTCCAA 2060 2070 2080 2090 2100 TCAAATCGGG TTAAAAACAG ATGTGACGGA TTATCATATT GATCAAGTAT 15 2110 2120 2130 2140 2150 CCAATTTAGT TGAGTGTTTA TCTGATGAAT TTTGTCTGGA TGAAAAAAAA 2160 2170 2180 2190 2200 GAATTGTCCG AGAAAGTCAA ACATGCGAAG CGACTTAGTG ATGAGCGGAA 2210 2220 2230 2240 2250 20 TTTACTTCAA GATCCAAACT TTAGAGGGAT CAATAGACAA CTAGACCGTG 2260 2270 2280 2290 2300 GCTGGAGAGG AAGTACGGAT ATTACCATCC AAGGAGGCGA TGACGTATTC 2310 2320 2330 2340 2350 AAAGAGAATT ACGTTACGCT ATTGGGTACC TTTGATGAGT GCTATCCAAC 97 2360 2370 2380 2390 2400 gtatttatat caaaa&atag atgagtcgaa attaaaagcc tatacccgtt 2410 2420 24.30 2440 2450 accsattaag agggtatatc gaagatagtc aagacttaga aatctattta 2460 2470 2480 2490 2S00 attcgctaca atgccaaaca cgaaacagta aatgtgccag gtacgggttc 2510 2520 2530 2540 2550 cttatggccg ctttcagccc caagtccaat cggaaaatgt gcccatcatt 2560 2570 2580 2590 2600 cccatcattt ctccttggac attgatgttg GATGTACAGA CTTA&ATGAG 2610 2620 2630 2640 26S0 gacttaggtg tatgggtgat attcaagatt aagacgCAAG atggccatgc 2660 2870 2680 2690 2700 aagactagga aatctagaat ttctcgaaga gaaaccatta gtaggagaag 2710 2720 2730 2740 2750 cactagctcg tgtgaaaaga gcggagaaaa aatggagaga caaacgtgaa 2760 2770 2780 2790 2800 aaattggaat gggaaacaaa tattgtttat aaagaggcaa aagaatctgt 2810 2820 2830 2840 2850 agatgcttta tttgtaaact ctcaatatga tagattacaa gcggatacca 2860 2870 2880 2890 2900 acatcgcgat gattcatgcg gcagataaac gcgttcatag cattcgagaa 2910 2920 2930 2940 2950 gcttatctgc ctgagctgtc tgtgattccg ggtgtcaatg CGGCTATTTT 38 2960 2970 2980 2990 3000 TGAAGAATTA GAAGGGCGTA TTTTCACTGC ATTCTCCCTA TATGATGCGA 3010 3020 3030 3040 3050 GAAATGTCAT TAAAAATGGT GATTTTAATA ATGGCTTATC CTGCTGGAAC 5 3060 3070 3080 3090 3100 GTGAAAGGGC ATGTAGATGT AGAAGAACAA AACAACCACC GTTCGGTCCT 3110 3120 ' 3130 3140 3150 TGTTGTTCCG GAATGGGAAG CAGAAGTGTC ACAAGAAGTT CGTGTCTGTC 3160 3170 3180 3190 3200 10 CGGGTCGTGG CTATATCCTT CGTGTCACAG CGTACAAGGA GGGATATGGA 3210 3220 3230 3240 3250 GAAGGTTGCG TAACCATTCA TGAGATCGAG AACAATACAG ACGAACTGAA 3260 3270 3280 3290 3300 GTTTAGCAAC TGTGTAGAAG AGGAAGTATA TCCAAACAAC ACGGTAACGT 15 3310 3320 3330 3340 3350 GTAATGATTA TACTGCGACT CAAGAAGAAT ATGAGGGTAC GTACACTTCT 3360 3370 3380 3390 3400 CGTAATCGAG GATATGACGG AGCCTATGAA AGCAATTCTT CTGTACCAGC 3410 3420 3430 3440 3450 20 TGATTATGCA TCAGCCTATG AAGAAAAAGC ATATACAGAT GGACGAAGAG 3460 3470 3480 34 90 3500 ACAATCCTTG TGAATCTAAC AGAGGATATG GGGATTACAC ACCACTACCA 3510 3520 3530 3540 3550 GCTGGCTATG TGACAAAAGA ATTAGAGTAC TTCCCAGAAA CCGATAAGGT m ■ 3560 3570 3580 3590 3600 ATGGATTGAG ATCGG2VjGAAA CGGAAGGAAG ATTCATCGTG GACAGCGTGG 3610 3620 3630 3640 36S0 AATTACTTCT TATGGAGGAA TAATATATGC TTTATAATGT AAGGTGTGCA 3660 3670 3680 3690 3700 AATAA&GAAT G&TTACTGAC TTGTATTGAC AGATAAATAA GGAAATTTTT 3710 3720 3730 3740 3750 ATATGAATAA AAAACGGGCA TCACTCTTAA AAGAATGATG TCCGTTTTTT 3760 3770 3780 3790 3800 GTATGATTTA ACGAGTGATA TTTAAATGTT TTTTTTGCGA AGGCTTTACT 3810 3820 3830 3840 3850 TAACGGGGTA CCGCCACATG CCCATCAACT TAAGAATTTG CACTACCCCC 3860 3870 3880 3S90 3900 AAGTGTCAAA AAACGTTATT CTTTCTAAAA AGCTAGCTAG AAAGGATGAC 3910 3920 3930 3940 3950 ATTTTTTATG AATCTTTCAA TTCAAGATGA ATTACAACTA TTTTCTGAAG 3960 3970 3980 3990 4000 AGCTGTATCG TCATTTAACC CCTTCTCTTT TGGAAGAACT CGCTAAAGAA 4010 4020 4030 4040 4050 TTAGGTTTTG TAAAAAGAAA ACGAAAGTTT TCAGGAAATG AATTAGCTAC 4060 4070 4080 4090 4100 CATATGTATC TGGGGCAGTC AACGTACAGC GAGTGATTCT CTCGTTCGAC 4110 4120 4130 4140 4150 TATGCAGTCA ATTACACGCC GCCACAGCAC TCTTATGAGT CCAGAAGGAC 100 4160 4170 4180 4190 4200 TCAATAAAGG CTTTGATAAA AAAGCGGTTG AATTTTTGAA ATATATTTTT 4210 4220 4230 4240 4250 tctgcattat GGAAAAGTAA ACTTTGTAAA acatcagcca tttcflagtgc 4260 4270 4280 4290 4300 agcactcacg TATTTTCAAC gaatccgtat tttagatgcg acgattttcc 4310 4320 4330 4340 4350 aagtaccgaa ACATTTAGCA CATGTATATC ctgggtcagg TGGTTGTGCA 4360 caaactgcag 2

7. A process according to any one of claims 1 or 2 wherein the cloning vector used in step (b) is a bifunetional vector that apart from being capable of replicating in bacterial cells selected from the group consisting of B. thuringiensis and B. cereus cells is capable of implicating at least in one other heterologous host organism, and that is identifiable in both the homologous and the heterologous host system. 2

8. A process according to claim 27, wherein the said heterologous host organisms axe a) prokaryoric organisms selected from the group consisting of the genera Bacillus, Staphylococcus, Streptococcus, Streptomyces, Pseudomonas, Escherichia, Agrobacterium, Salmonella, and Erwinia or b) eukaryotic organisms selected from the group consisting of yeast, animal and plant cells. 2

9. A process according to claim 28. wherein the said heterologous host organism is E. coli. 30. A process according to claim 27, wherein the bifunetional vector comprises under the control of expression sequences that are capable of functioning in bacterial cells selected from the group consisting of Bacillus thuringiensis and Bacillus cereus cells a structural gene encoding a 5-endotoxin polypeptide that occurs naturally in B. thuringiensis, or for a polypeptide that has substantial structural homologies therewith 1Q1 gad has sdll substantially the tonicity properties of the said crystalline 5-endotoxin polypeptide. 31. A process according #> ekta 30, wheaein die said expression sequences comprise a. sporc2!sai.oa-