PLASMIDS CODING FOR B. THURINGIENSIS CRYSTAL PROTEIN, THEIR PRODUCTION AND THEIR USE IN CREATING GENETICALLY ENGINEERED BACTERIAL STRAINS, THE AFORESAID STRAINS PER SE AND THEIR USE IN PRODUCING SAID PROTEIN, SAID PROTEIN SO-PRODUCED, INSECTICIDAL FORMULATIONS COMPRISING THE SAME AND A METHOD OF PRODUCING AN INSECTICIDAL EFFECT

Abstract: The crystal protein of Bacillus thuringiensis is an insecti­ cidal substance. Unfortunately growth phase limitations are exhibited by B. thuringiensis in producing this protein. This in­ vention, however, provides a plasmid capable of replication in a bacterial host species and containing expressible heterolo­ gous DNA coding for the crystal protein of Bacillus thuringien­ sis and including an expression mechanism for said heterolo­ gous DNA which is recognized by the host species system but does not exhibit substantial growth phase limitations in the bacterial host species. Genetically engineered bacterial host strains transformed by the plasmids of the invention express B. thuringiensis crystal proteins without exhibiting the afore­ said growth phase limitations.

Description: PLA5XRDS CODING FOR B. THURINGIENSIS CRYSTAL PROTEIN, THEIR PRODUCTION AND THEIR USE IN CREATING GENETTC.LLY ENGINEERED BACTERIAL STRAINS, THE AFORESAID STAINS PER SE AND THEIR USE IN PRODUCING SAID PROTEIN, SAID PROTEIN SO-PRODUCED',' INSECTICIDAL FORMULATIONS COMPRISING THE SAME AND A METHOD OF PRODUCING AN INSECTICIDAL' EFFECT The invention describes novel tenetically engineered plasmids. Examples of such plasmids have. been deposited with the American Type Culture Collection, Rockville, Maryland 20852 under the Budapest Treaty. Plasmid pESl has been assign'ed ATCC Number 31995, plasmid opJWK20 has been assigned ATCC Number 31997, and plasmid pJWK18 has been assigned ATCC Number 31998. This invention relates generally to the production of substances for the control of insects injurious to certain plants. More particularly, the invention relates to an improved means for producing substances toxic to larvae of the tobacco hornwo= Manduc'a' 'sexta and- related species. As is well know, the crystal protein made by B. thurinqien'sis is toxic to the larvae of a number of lepidopteran insects. Preparations containing crystals are used commercially as a highly selective biological insecticide. However, problems connected with the use of such insecticides, together with relatively high manufacturing costsr have made it difficult, in many cases, for such insecticides to compete effectively with other commercially available products. The fact that B. thuringiensis produces the crystal protein only during sporulation represents a significant disadvantage in connection with the manufacture and use of the crystals. Such a growth phase limitation, particularly'in an industrial process, can result in inconvenience and excessive time requirements during manufacture. In fact, certain pressures resulting from growth phase limitations or other factors may even result in strains of B. thuringensis losing their ability to produce the crystals Such acrystalliferous strains do not have insecticidal activity. The present invention discloses and embraces plasmids (and their manufacture) that are capable of replication in a bacterial host species and that contain expressible heterologous DNA encoding for the crystal protein of Be thuringiensis. The plasmids further include an expression mechanism for the heterologous DNA which is recognized by the host species' system but does not exhibit any substantial growth phase limitations in the bacterial host species. In another aspect, the. invention comprises genetically engineered bacterial strains transformed by such plasmids and, in a further aspect, the manufacture or creation of such strains. Bacterial strains genetically engineered to contain the recombinant plasmids of the present invention can be produced by transformation and can express' .3. thuringiensis crystal protein. A method of preparing the protein by growing such strains is included in the invention. The protein produced by these genetically engineered strains is toxic to the larvae of a number of lepidopteran insects. Such protein so-produced is included in the invention Preparations or formulations containing these crystals can be used as a highly selective biological insecticide and are also included in the invention as is a method of producing an insecticide-in an environment using such protein, preparations or formulations in a standard manner or by exposing the environment to growth of a genetically engineered strain a aforesaid. Thus, the invention provides an insecticidal formulation or preparation comprising B. thEr-ingiensis crystal protein produced as aforesaid. The invention, of course, provides 'a method of producing a plasmid of the invention which comprises isolating from a crystal protein-producing strain of 'Baci.l.L.us thu-rin'giensis. a first fragment of DNA comprising a expressible-DNA coding for the crystal protein, providing one or more other DNA fragments which contribute(s) an expression mechanism for heterologous DNA in a selected bacterial host other than B. thuringie-nsis ,the expression mechanism beign recognized by the host without exhibiting substantial growth phase limitations, and ligating said first DNA fragment and said other fragment(s) in the correct arrangement for expression of the heterologous crystal protein-producing DNA coding in the host. As indicated earlier, the invention also includes a method of creating a genetically engineered bacterial strain which is capable of producing the crystal protein of Bacillus thuringiensis comprising transforming a host bacterial strain other than B. 'thuringiensis by introducing thereinto a plasmid of the invention whereby the expressible genetic material in the host includes heterolocous DNA coding for the crystal protein of B. thuringiensis. '." v In accordance with a preferred form of the invention, novel recombinant plasmids are created sçhen the known cloning vector plasmid pBR322, Bolivar et al, Gene 2:95-113 (1977), is combined with plasmid fragments obtained from plasmids harbored by strains of B. thuringiensis. The "large plasmid fraction" is especially preferred. This "large plasmid fraction includes fragments having a size greater than about 10 megadaltons. The plasmids harbored by strains of B. thuringiensis are believed to be responsible for the production of the crystal protein in B. thuringiensis. The B. thuringiensis plasmid fragments used to construct the recombinant crystal protein coding plasmids of the present invention can be derived from a variety of B. thuringiensis strains, some of which are publicly available. For example, B. thuringnsis variety Kurstaki RD=7 is available from the Agricultural Research Culture Collection (NRRL) in Peoria, Illinois, U*S.A. It has been awarded NRRL Number B-3792. B. thuringiensis variety Kurstaki HD-73 is also available from the Agricultural Research Culture Collection. It has been awarded NRRL Number 3-4488. In the Examples given hereinafter the use of several B tharingiensis strains is illustrated. Such Examples are included for illustrative purposes only and are not intended to limit the scope of the invention to use of fragments derived rom such strains. The invention will now be further described and illustrated by the following Examples, and by reference to the accompanying drawings, wherein: FIGURE 1 is an agarose gel analysis of plasmid DNAs and marker DNA fragments comprising a photograph of 0.7% (lanes 1-5) and 0.35% (lanes 6 and 7) agarose slab gels stained with ethidium bromide; FIGURE 2 is a hybridization analysis of plasmid DNAs transferred to nitrocellulose comprising a photograph of a 0.7% ethidium bromide stained agarose gel (lanes 1-3) and autoradiograms of- 32P labeled plasmids (lanes 4-12); and FIGURE 3 is a photograph showing a radioimmune assay of crystal protein and proteins produced by bacterial strain ES12 before and after digestion with trypsin; and 1 FIGURE 4 is a photograph of an ethidium-bromide stained gel showing plasmids detected in extracts of different strains of B. thuringiensis. Example IV, given hereinafter, is a survey of B thuring1ens-'is strains for plasmid content and the ability of these plasmids to hybridize with a particular genetic probe for identification of the crystal protein gene. ¯ EXAMPLE I Use of B. thurintiensis variety kurstaki HD-l-Dipel plasmid DNA fragments in the construction of recombinant plasmids in accordance with the present invention Crystal-producing strains of B. thuringiensis vary-i-n the number and sizes of the plasmids they contain. Plasmids obtained from'B. thuringiensis variety kurstaki HD-l-Dipel range in molecular mass from approximately 47 to 1.32 megadaltons as shown in lane 1 of FIGURE 1, which shows the total plasmid complement normally obtained from this strain. Larger plasmids are present in this strain but were not readily detected as closed circular forms under the conditions of growth used in this study. However, plasmid fractions isolated from B. thuringiensis contained small amounts of the linearized forms of these large plasmids. Two plasmid fractions were obtained from the B. thuringiensis variety kurstaki HD-l-Dipel strain, as shown in lanes 2 and 3 of FIGURE 1. One fraction, shown in lane 2, contained the 47, 32 and 30 megadalton plasmids as well as trace amounts of linearized forms of the very large plasmids of B. thuringiensis (molecular weight x 106 shown on left). The second fraction contained smaller plasmids of 4, 9, 5.2, 5.5 and 9.6 megadaltons, as shown in lane 3. A preferred recombinant plasmid of the invention, designated pESl (ATCC Number 31995), is shown in lane 4 of FIGURE 1 in which the gel analysis of the plasmid may be compared with pBR322, shown in lane 5. After digestion by an enzyme which cuts the plasmid once, the linearized plasmid had a mobility corresponding to ca. 11 x 106 Mr when compared to Rind III digested lambda DliA (shown in lanes 6 and 7 of FIGURE 1). Molecular weights (x 10-6) on the right of the Figure refer to the Hind fII digest of lambda DNA. The arrow on the right in FIGURE l marks the origin for lanes 6 and 7. In constructing recombinant plasmids, such as pESI (ATCC Number 31995) according to the invention, the following detailed procedures were followed. These procedures are also outlined in Schnepf and Whiteley, Proc. Nat. Acad. Sci., USA, 78:28-93-2897 (1981). They are given only by way of example and are not intended to limit the scope of the claims herein. The two separated plasmid fractions, designated as "large" and "small", obtained from B. thuringiensis varv kurstaki HD-l-Dipel were digested with varying dilutions of Sau3Al. This was monitored by agarose gel electrophoresis. To generate a source of inserts, the "large plasmid fraction" was digested with the minimum amount of enzyme needed to convert all the closed covalent circular molecules to the linear form. Restriction endonucleases Sal I, Hind III, BamH 1 and Sau3Al were used as recommended by the manufacturer (New England Biolabs). This resulted in fragments with an average size greater than 10 megadaltons. For the small plasmid fraction", an amount of Sau3Al was used which left some closed covalent circular molecules, but produced few linear fragments under 2 x 106 Mr in size. Each fraction was then ligated to pBR322 that had been opened by digestion with the restriction enzyme Bam B1. Plasmid pBR322 was isolated from E. coli strain HB101 (pBR3Z2) as outlined by Blair et al Proc. Nat. Acad. Sci., USA, 69:2518-2522 (1972). Plasmid pBR322 is on deposit with ATCC, is publicly available and its accession number is ATCC 31071. DNA fragments (3 ug of B.- thuring iensis plasmid DNA and Q.15 ug of pBR322 DNA in a volume of lO ul) were ligated as described for cohesive ends by Maniatis- et al in Cell 15:667-701 (1978). Recombinants were then screened and selected, Staphylococcus Protein A (Pharmacia) was labeled with 125I (Amersham) using chloramine T as described by 'Erlich et al, Cell 13:681-689 (1978). The DNA 'polymerase I-catalysed fill-in reaction described by Maniatis t al Proc. Nat. Acad. Sci., USA, 72:1184-1188 (1975) was used to label DNA fragments of Sau3Al digests with [.a-32P]dCTP (Amersham). Plasmids were obtained from B. thuringiensis var. kurstaki HD-l-Dipel, kindly provided by Dr. Lee A. Bulla, Jr., according to the method for plasmid screening of White and Nester, J. Bacteriol. 141:1134-114t (1980). All plasmid preparations were additionally purified by centrifugation in cesium chloride-ethidium bromide gradients. Samples (100 ug DNA per gradient) for cloning experiments were fractionated by centrifugation through 5-25% sucrose gradients for 2.5 hours at 35,000 RPM in an International B-60 centrifuge using the SB-283 rotor. Electrophoresis in agarose gels was used to analyze plasmid DNAs as described by eyers et al, J. Bacteriol. 127:1529-1537 (1976j. Fragments of the plasmids were produced by digestion of DNA with restriction enzymes as is known in the art. Hybridization to plasmid DNAs was performed after partial depurination as described by Wahl et al, Proc. Nat. Acad. Sci., USA, 76:3683-3687 (1979) and transfer of DNA from gels to nitrocellulose was done as described by Thomashow et al Cell 19:729-739 (1980). The recombinant plasmids thus produced were then transformed into E. coli. Transformation was carried out as described by known procedures and transformants were selected on media containing 100 ugZml ampicillin. Since cloning was performed by insertion of messenger DNA into the BamEI 1 site of pBR322, which is located in a gene coding for tetracycline resistance, ampicillin resistant transformants were screened for sensitivity to tetracycline (25 ug/ml). Colonies resistant to ampicillin but sensitive to tetracycline were presumed to contain inserts. Those transformed colonies presumed to carry B. thuringiensis DNA inserts were then screened for the production of crystal protein antigen using antibodies and 1251-Protein t To prepare antibodies to the crystals, the crystals were first purified from sporulated cultures of 3. thuringiensis grown in modified G medium, Aronson et al, J. Bacteriol. 106:1016-1025 (1971)r by four successive centrifugations in Renograffin (Squibb) gradients. Contavnination with spores was estimated at less than 0.1% oy phase microscopy. Solubilized crystals were electrophoresed on preparative 108 polyacrylamide slab gels containing NaDodSoq, r and the portion of the gel containing the major crystal protein polypeptide was sliced from the gel, crushed, mixed with an equal amount of Freund's Complete Adjuvant and used to immunize rabbits. The immunoglobulin G fraction was purified from the serum of the immunized rabbits by precipitation with ammonium sulfate and chromatography on DEAE cellulose and analyzed by Ouchterlony immunodiffusion, as is known in the art. To screen by detection of antigens, colonies were first transferred from agar plates to filter paper and denatured with phenol-chloroform-heptane and chloroform-methanol as described by Henning et al, Anal. Biochem 97:153-157 (1979). The filters were soaked in 1% bovine serum albumen (Sigma, fraction V) and incubated with antibody and 125I-Protein A as described by Renart et al, Proce Nat. Acad. Sci., USA, 76:3116-3120-, (1979). Colonies containing material capable of reacting with the crystal protein antibodies were detected by autoradiography. The concentrations of antibody (l0-- to 10-4 dilution) and 1251-protein A (0.2 to 1 x 106cm) were varied to obtain conditions permitting the detection of 5 ng crystal protein in 1 ul spotted on a filter while colonies of E. coli HB101 (pBR322) were either unreactive or appeared light against a grey background. Protein samples were electrophoresed on 109 NaDodSO4/polyacrylamide slab gels, transferred electrophoretically to nitrocellulose, and incubated with antibody (5 x 10-3 dilution) and 125I-Protein A (6 x lO5cpm) in accordance with known procedures. The reaction of transferred peptides with antibody and 1251-Protein A was detected by radioautography Cells grown for 16 hours in L broth containing 100 ug/ml ampicillin were harvested by centrifugation, suspended in 0.1 M Tris buffer at pH 7.0, lmM EDTA, 200 ug/ml phenylmethylsulfonyl fluoride and disrupted by sonication. Insoluble material, obtained by centrifugation of the sonicates at 100,000 x g for 30 minutes, was-thoroughly suspended in 4M urea, 0.285M 2-mercaptoethanol, 0.05M Narc03, pH 9 5, and dialysed against O.OSM Tris buffer pH 7.4 for insect toxicity assays or for electrophore.ic analysis. For proteolyis with subtilisin or trypsin, the samples were prepared as above substituting 0.05M cyclohexylaminoethane sulfonic acid (CHES), pH 10.0, 0.285M 2-mercaptoethanol for pH 9.5 urea buffer; final dialysis was against Q.OlM CHES buffer, pH lO.O. For trypsin digestion, O.O1M CHES was used as the buffer and digestion was carried out at room temperature during dialysis against that buffer; digestion with subtilisin was performed according to Cleveland et al, J. Biol. Chem. 252:1102-1106 (1977). The reactions were stopped by boiling the samples in electrophoresis sample buffer and polypeptides capable of reacting with antibody to the crystal protein were detected using 125I-Protein A and radioautography as described above. Protein concentrations were determined according to Bradford, Anal. Biochem. 72:248-254 (1976). The recombinant plasmid pESl (ATCC Number 31995)- consists of the plasmid vector pBR322 and DNA homologous to the 30, 32 and 47 megadalton plasmids, as well as DNA homologous to linearized forms of the very large plasmids of B. thuriniensis. One explanation for this result is that several Sau3Al partial digest fragments from each of the large B. thuringiensis plasmids, were ligated together; this heteromultimer could then have been ligated into the vector. Another possibility is that the apparent hybridization to the 47 megadalton plasmid as shown in FIGURE 2, is actually a trailing artifact from the 30 or 32 megadalton plasmids. EtANPLE II Evidence that E. coli transformed by recombinant plasmids of the present invention produce B. thurinqiensis crystal protein. The recombinant plasmids of the present invention can be used to transform E. coli cells. These transformed cells produce B. thuringiensis crystal protein. Such transformation is possible because the recombinant plasmids of the present invention contain both vector DNA and DNA from B. thuringiensis plasmids that codes for the crystal protein. -Plasmid pESl (ATCC Number 31995) was used to demonstrate this. As illustrated in FIGURE 2, plasmid pESl (ATCC Number 31995) contains substantial homology with DNA from the plasmid pBR322 as well as DNA homologous to the 30, 32 and 47 megadalton plasmids as well as DNA homologous to linearized forms of the very large plasmids of B. thuringiensis. In FIGURE 2, lanes 1-3 show a photograph of. a 0.7* ethidium bromide-stained agarose gel: Lane 1 shows BamH 1 digested pBR322 DNA; lane 2 shows total plasmid complement of B. thuringiensis and lane 3 shows the "large plasmid fraction" from B. thuringiensis. Lanes 4-6 show an autoradiogram of 32P-labeled pBR322 DNA hybridized with: BamH 1 digested pBR322 DNA, as shown in lane 4; total plasmid complement, as shown in lane 5; and the 'large plasmid fraction" from B. thuringiensis, as shown in lane 6. Lanes 7-9 show an autoradiogram of 32P-labeled pESI (ATCC Number 31995) DNA hybridized with: BamH 1 digested pBR322, as shown in lane 7; total plasmid complement, as shown in lane 8; and "the large plasmid fraction" from B. thuringiensis, as shown in lane 9. Lanes 1-12 show an autoradiogram of 32P-labeled total B. thuringiensis plasmid DNA hybridized with: BamH 1. digested pBR322 DNA as shown in lane 10; total plasmid complement, as shown in lane 11; and the "large plasmid fraction" from B. thuringiensis, as shown in lane 12. The arrows in this Figure show the position of the linearized fragments and the hybridization of the probe to such fragments. FIGURE 3 presents a radio immune assay of crystal protein produced by transformed E. coli strain ES12 before and after digestion with trypsin. Autoradiograms of a solid-phase radio immune assay for polypeptides are shown in lanes 1-3 of FIGURE 3. These fragments react with anti-crystal antibody following NaDodSO4/polyacrylamid.e gel electrophoresis. Lane 1 shows 1 ug B. thuringiensistcrystal protein. Lane 2 shows 100 ug S. coli HB101 (pBR322) protein extract. Lane 3 shows 100 ug ES12 protein extract. Lane 4 is derived from dissolved B. thuringiensis crystals reacted with 5% (wt/wt) trypsin at pH 10 for 3 hours at room temperature, Lanes 5-7 show the ES12 extract reacted with: 2% (wt/wt) trypsin, as shown in lane 5, 4% : (wt/wt) trypsin, as shown in lane 6; and 68 (wt/wt) trypsin at pE-10 for 3 hours at room temperature, as shown in lane 7. -- Lanes 1-3 df FIGURE 3 show that the antigen made by E512, which reacted with crystal protein antibodies, had the same electrophoretic mobility as the B. thuringiensis crystal protein. Dissolved- B.thuringiensis crystals and cell extracts of BB101 (pBR322) and ES12 were electrophoresed on a NaDodSO4/polyacrylamide gel and reacted with anti-crystal antibody and 125I-Protein A after transfer: to nitrocellulose. The ES12 extract, shown in lane 3 of FIGURE 3, contained a polypeptide antigen having the same (or very similar) electrophoretic mobility as the dissolved B. thuringiensis crystals shown in lane 1 of FIGURE 3. This polypeptide antigen was missing from a similar extract of HB101 (pBR322), as shown in lane 2 of FIGURE 3, and was not detected when pre-immune serum was substituted for anti-crystal antibody, or when 125I-Protein A was used without prior antibody treatment (data not shown). Comparison of the bands shown in FIGURE 3 (1 ug crystal protein in lane 1) with the total amount of protein applied to lane 3 (100 ug) indicates that the crystal protein antigen accounts for a small amount of the protein (1% or less) in ES12. When the radioimmune detection of polypeptide was used to monitor the fractionation of ES12 extracts it was found that a reducing agent plus a denaturant or an alkali pE was required to solubilize the crystal protein antigen. These conditions are also required to solubilize B. thuringiensis crystals. It has been reported by Lilley et al, J. Gen. Microbial. 118:1-11 to980) that the crystal protein can be digested by a number of proteases at pH 10 to produce primarily a single polypeptide. Lanes 4-7 of FIGURE 3 show the results of an experiment where dissolved B. thuringiensis crystals and an eluate from the particulate fraction of ES12 were subjected to-partial digestion at pH 10 with the indicated amounts of trypsin. As seen in lanes 5, 6, and 7 of FIGURE 3, digestion of the ES12 extract with increasing amounts-of trypsin yielded a pattern, as shown in lane 7 of FIGURE 3, which was similar to the pattern produced by trypsin digestion of the crystal protein of B. thuringiensis, as shown in lane 4 of FIGURE 3. Qualitatively the patterns of the bands generated from the two preparations were similar. The quantitative, and minor qualitative, differences may reflect a less efficient digestion of the crystal protein antigen in ES12 extracts due to the presence of numerous other polypeptide species. The larger number of polypeptides produced by trypsin digestion of the crystal protein in these experiments as opposed to the number reported by Lilley et al, supra, may be due to differences in the conditions of trypsin treatment. Similar experiments using subtilisin also showed agreement in the electrophoretic mobilities of the bands produced from the crystal protein of B. thuringiensis was obtained by assaying for insect toxicity. Extracts of particulate fractions obtained from E. coli HB1D1 (pBR322) and ES12 were mixed with feed meal supplied to neonate caterpillars of the tobacco hornworn Manduca sexta. Neonate larvae of the Manduca sexta were supplied by Drs. J. Truman and L. Riddiford, Department of Zoology, University of Washington. Extracts were prepared as described above from 8 liters of the appropriate E. coli strains; 6-8 mls of extract were mixed with 50 ml of molten agar-based diet and quickly poured to give a shallow layer. Strips of the solidified diet were placed in glass vials (3-4 ml/vial) with one neonate larva for 10 days at room temperature. The results indicated that the extracts of the genetically engineered recombinant strain were toxic to caterpillars. The larvae exposed to the 5512 extracts did not complete the first instar before death. An equivalent amount of extract from control strain, E. coli HB101 (pBR322), had no noticeable effect on the growth and development of 15 larvae through at least the third instar when compared to larvae grown on feed meal without any extract added. Identical results were obtained when this experiment was repeated with another set of E. coli extracts. The minimal amount of extract of ES12 required to kill the caterpillar larvae has not yet been determined. Assuming that the crystal protein antigen in the ES12 extracts was 0.5-18 of the total protein, then the feed meal prepared with the extract'from ES12 contained 12-25 ug crystal protein per ml whereas a concentration of 2 ug/ml of pure crystal protein is sufficient to achieve 100% killing of the larvae. The resulting transformed strain of E. coli, Yes12, carries a recombinant plasmid and produces a protein antigen that reacts with antibodies specific for the crystal protein of B. thuringiensis. This is confirmed bp the above described tests wherein the recombinant plasmid isolated from this strain, pESl (ATCC Number 31995) simultaneously transforms cultures of E. coli strain HBl0l to both ampicillin resistance and the production of the crystal protein antigen, whereas the plasmid vector pBR322 transforms 113101 to ampicillin and tetracycline resistance -but no crystal antigen can be detected. Test results indicate that the DNA insert of the recombinant plasmid pESl (ATCC Number 31995) encodes the ability to make a polypeptide possesing properties similar to those of the crystal protein of B. thuringiensis. In fact, the results strongly suggest that the DNA insert of pESI (ATCC Number 31995) encodes the gene for the crystal protein of B. thuringiensis and that this gene is expressed in a biologically- active form in E. coli. - Preparations made from this new genetically engineered strain, ES12, may therefore be used as an insecticide for appropriate cases, c EXAMPLE III Use of B. thuringiensis var. kurstaki ED-73 (NRRL 3-4488) plasmid DNA fragments in the construction of recombinant plasmids in accordance with the present invention. B. thuringiensis var. kurstaki HD-73 contains fewer so-called large plasmids than do some of the other B. thuringiens,s strains. However, as this Example illustrates, this strain can be used to generate the DNA fragments utilized in constructing the recombinant plasmids of the present invention. The "large plasmid fraction" from B. thuringiensis var. kurstaki HD-73 was obtained by sucrose gradient centrifugation in the same way as the "large plasmid fraction" from B. thuringiensis var. kurstaki ED-l-Dipel described in Example I. Digestion of this fraction with restriction - enzyme Bgl II yielded approximately six fragments1 one of- which hybridized with a Pvu II-C DNA probe obtained from pESI (ATCC Number 31995). The probe is discussed more fully in Example IV.- The "large plasmid fraction" was digested with Bgl II, ligated to BamS I-digested pBR322 and transformed into E. coli. Colonies which were ampicillin-resistant and tetracycline-sensitive were screened for- hybridization with the Pvu II-C DNA probe and for their ability to synthesize the crystal protein antigen A strain was isolated designated JWKl, having a plasmid, pier20 (ATCC Number 31997), whose linearized mass is approximately 13.5 megadaltons. The results of preliminary analyses using Bgl II, Sal I, and Hind III digestion of pJWK20 and the "large plasmid fraction" from HD-73 suggest that pJWK20 has a single insert of approximately 10.9 megadaltons derived from the 45 megadalton plasmid of B. thuringiensis var. kurstaki HD-73. Extracts of genetically engineered strain JWKl were found to contain a polypeptide of approximately 130,000 Mr which reacted with the antibody to the crystal protein from RD-l-Dipel. The mobility of this peptide matched that of the crystal protein from HD-l-Dipel. Tests for insect toxicity showed that feed meal supplemented with extracts of transformed E. coli strain JWKl where lethal for larvae of the tobacco hornworm. The onset of toxic symptoms was similar to that observed for larvae fed with meal containing solubilized crystal protein. As in previously described toxicity tests, meal containing extracts of E. coli (pBR322) did not retard the growth of the larvae. Thus it can be seen that genetically engineered E. coli strain JWK1 produces a protein which is similar in size, antigenicity and biological activities to the crystal protein of B. thuringiensis HD-1-Dipel. EXAMPLE IV Survey of B. thuringiensis strains for plasmid content and the ability of these plasmids to hybridize with a probe from pESl (ATCC Number 31995). , Restriction enzyme analysis of transposon Tn5 inserts into pESI (data not shown) indicates that a major portion of the presumed crystal protein gene is contained within two Pvu II cleavage sites on the B. thuringiensis plasmids. The cleavage sites define a Pvu II-C DNA fragment that can be used as a probe to analyze plasmid profiles of the various strains of B. thuringiensis to determine which plasmids contain the gene. The Pvu II-C probe fragment was purified by sucrose gradient sedementation. The DNA fragments were labeled with alpha-32-P-dCTP using either the DNA polymerase-catalyzed. fill-in reaction described by Maniatias et al, Cell 15;667-701 (1978), or by nick translation, described by Maniatias et al, Proc. Nat. Acad. Sci., USA 72:1184-1188 (1975). The probe was then hybridized to plasmids from various B. thuringiensis strains after- electrophoresis and transfer to nitrocellulose. At the time of filing, about twenty cry-stal-producing strains of B. thuringiensis had been examined for plasmid content and the ability of these p1asmids to hybridize with the Pvu II-C probe fragment from pESl (ATCC. Number 31995). An example of one of the gels run as part of this survey is shown in Figure 4. The photograph is of an ethidium bromide stained gel that shows plasmids detected in extracts of various Bthuringiensis strains. Lane a is subspecies tolworthii; lane b is subspecies darmstadiensis; lane c is subspecies sotto; lanes d-g are var. thuringiensis F-10, eD-290, HD-120, and HD-2 respectively; lanes h-j are var. Kurstaki HD-244, HD-73 and 11D-l, respectively. The numbers in the margin indicate plasmid size in megadaltons. Other gels were run (data not shown) with extracts from B. thuringiensis subspecies alesti HD-4, subspecies toumanoffi F-9, subspecies galleriae HD-8, subspecies wuhnanesis F6s- and subspecies morrisoni F-5. The results of the electrophoresis shown in FIGURE 4 demonstrated the presence of several plasmid bands in each one. Presumably, most of the bands consist of closed circular molecules although some bands may represent open circular forms. The strains varied greatly in the number and sizes of the plasmids they contained; however some bands of the same or similar -- mobilities were present in several strains. It is pointed . out that the data presented in FIGURE 4 represent the analysis of routine CsCl-purified plasmid preparations and that no attempts have been made to verify conditions of growth, plasmid extraction or electrophoresis in order to detect all possible plasmids or to duplicate electrophoretic conditions used by other investigators. In addition, the sizes have been roughly estimated by comparisons with the mobilities of plasmids whose contour lengths have been determined by electron microscopy by Stahley et al, Biochem. Biophys, Res. Comm., 84:581-588 (1978). The strains examined in the experiment shown in FIGURE 4 differ in flagellar serotype and in their ability to cross-react with the antibody to the crystal protein of strain ED-l-Dipel. The serotype of the kurstaki strains is 3a, 3b;'the thuringiensis strains are type 1; the subspecies sotto is type 4a, 4b; subspecies darmstadicnsis is type 10 and subspecies tolworthii is type 3. See De Barjac and Bonnefoi, C.R. Acad. Sci., 264:1811-1813 (1967) Fourteen of the twenty strains tested reacted with the antibody to the crystal protein of strain HD-l-Dipel and showed hybridization with Pvu II-C probe. EXAMPLE V Use of B. thurinciensis subspecies sotto plasmid DNA fragments in the construction of the recombinant plasmids of the present invention The procedures used to clone the crystal protein gene from B. thuringiensis subspecies sotto are essentially the same procedures used to clone the gene from B. thuringiensis var. Kurstaki HD-73. The procedures are outlined in Example III. The subspecies sotto was selected for cloning the crystal protein gene because it contains only two plasmids and belongs to a different serotype. To clone the gene, total plasmid DNA purified by centrifugation in a CsCl-ethidium bromide- gradient was partially restricted with endonuclease Mbo I. The resulting fragments were ligated into the Bamil I site of pBR322. After transformation into E. coli, colonies were selected which were ampicillin-resistant and tetracycline-sensitive. These colonies were screened for their ability to hybridize with the Pvu II-C fragment and for their ability to produce the crystal protein. One recombinant strain, JwK 11, ; - contained the plasmid pJF7EC18; the size of the inserted B. thuringiensis plasmid DNA has not yet been determined accurately. Strain iR 11 produces a 130,000 Mr protein which cross-reacts with antisera prepared against the crystal protein isolated fro B thuringiensis kurstaki HD-l-Dipel. Extracts of 3WK t are toxic to-larvae of Manduca sexta. It may be seen therefore that, in accordance with the invention, crystal protein of B. thuringiensis is produced by a host strain transformed with recombinant plasmids. In vivo, B. thurinqiensis only produces crystal protein during sporulation. An advantage of this invention is the absence of the necessity for the host to sporulate in order for expression of the crystal protein to occur. Expression of the genetic information in the plasmids of the invention is not limited to a specific growth phase in the host because the crystal protein is expressed in accordance with an expression mechanism that is recognized by the host species in all major growth phases. By removing growth phase restrictions, - production of the toxin is now possible by continuous culture rather than batch culture. This can permit high 7 rate, high productivity fermentations with a decrease in capital equipment requirements. Various modifications and variations within the- scope of the invention in addition to the precise procedures shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. REFERENCES 1. Aronson, A. I., Angelo, N. t and Holt, S. C., J. Bacteriol. 106:1016-1025 (1971). -2. Blair, D. C., Sherratt, D. J.,- Clewell, D. B., and Helinski, D-. R., Proc. Nat. Acad Sci., USA, 69:2518-2522 (1972). 3. Bolivar, F., Rodriguez, R. L.; Greene, P. J., Betlach, M. C., Heyneker, H. L., and Boyer, B. W., Gene 2:95-113 (1977). 4. Bradford, M., Anal. Biochem. 72:248-254 (1976). 5. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K., J. Biol. Chem. 252(3):1102-1106 (1977). 6. DeBarjac, H., and Bonnefoir A., C. R. Acad. Sci. 264:1811-1813 (1967). 7. Erlich, H. A., Cohen, S. N., and McDevitt, H. O., Cell 13:681-689 (1978) 8. Henning, U. Schwarz, H., and Chen, R., Anal. Biochem. 97:153-157 (1979). 9. Lilley, M., Ruffell, R. N., and Somerville, H. J. J. Gen. Microbiol. 118:1-11 (19-80). 10. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G. K.t and Efstratiadis, A. Cell 15:667-701 (1978). 11. Maniatis, T., Jeffrey, S and Rleid, D. G., Proc. Nat. Acad. Sci., USA, 72:1184-1188 (1975). 12. Meyers, J. A., Sanchez, D., Elwell, L. P., and Falkow, S , J. Bacteriol. 127:1529-1537 (1976). 13. Renart, J., Reiser, J., and Stark, G. R., Proc. Nat. Acad. Sci., USA 76:3116-3120 (1979). 14. Schnepf, H. E., and Whiteley, H. R., Proc. Nat. Acad. Sci., USA 78:2893-2897 (1981). 15. Stahly, D. P., Dingman, D. W., Bulla, L. A., and Aronson,'A. I., Biochem. Biophys. Res. Comm. 84:581-588" (1978). 16. Thomashow, M. F., Nutter, R., Montoya, A. L., Gordon, M. P., and Nester, E. W., Cell 19:729-739 (1980). 17. Wahl, Go M., Stern, N., and Stark, G. H., Proc. - Nat. Acad. Sci., USA, 76:3683-3687 (1979). 18. White, F. F., and Nester, E. W., J. Bacteriol. 141:1134-1141 (1981).

Claims: 'CLAIMS: 1. A plasmid capable of replication in a bacterial host species and containing expressible heterologous DNA coding for the crystal protein of Bacillus thuringiensis and including an expression mechanism for said heterologous DNA which is recognized by the host species system but does not exhibit substantial growth phase limitations in the bacterial host species. 2. A plasmid as claimed in claim 1 comprising a DNA portion having substantial sequence homology to the DNA coding for the crystal protein of B. thuringiensis. 3. A plasmid as claimed in claim 1 comprising a DNA portion having substantial sequence homology to plasmids of B. thuringiensis having a molecular mass greater than 10 x 106 Mr. 4. A plasmid as claimed in claim 3, wherein said DNA portion is identified with a Pvu II-C DNA fragment probe or a probe having substantial sequence homology with a Pvu II-C probe. 5 A plasmid as claimed in claim 4, wherein said DNA portion is derived from B. thuringiensis subspecies tolworthi; subspecies darmstadiensis; subspecies sotto; subspecies thuri'ngiensis; subspecies' 'thuringiensis, HD-290; subspecies thuringiensis, HD-120; subspecies thuringiensis, HD-2; subspecies kurstaki, HD-244; subspecies '-ku'rstaki, HD-73; subspecies- kurstaki, HD-1; subspecies alesti, HD-4; subspecies toumanoffi, F-9; subspecies galleriae, HD-8; subspecies wuhnanesis, F-6; or subspecies -morrisoni, F-5. 6. A plasmid according to claim 1 as claimed in any one of claims 1 to 5 including a DNA portion having substantial sequence homology to plasmid pBR322. 7. The plasmids pESl (ATCC Number 31995), pJ2O (ATCC Number 31997), and pJWK18 (ATCC Number 31998), a mutant plasmid having the B. thuringiensis crystal protein coding characteristics of said plasmid PESl, or pJWK20f or pFçK18, and progeny of any of said plasmids or mutant plastnids. - .; - 8. A bacterial strain containing a plasmid as claimed in any one of claims 1 to 6 or a plasmid, mutant plasmid or progeny thereof as claimed in claim 7. 9. The bacterial strains ES12, JWK1, and JWKll, in each case transformed by a plasmid as claimed in claim 1, and B. thuringiensis crystal protein antigen-producing mutants thereof. 10. A genetically engineered bacterial strain which has been transformed to introduce there into heterologous DNA which, upon expression, results in a polypeptide of 130,000 Mr which reacts specifically with antibody for the crystal protein of B. thurln'glefl's'is. host other than B. thuringiensis, the expression mechanism being recognized by said host without exhibiting. substantial growth phase limitations, and ligating said first DNA fragment and said other fragment(s) in the correct arrangement for expression of the heterologous. crystal protein-producing DNA coding in said host.. - - 15. A method of creating a genetically engineered - bacterial strain which is capable of producing the crystal protein of Bacillus thuringiensis comprising transforming a host bacterial strain other than B.. thuringiensis by introducing there into a plasmid as claimed in any one of claims 1 to 6, or a plasmid, mutant plasmid or progeny thereof as claimed in claim 7, whereby the expressible genetic material in the host includes. heterologous DNA coding for the crystal protein of B. thuringiensis 16. A method as claimed in claim 15, wherein the host is E. - coli. 17. A method as claimed in claim 16, wherein the host is ES12, JWKl or JWKll. 18. A method of preparing Bacillus thuringiensis crystal protein comprising growing a bacterial strain as claimed in any one of claims 8 to 10 or which has been created by a method as claimed in any one of claims 15 to l7 17. - -' - 11. B. thuringiensis crystal protein which has been produced by a genetically engineered bacterial strain transformed by introduction thereinto of a plasmid as claimed in any one of claims 1 to 6 or a plasmid, mutant plasmid or progeny thereof as claimed in claim 7, or by a bacterial strain as claimed in claim 9 or by a genetically engineered bacterial strain as claimed in claim 10 12. An insecticidal formulation or preparation comprising a protein as claimed in claim 11 in a form suitable for insecticidal use, optionally including a diluent, carrier or excipient. -13 A method of producing an insecticidal effect in an environment which comprises treating the environment with a protein as claimed in claim 11 or with a formulation or preparation as claimed in claim 12 or exposing the environment to growth of a bacterial strain as claimed in claim 9 or growth of a genetically engineered bacterial strain as claimed in claim 10. 14. A method of producing as plasmid as claimed in any of claims 1 to 7 which comprises isolating from a crystal protein-producing strain of Bacillus thuringien sis a first fragment of DNA comprising an expressible DNA coding for the crystal protein, providing one or more other DNA fragments which contribute(s) an expression mechanism for heterologous DNA in a selected bacterial Additional set of claims CLAIMS: for AUSTRIA (Art. 167) 1. A method of producing a plasmid capable of replication in a bacterial host species and containing expressible heterologous DNA coding for the crystal protein of Bacillus¯ thuringiensis and including an expression mechanism for said heterologous DNA which is recognized- by the host species system but does not exhibit substantial growth phase limitations in the bacterial host species, which method comprises isolating from a crystal protein-producing strain of Bacillus thuringiensis a first fragment of DNA comprising an expressible DNA coding for the crystal protein, providing one or more other DNA fragments which contribute(s) an expression mechanism for heterologous DNA in a selected bacterial host other than Bz thuringiensis,- the expression mechanism being recognized by said hose without exhibiting substantial growth phase limitations, and ligating said first DNA fragment and said other fragment(s) in the correct arrangement for expression of the heterologous crystal protein-producing DNA coding in said host. 2. A method as claimed in claim 1, wherein the plasmid comprises a DNA portion having substantial sequence homology to the DNA coding for the crystal protein of B thuring-tensis 3. A method as claimed in claim 1, wherein the plasmid comprises a DNA portion having substantial sequence homology to plasmids of B. thur-ingiensis having a molecular mass greater than 10 x 106 Mr 4. - A method as claimed in claim 3, wherein said DNA portion is identified with a Pvu II-C DNA fragment probe or a probe having substantial sequence homology with a Pvu II-C probe. 5. A method as claimed in claim 4, wherein said DNA portion is derived from B. B thuringiens-is subspecies -tolworthi; subspecies' darmstadiensis; subspecies sotto; subspecies thuringiensis, subspecies thuringien-sis, HD-290, subspecies thuringiensis, HD-120; subspecies thuringiensis, HD-2; subspecies kurstaki,- HD-244; subspecies' kurstaki, HD-73; subspecies kurstaki, HD-1; subspecies- alesti, HD-4; subspecies toumanoffi, F-9; subspecies galleriae, HD-8; subspecies wuhlanesis, F-6; or subspecies morrisoni, F-5. 6. A method as claimed in any one of claims 1 to 5, wherein the plasmid includes a DNA portion having substantial sequence homology to plasmid pBR322 7. A method as claimed in claim 1, wherein the thus-produced plasmid is plasmid pESl (ATCC Number 31995), plasmid pJRK20 (ATCC Number 31997) or plasmid pJWK18 (ATCC Number 31998), said plasmid optionally being mutated but retaining said expressible heterologous DNA coding for the crystal protein of Bacillus thurinsiensis. 8. A method of creating a genetically engineered bacterial strain which is capable of producing the crystal protein of Bacillus thuringiensis comprising transforming a host bacterial strain other than B. thflriflgiensis by introducing thereinto a plasmid which has been produced by a method as claimed in any one of claims 1 to 7 or progeny thereof whereby the expressible genetic material in the host includes heterologous DNA coding for the crystal protein of B. tharingiensis. 9* A method as claimed in claim 8, wherein the host is E. coli. 10. A method as claimed in claim 9, wherein the host is ES12, JWK1 or JWKll. 11. A method of preparing Bacillus thuringi-ensis crystal protein comprising growing a. bacterial strain which has been created by a method as claimed in any one of claims 8 to 10. 12. A method of producing an insecticidal effect in an environment which comprises treating the environment with the crystal protein prepared by a method as claimed in claim 11, said crystal protein optionally being in the form of a formulation including a diluent, carrier or excipient.

Inventors: SCHNEPF, HARRY ERNEST, WHITELEY, HELEN RIABOFF
Applicants: WASHINGTON RES FOUND
Application year: 1982
Main classification: C12N15
IPC codes: C07K14, A01N63