DNA Constructs Encoding Bacillus Thuringiensis Toxins From Strain A20

This invention relates to recombinant DNA, and in particular to such DNA coding Bacillus thuringiensis endotoxin.

The organism Bacillus thuringiensis carries genes which encode protein endotoxins which are insecticidal to a variety of agronomically important insects. They are not however harmful to many benign insects, or to earthworms, birds, fish or mammals. These endotoxins are thus useful as agricultural insecticides, particularly against Lepidopteran pests. Bacillus thuringiensis strains have been used as agricultural insecticides for many years.

Recently, Bacillus thuringiensis endotoxin genes have been engineered into dicotyledonous plants and shown to confer protection against insect attack. The Bacillus thuringiensis kurstaki strain, A20, is the subject of our prior unpublished copending UK application No 8730132 filed 24 Dec. 1987. Strain A20 has been deposited at the National Collection of Industrial and Marine Bacteria (NCIMB) at Aberdeen, Scotland on 20 Oct. 1987 under the NCIMB Accession Number 12570. It is more active than the commonly used variety kurstaki strain, HD-1. Strain A20 carries three different, but highly related, endotoxin genes similar to those described in the scientific literature as 6.6-, 5.3-, and 4.5-type genes. The first two genes are carried in one or more copies on plasmids and also on the bacterial chromosome, while the 4.5- type gene is only found on the bacterial chromosome.

According to the present invention we provide recombinant DNA coding for an insecticidally active form of the Bacillus thuringiensis endotoxin which DNA is derived from the A20 strain of Bacillus thuringiensis. The source of the DNA maybe either the chromosome or a plasmid.

In a further aspect, our invention comprises recombinant DNA coding for an insecticidally-active form of the Bacillus thuringiensis endotoxin which is derived from a 6.6-type endotoxin gene carried on a plasmid harboured by strain A20. The maximum molecular weight of such endotoxins is about 103,000 Dalton.

We have made such recombinant DNA comprising the first 2910 basepairs (970 amino acid codons) of the N-terminal coding region of a plasmid-derived 6.6-type endotoxin gene from Strain A20. The 6.6-type construct we have made encodes a fusion protein that includes 28 amino acid codons derived from the pUC19 vector DNA. The Bacillus thuringiensis-derived portion of the recombinant DNA has 10 basepair changes as compared with the analogous plasmid-derived 6.6-type gene from Bacillus thuringiensis strain HD 73 (Adang et al, Gene 36, 1985, 289-300). Surprisingly, all of the base changes occur within the generally conserved N-terminal portion of the gene previously designated as a "highly-conserved" region (Whiteley and Schnepf, Ann. Rev. Microbiol., 40, 1986, pp549-576). Nine of the ten changes are clustered in a 0.35 kilobase segment which does not overlap with any of the 5 regions shown to be conserved in all types of endotoxin genes analysed to date (Sanchis et al., Molecular Microbiol., 3, 1989, pp229- 238). Four of the 10 base changes result in amino acid substitutions.

The invention further comprises recombinant DNA coding for an insecticidally-active form of the Bacillus thuringiensis endotoxin which is derived from a 5.3-type endotoxin gene carried on a plasmid harboured by strain A20. The maximum molecular weight of the endotoxin is about 78,000 Daltons.

We have made such recombinant DNA comprising the first 2161 basepairs (696 amino acid codons) of the N-terminal coding region of a plasmid-derived 5.3-type endotoxin gene from Strain A20. This endotoxin gene differs from the analogous plasmid-derived 5.3-type gene from Bacillus thuringiensis strain HD-1 (Geiser etal., Gene 48, 1986, pp109-118) in that a deoxyguanosine base present in the published sequence at nucleotide 2024 is not present, resulting in a reading frame shift relative to the published structural gene. This frame shift results in a termination of the endotoxin gene product at amino acid residue 696, within the Bacillus thuringiensis-derived portion of the recombinant DNA. The recombinant DNA, however, encodes an insecticidally-active 5.3 type endotoxin protein.

In a further aspect, our invention comprises recombinant DNA coding for an insecticidally-active Bacillus thuringiensis endotoxin which is a chimera derived from sequences from at least two separate Bacillus thuringiensis genes. The molecular weight of the chimera may be of the order of 110,000 Daltons. Preferably the link or links between the sequences fall outside the hypervariable regions of such genes. In a more specific aspect, our invention comprises recombinant DNA coding for an insecticidally-active form of the Bacillus thuringiensis endotoxin comprising the first 1692 basepairs (564 amino acid codons) of the amino-terminal coding region from a 5.3-type endotoxin gene derived from Strain A20 and a restriction endonuclease Hind III-generated internal fragment of 1131 basepairs (377 amino acid codons) from a 4.5-type endotoxin gene derived from strain A20.

This chimetic gene encodes a novel fusion protein which also includes 28 amino acid codons derived from the pUC19 vector DNA. The Bacillus thuringiensis portion of the recombinant DNA has 97.1% DNA sequence homology with the 5.3-type gene from which it was largely derived. The internal Hind III-generated fragment used in the chimera and derived from the 4.5-type gene only differs from the analogous Hind III fragment from the 5.3-type gene by a 78 basepair insertion and 7 other physically separate base changes. These differences result in the in-frame addition of 26 amino acids due to the insertion, and in seven other amino acid changes; thus the novel protein has 91% amino acid homology with the 5.3-type gene product.

Recombinant genes according to our invention encoding insecticidally-active endotoxins may be of varying lengths. When cloning DNA from the A20 strain chromosome, it is convenient to use bacteriophage .lambda. vectors or other cloning vectors that sequester the recombinant DNA from host cell enzymes that might cause homologous recombination.

Three Escherichia coli strains containing the cloned 6.6- and 5.3-type endotoxin genes from A20 and the chimetic endotoxin gene have been deposited at the National Collection of Industrial and Marine Bacteria at Aberdeen, Scotland. These are:

The first two deposits were made on 23 Sep. 1988 and the third on 15 Sep. 1989.

The invention may be further understood by reference to the drawings, in which:

FIGS. 1A, 1B and 1C show diagramatically the structure of 6.6, 5.3 and chimetic-type genes, respectively as cloned;

FIG. 2 illustrates schematically the construction of pJH1 (=pIC49), pJH2(=pIC47) from publicly available vector pUC19 and DNA obtained from A20 (NCIMB 12570);

FIGS. 3A1, 3A2, 3A3, 3B1, 3B2, 3B3, 4A1, 4A2, 4A3, 4B1, 4B2, 4B3, 5A1, 5A2, 5A3, 5B1, 5B2 and 5B3 show the base sequence, the amino acid sequence and the main restriction sites of the pJH10 endotoxin gene;

FIGS. 6A1, 6A2, 6A3, 6B1, 6B2, 6B3, 7A1, 7A2, 7A3, 7B1, 7B2, 7B3, 8A1, 8A2, 8A3, 8B1 and 8B2 show the base sequence, the amino acid sequence and the main restriction sites of the pJH1 endotoxin gene;

FIGS. 9A1, 9A2, 9B1, 9B2, 10A1, 10A2, 10B1, 10B2, 11A1, 11A2, 11B1, 11B2, 12A1 and 12A2 show the base sequence, the amino acid sequence and the main restriction sites of the chimetic pJH2 endotoxin gene;

FIG. 13 shows the characteristics of the pJH10 endotoxin gene product;

FIG. 14 shows the characteristics of the pJH1 endotoxin gene product;

FIG. 15 shows the characteristics of the pJH2 endotoxin gene product.

With further reference to FIG. 1, in this diagram H represents HindIII, D=DraI, X=XmnI, C= ClaI, E=EcoR1, R=EcoRV, P=Ps+I, B=BclI and K =KpnI. A dot shows a base change, while x indicates a base change and amino acid substitution in the 6.6-type gene of the invention as compared with the 6.6-type gene of strain HD73. The line under the 6.6-type gene indicates the 2.2 kilobase DraI fragment sequenced, while the line under the 5.3-type gene indicates the approximate length of the coding sequence. The heavy broken line indicates vector DNA. In FIG. 2, H is again HindIII, while N represents NdeI.

With reference to FIGS. 5A1, 5A2, 5A3, 5B1, 5B2 and 5B3, it should be noted that the sequence from nucleotide 2228 to nucleotide 2907 (internal DraI to HindIII) has been taken from the published literature. All other sequence data given for constructs of the inventions has been obtained independently by us.

In Heliothis virescens bioassays the chimetic gene product is of comparable potency to that of the 5.3-type gene over the normal bioassay time-course of six days.

The preferred method of producing recombinant DNA according to the invention is by culturing a sample of deposited strains NCIMB 40049, 40050 or 40211, obtainable from the National Collection of Industrial and Marine Bacteria, under suitable conditions in appropriate media. Such conditions and media are well-known to the art. Variant forms of DNA according to the invention may be made by cloning suitable DNA sequences from bacteria of strain A20 (also deposited at the NCIMB, under No 12570).

The invention further comprises insecticidal compositions comprising Bacillus thuringiensis endotoxin produced by expression of recombinant DNA according to the invention: as well as a process of combating insects which comprises exposing them to effective amounts of such endotoxin.

Insecticidal compositions according to the invention may be obtained from cultured cell suspensions by appropriate lysis techniques. Preparations of lysed cells (i.e. cell extracts) may be used directly, or concentrated by lyophilisation, followed by reconstitution.

The process of the invention is generally carried out by incorporation of the nonviable cell extracts into, or onto, the insect food source. Alternatively the novel insecticidal gene can be introduced into the genome of food plants normally attacked by Lepidopteran pests. For this purpose suitable plant regulatory DNA sequences are inserted adjacent the recombinant DNA in operative relation therewith so as to allow expression of the endotoxin in the plant. Specific examples of commercially important plants to be protected in this manner are maize, cotton, and tobacco. Various plant transformation methods are known to the art, for example the use of Ti plasmid vectors (for dicots) protoplast regeneration, embryo microinjection and microprojectiles.

Insects which are combated by the invention include various Lepidopteran pests, for example, those in Table 1.

The following Examples illustrate the invention.

Cloning of the plasmid-derived 6.6-type endotoxin gene carried on pJH10 and of chromosomally-derived endotoxin genes.

Covalently-closed circular (ccc) plasmid DNA was prepared from Bacillus thuringiensis Strain A20 (NCIMB 12570) by techniques well-known in the art. Large molecular weight (greater than 40 kilobases) plasmid ccc DNA was isolated by size fractionation on 10%-40% sucrose step gradients prior to digestion with restriction endonuclease Hind III and ligation into the Hind III--digested plasmid vector pUC19. The ligation reacted mixture was transformed into E. coli Strain MC1022, an ampicillin-sensitive strain with the genotype ara D139, .DELTA.(ara, leu) 7697, .DELTA.(lac Z) M15, gal U, gal K, str A. Amipicillin-resistant transformed colonies containing the 6.6 kilobase Hind III fragment were detected by hybridisation of lysed colonies fixed to nitrocellulose using a radioactively-labelled fragment of the Strain HD73 6.6-type endotoxin gene as a probe. Isolate pJH10 was further characterised by DNA hybridisation, restriction endonuclease mapping, and DNA sequence analysis techniques, all of which are well known in the art.

Endotoxin genes were cloned from chromosomal DNA prepared from Strain A20 (NCIMB 12570) as follows:

A 500 ml culture of Strain A20 was grown in L-broth at 37.degree. C., with shaking, until an O.D..sub.600 =1.00. Cells were harvested by centrifugation at 8000 rounds per minute (rpm) for 10 minutes at 4.degree. C., then re-suspended in 5 ml TES buffer (50 mM TriS-HCl pH7.5, 50mM NaCl, 5 mM EDTA). Cells were treated for 30 minutes at 37.degree. C. with lysozyme (0.5 mg/ml final concentration) and RNase (0.1 mg/ml final concentration taken from a stock solution of 5 mg/ml boiled at 100.degree. C for 5 minutes prior to use). Lysis was completed by the addition of Sarcosyl to give a final concentration of 0.8% and incubation at 37.degree. C. for 60 minutes in the presence of Pronase (0.5mg/ml final concentration taken from a stock solution of 5 mg/ml pre-incubated at 37.degree. C. for 60 minutes prior to use). Lysate volume was adjusted to 9.0 ml in the 50 mM Tris-HCl pH 7.6, 10 mM EDTA, prior to the addition of 9.2 g caesium chloride (CsCl). After the CsCl dissolved, 1.25 ml of a 5 mg/ml solution of ethidium bromide was added prior to isopyonic centrifugation of the mixture at 40,000 rpm for 48 hours at 15.degree. C.

After removal of CsCl and ethidium bromide by conventional techniques, an aliquot of purified chromosomal DNA was partially digested with the restriction endonuclease EcoR1 prior to ligation into EcoR1-digested bacteriophage .lambda. EMBL4 vector DNA. Ligation reaction mixtures were packaged into viable phage particles using a commercially-available kit from Amersham International PLC. The resultant recombinant phage particles were selected by growth on E. coli host strain PE392, a P2 lysogen of strain LE392 which has the genotype hsd R514 (r.sub.k.sup.-, M.sub.k.sup.+), sup E44, SUP F58, lacYl or .DELTA.(lac12Y), gal K2, gal T22, met B1, trp55. Recombinant phage carrying one or more endotoxin genes were detected by hybridisation of lysed plaques fixed to nitrocellulose using a radiolabelled fragment of the Strain A20 6.6-type plasmid-derived endotoxin gene as a probe.

Plaques containing endotoxin genes were purified and characterised by restriction endonuclease mapping techniques well known in the art.

Construction of plasmid pJH2 containing the chimeric endotoxin gene.

DNA was prepared from plasmids pIC41 and pIC45, the derivation of which is shown in FIG. 2. pIC41 DNA was digested with restriction endonuclease HindIII and joined to the isolated, internal 1131 basepair HindIII-generated fragment from plasmid pIC45 using T4 DNA ligase. Methods for DNA preparation, DNA fragment purification and in vitro ligation reactions are all well known to the art. The ligation reaction mixture was introduced by transformation into E. coli strain MC1022, an F.sup.- strain with the genotype ara D139, .DELTA.(ara, leu) 7697, .DELTA.(lacZ) M15, galU, gal, str A. Ampicillin-resistant transformed colonies containing the 1131 basepair fragment were detected by hybridisation of lysed colonies fixed to nitrocellulose filters using the radioactively labelled 1131 basepair fragment as a probe. DNA samples from colonies reacting to the probe were checked for the presence of the introduced 1131 basepair fragment in the correct orientation. Transformation of E.coli cells made competent by treatment with calcium chloride, colony blotting, nick-translation of DNA to prepare radioactive probes, DNA hybridisation techniques, and restriction endonuclease "mapping" techniques are all well known in the art.

pJH1 was made in a similar manner from pIC34 and pIC41, as shown in FIG. 2.

Preparation of insecticidal extracts according to the invention.

E.coli strain MC1022/pIC47 was grown in 400 ml Luria-broth supplemented with 50 .mu.g Ampicillin per ml overnight at 37 C. with shaking. Cells were harvested by centrifugation at 8000 rpm for 10 minutes at 4 C. and resuspended in 26.4 ml lysing buffer (150 mM NaCl, 20 mM Tris pH 8, 5 mM EDTA, 7 mM .beta.-mercaptoethanol). The resuspended cells were placed on ice and then 0.3 ml 0.1M phenylmethylsulphonylfluoride (PMSF) was added, followed by 3 ml of a freshly prepared solution of 10 mg lysozyme per ml, followed by a further addition of 0.3 ml PMSF. The mixture was left for 30 minutes on ice. The cell suspension was subjected to sonic disruption by treatment for 45 seconds with a one centimeter diameter sonicator probe operated at a frequency of 12 microns on an MSE Soniprep 150 sonifier. The sonicated sample was then centrifuged at 11000 rpm for 20 minutes at 4.degree. C. (using 50 cc capacity sterile centrifuge tubes). The supernatant was recovered by pouring off into a clean, sterile plastic container then dialyzed overnight at 4.degree. C. in 20 mM phosphate buffer (mono- and di-basic) pH 7.2. Insecticidal extracts can be stored frozen, or lyophilised and stored frozen.

Efficacy of insecticidal extracts prepared from E.coli strains MC1022/pJH1 and MC1022/pJH2 in controlling Heliothis virescens and MC1022/pJH10 in controlling Heliothis virescens and Ostrinia nubilalis.

Insecticidal extract was added to molten insect diet (e.g. standard Heliothis diet) to give a final concentration of, for example, 1 ml per 10 g of diet (equivalent to approximately 510 ppm freeze-dried extract per treatment). 2.5 ml of treated diet was added to each of 10 plastic pots, covered and allowed to solidify. One first-instar larva was added to each pot and incubated at 27.degree. C. for six days, at which time the results were recorded. In tests with lower treatment concentrations (e.g. 0.1 ml extract per 10 g of diet), surviving larvae at six days were left for longer periods of observation to allow documentation of altered stability. Results of typical experiments are shown in Tables 2 and 3.

Efficacy of insecticidal extracts prepared from strains MC1022/pJH2 and pJH1 on Lepidopteran larvae.

Lyophilised extracts from E.coli strains MC1022/pJB2 and MC1022/pdH1 were reconstituted in distilled water to give a stock solution which was then added, in varying amounts, to molten diet as described above in Example 4. Results of a typical bioassay experiment on Plutella xylostella (Diamondback moth, DBM), Heliothis zea (Corn earworm, CEW) and Trichoplusia ni (Cabbage looper, CL) are given in Table 4.

The work described herein was all done in conformity with physical and biological containment requirements specified in the NIH and GMAC guidelines.

The specification refers to four microorganisms that have been deposited. These are shown below. All four were deposited at the National Collections of Industrial and Marine Bacterial Limited (NCIMB) of PO BOX 31, 135 Abbey Road, Aberdeen, AB9 8DG, Scotland.

The Bacillus thuringiensis strain A20 is fully described in our European Patent Publication no 325037, the disclosure of which is incorporated herein by reference. The three E. coli strains are of well known type, differing only in the plasmids they carry, full details of which are given elsewhere in the present specification.