AU1782388A – Coleopteran active microorganisms, related insecticide compositions and methods for their production and use
– Google Patents
AU1782388A – Coleopteran active microorganisms, related insecticide compositions and methods for their production and use
– Google Patents
Coleopteran active microorganisms, related insecticide compositions and methods for their production and use
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Publication number
AU1782388A
AU1782388A
AU17823/88A
AU1782388A
AU1782388A
AU 1782388 A
AU1782388 A
AU 1782388A
AU 17823/88 A
AU17823/88 A
AU 17823/88A
AU 1782388 A
AU1782388 A
AU 1782388A
AU 1782388 A
AU1782388 A
AU 1782388A
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Australia
Prior art keywords
gene
protein
microorganism
plasmid
coleopteran
Prior art date
1987-05-06
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Granted
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AU17823/88A
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AU617110C
(en
AU617110B2
(en
Inventor
William Preston Donovan
Jose Manuel Gonzales Jr.
Barry Lewis Levinson
Anthony Macaluso
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Ecogen Inc
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Ecogen Inc
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1987-05-06
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1988-05-04
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1988-12-06
1988-05-04
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Ecogen Inc
1988-12-06
Publication of AU1782388A
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patent/AU1782388A/en
1991-11-21
Application granted
granted
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1991-11-21
Publication of AU617110B2
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patent/AU617110B2/en
1991-11-28
Assigned to ECOGEN INC.
reassignment
ECOGEN INC.
Amend patent request/document other than specification (104)
Assignors: ECOGEN, INCORPORATED
1992-08-20
Publication of AU617110C
publication
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patent/AU617110C/en
2008-05-04
Anticipated expiration
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Status
Ceased
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Classifications
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
A—HUMAN NECESSITIES
A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
A—HUMAN NECESSITIES
A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
A—HUMAN NECESSITIES
A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
A01N25/30—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests characterised by the surfactants
A—HUMAN NECESSITIES
A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
A01N63/22—Bacillus
A01N63/23—B. thuringiensis
A—HUMAN NECESSITIES
A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
A01N63/50—Isolated enzymes; Isolated proteins
C—CHEMISTRY; METALLURGY
C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N15/09—Recombinant DNA-technology
C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
C12N15/62—DNA sequences coding for fusion proteins
C—CHEMISTRY; METALLURGY
C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N15/09—Recombinant DNA-technology
C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
C12N15/70—Vectors or expression systems specially adapted for E. coli
C—CHEMISTRY; METALLURGY
C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N15/09—Recombinant DNA-technology
C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
Abstract
This invention relates to Bacillus thuringiensis strains that have insecticidal activity against lepidopteran and coleopteran insects, the coleopteran-active endotoxin being produced by an acquired plasmid. This invention also relates to the crystalline protein toxin useful as a biological insecticide against Coleoptera which toxin is produced by the strain of Bacillus thuringiensis. This invention also relates to the expression in various microorganisms of the gene, known as cryC, which codes for this toxin, and for related novel insecticide compositions and methods for their use.
Description
COLEOPTERAN ACTIVE MICROORGANISMS, RELATED INSECTICIDE COMPOSITIONS AND METHODS FOR THEIR PRODUCTION AND USE
1.0 INTRODUCTION This invention relates to biologically pure cultures of Bacillus thuringiensis strains which have insecticidal activity at least against insects of the order Coleoptera. This invention also relates to the crystalline protein toxin which is useful as a biological insecticide against coleopteran insects. The toxin is naturally produced by this strain of Bacillus thuringiensis. This invention also relates to the expression in various microorganisms of the gene, herein referred to as cryC, which codes for the coleopteran active toxin and for related novel insecticide compositions incorporating the toxin itself and microorganisms transformed with the cryC gene.
2.0 BACKGROUND OF THE INVENTION
2.1 COMMERCIAL PESTICIDES: GENERAL CONSIDERATIONS
Each year, significant portions of the world’s commercially important agricultural crops are lost to insects and other pest infestation. The damage wrought by these pests extends to all areas of commercially important plants including foods, textiles, and various domestic plants, and the economic damage runs well into the millions of dollars. Thus, protection of crops from such infestations is of paramount concern. f Broad spectrum pesticides are most commonly used for crop protection, but indiscriminate use of these agents can lead to disruption of the plant’s natural defensive agents. Furthermore, because of their broad
spectrum of activity, the chemical pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. These are also frequently toxic to animals and humans and, thus, pose environmental hazards when applied.
Additionally, insects and other organisms have frequently developed resistance to these pesticides when repeatedly exposed to them. In addition to reducing the utility of the pesticide, resistant strains of minor pests may become major infestation problems due to the reduction of beneficial parasitic organisms.
This is a major problem encountered in using broad spectrum pesticides. What is needed is a biodegradable pesticide that combines a narrower spectrum of activity with the ability to maintain its activity over an extended period of time, i.e., to which resistance develops much more slowly, or not at all. Biopesticides appear to be useful in this regard.
2.2. BIOLOGICAL PESTICIDES
Biopesticides, also called biorationals, make use of naturally occurring pathogens to control insects, fungal, and weed infestations of agricultural crops. Such, substances may comprise a bacterium which produces a substance toxic to the infesting agent (such as a toxin), with or without a bacterial growth medium. Such bacteria, which can be applied directly to the plants by standard methods of application, are typically less harmful to non-target organisms, and to the environment as a whole, in comparison to chemical pesticides.
The use of biological methods of pest control was first suggested in 1895 when a fungal disease was discovered in silkworms. It was not until 1940, however, when spores of the milky disease bacterium Bacillus popilliae were used to control the Japanese beetle, that successful biological pest control was first achieved. The bacterium, named Bacillus thuringiensis (hereinafter referred to alternatively as «B.t. «or «BT»), a bacteria that produces a toxin fatal to caterpillars and other insects, is currently the most widely used biopesticide. In the late 1960,s, the discovery of HD-1, a highly toxic strain of B.t., set the stage for commercial use of biopesticides.
2.3 BACILLUS THURINGIENSIS AND DELTA-ENDOTOXINS
Bacillus thuringiensis is a widely distributed, rod shaped, aerobic, spore-forming microorganism. During its sporulation cycle B.t. forms proteins known as protoxins or delta-endotoxins. These protoxins are deposited in B.t. as parasporal, crystalline inclusions or as part of the spore coat. The pathogenicity of B.t. to a variety of sensitive insects, such as those in the orders Lepidoptera and Diptera, is essentially due to this parasporal crystal, which may represent over 20% of the dry weight of the B.t. cell at the time of sporulation.
The parasporal crystal is active in the insect only after ingestion. For instance, after ingestion by a lepidopteran insect, the alkaline pH and proteolytic enzymes in the mid-gut activate the crystal allowing the release of the toxic components. These toxic components poison the mid-gut cells causing the insect to cease feeding and, eventually, to die. In fact, B.t. has
proven to be an effective and environmentally safe insecticide in dealing with lepidopteran pests.
It has been reported that different strains of B.t. produce serologically different parasporal crystals. However, one of the predominant crystal forms produced by many of the B.t. strains is a form known as P-1. P-1 has a molecular weight of about 130,000-daltons and may also be present in the spore coat. The genes for the parasporal crystal P-1 and those of most of the other protein crystals, have been discovered to reside on any one of a large number of different plasmids of varying size in B.t.
2.4 COLEOPTERAN-ACTIVE Bacillus thuringiensis
The first isolation of coleopteran-toxic B.t. was reported in 1983. (A.Krieg et al. (1983) Z.ang.Ent. 96, 500-508; Ibid. (1984) Anz. Schaedlingskde, Pflanzenschutz, Umweltschutz 57, 145-150) This strain makes a single crystal reported to be comprised of proteins of 68 and 50 kDa (K. Bernhard FEMS Microbiol. Lett. 33, 261-265 (1986). This strain was given the designation Bacillus thuringiensis var. tenebrionis. It was reported that larvae of Lepidoptera and Nematocera were not sensitive to spores and crystals of this strain. A similar strain reported by Mycogen Corp. (San Diego, CA), produces a 64 kDa protein. (C. Herrnstadt et al. Bio/Technology 4, 305-308 (1986)).
2.5 DELTA-ENDOTOXIN GENE CLONING
Since B.t. toxin genes typically reside on plasmids and their products have proven to be effective insecticides which are readily isolated when in
crystalline form or when associated with spore formation, they have been the subject of a great deal of scientific study, particularly with regard to gene isolation and cloning procedures.
The gene which codes for P-1 has been isolated from B.t. subspecies kurstaki strain HD-1-Dipel, and cloned and expressed in E. coli [Schnepf et al., U.S. Patent 4,467,036]. The protein product, P-1, was determined to be toxic to a lepidopteran insect (tobacco hornworm larvae). The nucleotide sequence of the promoter region and part of the coding region of the crystal protein gene for P-1 have also been determined [H.P. Wong et al., The Journal of Biological Chemistry, Vol. 258, No. 3, pp.1960-1967 (1983)]. The entire nucleotide sequence of this gene has also been determined and the delta-endotoxin protein itself has been expressed in a transformed E. coli strain. [M.J. Adang et al., Gene, Vol, 36, pp.298-300 (1985) and PCT application PCT/US85/01665, for: B.t. Crystal Protein Gene Toxin Segment, (1985)].
The genes for other delta-endotoxin forms have also been cloned and expressed in E. coli. Recombinant plasmids containing a mosquitocidal delta-endotoxin gene from B.t. var. israelensis was inserted into an E. coli vector. A 26,000-dalton polypeptide was synthesized by E. coli transformed with this vector. This polypeptide was shown to be lethal to insects in the order Diptera (mosquitos). [E.S. Ward et al., FEBS Vol. 175, 2 , pp.377-382, 1984]. The nucleotide sequence of the gene coding for this crystal protein was also determined along with the resultant protein sequence [C. Waalwijk et al., Nucleic Acids Research, Vol.13, No. 22, pp.8207-8217, (1985)]. Another B.t. var. israelensis gene encoding a
130 KDa crystal protein was cloned and used to transform Bacillus megaterium and Bacillus subtilis. Both B. megaterium and B. subtilis expressed crystalline inclusions during sporulation which inclusions were determined to be toxic to the larvae of Aedes aegypti. [V. Sekar et al., Gene, Vol. 33, pp.151-158, (1985)].
Another delta-endotoxin protein crystal was derived from B.t. subspecies sotto. The gene coding for this crystalline protein was cloned in a vector and then expressed in a transformed E. coli. This gene codes for a 144,000 dalton peptide (934 amino acid residues). The nucleotide sequence for the gene and the amino acid sequence of the corresponding protein (as deduced from the DNA sequence) have been reported. [Y. Shibano et al., Gene, Vol. 34, pp.243-251, (1985)].
It has also been recognized that another major delta-endotoxin protein is produced by several subspecies of B.t. [T. Yamamoto, Biochem. and Biophys. Res. Comm. Vol. 103, No. 2, pp.414-421 (1981); T. Yamamoto et al. Archives of Biochemistry and Biophysics, Vol. 227, No. 1, pp.233-241 (1983)]. This delta-endotoxin has been identified as P-2 and isolated from B.t. var. kurstaki (HD-1). This delta-endotoxin has a molecular weight of approximately 65,000 and is known to be toxic to lepidopteran and dipteran insects. In contrast, P-1 is active only against insects of the order Lepidoptera.
To date, although the rare coleopteran active organisms have been isolated neither the toxin protein nor the gene coding for it have been purified or sequenced. This fact has rendered it impossible to provide a means for expressing this uniquely active delta-endotoxin protein in an organism other than B.t.
The availability of a cloned gene coding for coleopteran-active protein toxin would enable the enhanced production of this protein in heterologous organisms free of other delta-endotoxins.
3.0 SUMMARY OF THE INVENTION
This invention relates to a biologically pure culture of a Bacillus thuringiensis strain which has insecticidal activity against insects of the order Coleoptera. This invention also relates to a coleopteran active delta-endotoxin produced by a strain of Bacillus thuringiensis, the DNA sequence for the gene which codes for this protein and novel insecticides incorporating this protein and/or organisms producing it. More specifically, this invention relates to the cloning and transformation of microorganisms with the cryC gene coding for the coleopteran active delta-endotoxin. In addition, this invention is useful in permitting the transformation of a non-sporulating microorganism with the gene coding for the coleopteran active toxin so that it may be produced during virtually all stages of microorganism growth and, thereby, not be limited to production only during a sporulation stage.
It is, therefore, an object of this invention to provide a biologically pure culture of a Bacillus thuringiensis strain which has insecticidal activity against insects of the order Coleoptera. It is an additional object of this invention to provide a homogeneous coleopteran active protein produced by the isolated gene referred to herein as cryC. This protein may be produced by the process of transforming a microorganism, sporulating or non-sporulating, such as Bacillus megaterium or E. coli or a different strain of
B.t. with the cloned cryC gene. This process, by virtue of selection of the appropriate host and vector, would permit high yield production of the delta-endotoxin such that it is possible to derive a substantially homogeneous preparation of it, i.e. minus any contamination by other varieties of delta-endotoxins. The coleopteran active protein and/or the transformed host may be utilized in a variety of insecticidal compositions.
It is further an object of this invention to provide an organism, other than the native B.t. host, transformed with the cryC gene. This foreign transformed host enables the production of the coleopteran active delta-endotoxin under more desirable and/or selective culturing conditions.
It is an additional object of this invention to provide strains of Bacillus thuringiensis which have a dual activity not found in nature, that is, an insecticidal activity against insects in the orders Lepidoptera and Coleoptera.
It is another object of this invention to provide a DNA probe useful for detecting the presence of the cryC gene in the various Bacillus thuringiensis strains. This DNA probe also enables the screening of various strains of B.t. for the possible presence of related genes coding for proteins sharing a common homology with the coleopteran active protein and the isolation of these related genes. It is a further object of this invention to provide a method for controlling insects of the order Coleoptera with coleopteran active
Bacillus thuringiensis or organisms transformed with the cryC gene, which renders that strain active against
Coleoptera.
It is also an object of this invention to provide a method for controlling insects in both the orders Lepidoptera and Coleoptera with transconjugant Bacillus thuringiensis strains which are active against both types of insects, strains which are unknown in the wild. All of the above embodiments of this invention will be described in greater detail in the description of the invention which follows.
4.0 BRIEF DESCRIPTION OF THE FIGURES
FIGURE 1 shows a comparison of the crystal types produced by strain EG2158 and their appearance within the microorganism.
FIGURE 2 is a photograph of a gel electrophoresis showing the respective plasmid arrays of HD-1 and EG2158.
FIGURE 3 is a photograph of a gel electrophoresis showing the respective plasmid arrays of transconjugants harboring coleopteran and lepidopteran active toxin plasmids.
FIGURE 4 is a photograph of a gel electrophoresis showing a comparison of the crystalline proteins from EG2158 to other strains producing the F-1 crystal.
FIGURE 5 is comprised of 5(A), 5 (A’) and 5(B’). 5(A) is a photograph of a gel electrophoresis of the R-1 and F-1 crystal proteins. 5 (A’) and 5(B’) are also photographs of electrophoresis gels which show the differential production of 77 and 71 kDa proteins in EG2158 and derivatives of EG2158.
FIGURE 6 is comprised of 6(A) and 6(B), both of which are photographs of a gel electrophoresis showing the productions of the 71 kDa protein in transconjugant strains having the 88-Md plasmid from EG2158.
FIGURE 7 is a restriction map of the recombinant plasmids pEG212 and pEG213 that contain the cloned cryC gene. The location and direction of transcription of the cryC gene are indicated by the large arrow.
FIGURE 8 shows the DNA sequence of the cryC gene (including nucleotides 569 to 2500 which code for the structural toxin protein and nucleotides 2501-2503 code for the «stop» signal) and also the amino acid sequence of the coleopteran toxin encoded by the cryC gene (nucleotides 569-2500).
FIGURE 9 is comprised of 9a and 9b. 9a is a photograph of an ethidium bromide stained Eσkhardt gel. The native plasmids that are present in Bacillus thuringiensis strains HD1 and EG2158 are visible illustrating that certain B.t. strains contain several native plasmids. 9b is a photograph of an autoradiogram that was made by hybridizing the radioactively-labeled cloned cryC gene with the plasmids shown in 9a. 9b illustrates that the cloned cryC gene hybridized exclusively to a plasmid of 88 MDa in the coleopterantoxin strain EG2158 but failed to hybridize to any plasmids in strain HD1, a strain that is not toxic to coleopterans.
FIGURE 10 is a photograph of an SOS/ polyacrylamide gel which shows that a recombinant host strain of Bacillus megaterium (EG1314) harboring the
cloned cryC gene synthesizes large quantities of a protein having a size similar to that of authentic coleopteran (cry) toxin.
5.0 DESCRIPTION OF THE INVENTION
Generally stated, the present invention provides a newly isolated Bacillus thuringiensis strain which has insecticidal activity against insects of the order Coleoptera. A biologically pure culture of this strain has been deposited with the NRRL. Bioassays described below have confirmed the coleopteran activity of this strain. This strain of B.t., therefore, is preferred for use as at least one of the active ingredients in an insecticide composition useful against coleopteran insects.
The present invention further provides for transconjugant derived Bacillus thuringiensis strains which have insecticidal activity against both lepidopteran and coleopteran insects. This dual activity in B.t. is unknown in the wild. A B.t. strain having this dual activity would also, therefore, be preferred for use as at least one of the active ingredients in an insecticide composition useful against both coleopteran and lepidopteran insects.
Additionally, this invention provides, generally stated, a method for producing Bacillus thuringiensis strains having insecticidal activity against both coleopteran and lepidopteran insects comprising:
(a) providing a Bacillus thuringiensis strain having insecticidal activity against coleopteran insects
conferred by a gene coding for coleopteran active toxin protein said gene being located on a plasmid said strain being in admixture with a Bacillus thuringiensis strain having insecticidal activity against lepidopteran insects under conditions favoring conjugation and
(b) isolating from the culture admixture of step (a) a transconjugant having activity against both lepidopteran and coleopteran insects.
This method, in a preferred embodiment, also utilizes intermediate strains to transfer either the coleopteran or lepidopteran toxin-coding plasmid to another intermediate recipient strain or directly to the ultimately desired transconjugant host which already would contain at least one other of the toxin encoding plasmids.
The general method described above also encompasses the embodiment wherein said Bacillus thuringiensis strain of step (a) having activity against coleopteran insects additionally has activity against lepidopteran insects conferred by at least one gene coding for a lepidoperan-active toxin, whereby said tansconjugant of step (b) has lepidopteran and coleopteran activity conferred by at least three toxin genes.
The general method described above additionally encompasses the embodiment wherein said Bacillus thuringiensis strain of step (a) has activity against lepidopteran insects conferred by more than one toxin gene, whereby said transconjugant of step (b) has lepidopteran and coleopteran activity conferred by at least three toxin genes.
For instance, in the practice of this invention a strain having coleopteran activity would be provided in admixture first with a Bacillus thuringiensis strain whereby said Bacillus thuringiensis strain acquires (by conjugation) the plasmid conferring insecticidal activity against Coleoptera and then providing the transconjugant strain in admixture with said Bacillus thuringiensis having lepidopteran activity under conditions favoring conjugation whereby said Bacillus thuringiensis strain having lepidopteran activity acquires the plasmid conferring coleopteran activity by conjugation from said transconjugant strain.
The present invention also provides for a cloned gene coding for Bacillus thuringiensis coleopteran active toxin comprising the DNA nucleotide sequence shown in FIG. 8. This gene (which comprises double stranded DNA wherein the nucleotide strands have a complementary base sequence to each other) codes for a protein (or as also used herein equivalently, polypeptide) having the amino acid sequence of the coleopteran active toxin which amino acid sequence is shown in FIG. 8. The coleopteran active toxin encoded by the cloned gene has insecticidal activity against coleopteran insects.
Methods of producing the coleopteran active protein are also provided by this invention. In this method of production the cryC gene is inserted into a cloning vector or plasmid which plasmid is then utilized to transform a selected microorganism.
The cloning vectors, as described herein, are generally known in the art and are commercially available. The choice of a particular plasmid is within the skill of the art and would be a matter of personal
choice. Plasmids suitable for use in this invention are, for instance, pBR322, plasmids derived from B.t., and plasmids derived from Bacillus and Staphylococcus microorganisms, preferrably. Bacillus megaterium. Microorganisms suitable for use with this invention are both εporulating and non-sporulating microorganisms such as E. coli, B.t., and Bacillus megaterium. The microorganisms utilized are also known in the art and are generally available. The choice of any particular microorganism for use in the practice of this invention is also a matter of individual preference. In a preferred embodiment of this invention the microorganism would comprise Bacillus megaterium.
Generally stated, the coleopteran active toxin protein can be produced by a transformed organism and later purified into a homogenous preparation having an amino acid sequence as shown in FIG. 8. More specifically, this protein may be produced by transforming a microorganism with a plasmid containing the cryC gene, growing the transformed microorganism so that the protein coded for by the cryC gene is expressed in the microorganism and by extracting the protein from the organism with standard protein purification techniques. It is also within the scope of this invention that the protein not be separated from the transformed microorganism but that this organism, including the expressed coleopteran active protein, be utilized as or in an insecticidal composition.
This invention also provides for a novel insecticide for use against Coleoptera comprising a mixture of B.t. coleopteran active toxin and a suitable carrier. The toxin may be contained in the organism or associated with spores, or be a homogeneous protein
preparation or in a mixture of spores with cultured transformed organisms. The toxin may also be contained in a non-sporulating microorganism or a sporulating microorganism such as Bacillus megaterium or B.t. A suitable carrier may be any one of a number of solids or liquids known to those of skill in the art.
This invention also comprises the recombinant vectors or plasmids including the cryC gene and the particular microorganisms which have been transformed with this gene. In addition, this invention also provides for oligonucleotide probes for the gene coding for the coleopteran active delta-endotoxin. All of these aspects of the inventions are described in detail below and illustrated in the following examples.
5.1 COLEOPTERAN ACTIVE Bacillus thuringiensis
EG2158 is a B.t. strain isolated (deposited and maintained as a biologically pure culture) from soybean grain dust from Kansas. EG2158 produces two types of intracellular inclusion during sporulation (FIG. 1): A somewhat rhomboid crystal (referred to below as R1) and a flat, diamond-shaped crystal (referred to below as F1). Bioassays set forth in the Examples below show that sporulated cultures of EG2158 (consisting of a mixture for spores, R1 and F1 crystals) were toxic to larvae of the Colorado potato beetle (hereinafter alternatively referred to as CPB.), Leptinotarsa decemlineata (Say), but not toxic to lepidopteran larvae of several species (Trichoplusia ni and others).
EG2158 contains a unique plasmid array (FIG. 2) of 5 plasmids of approximate sizes of 35, 72, 88, 105 and 150 megadaltons (Md).
Table I below describes which plasmid codes for a particular toxin.
TABLE I
STRAIN EG2158 COLEOPTERAN ACTIVITY
IS ENCODED BY A TRANSMISSIBLE PLASMID
TOXIN PLASMID PROPERTIES
150 Md Encodes «flat diamond» crystal.
Loss has no effect on coleopteran activity.
88Md Encodes rhomboid crystal and coleopteran activity.
Transfers into B.t. and B. cereus recipient strains. Transconjugant made rhomboid crystal and is toxic to CPB larvae.
Loss of the 150 Md plasmid eliminated production of F1 crystal without affecting toxicity to CPB, while loss of the 35-Md plasmid had no effect on R1 or F1 production or toxicity. (Table II)
(Strains of EG2158 and its variants, and all B.T. and B. Cereus strains were grown for bioassay as follows: spores were inoculated into 5 mis of M27 broth
in a 50 ml sterile flask. M27 broth is composed of 33 mM each of HPO4= and H2PO4- anions; 98 mM K+; 0.17% peptone; 0.1% beef extact; 150mM NaCl; 5.5 mM glucose; 330 uM Mg++, 230 uM ca++, and 17 uM Mn++ (added as the chloride salts). (As used herein, the letter «u» when used as part of a term of measurement or quantity is synonomous with the prefix «micro».) The cultures were incubated at 30ºC with shaking for 3 days, at which time sporulation and crystal formation were complete. Five ul of sterile 1-octanol were added as an anti-foaming agent and the cultures were transferred to sterile plastic tubes, sealed, and stored at 5ºC.)
TABLE II
Mortality and leaf consumption by first instar Colorado Potato Beetle larvae on potato leaf discs treated with BT
Number Alive Approximate /10 at Leaf
Strain 24 h 48 h Consumption %
(Control) 10 10 90
1 EG2158 10 2 10
2 EG2158 (-150 Md; 10 1 15 -flat diamond (F-1))
When EG2158 was grown in mixed culture with other strains of B.t. or B. cereus, the 105- and 88-Md plasmids were transmitted, by conjugation, into the other strains. B.t. or B. cereus stains which acquired the 105-Md plasmid were not altered detectably (that is, the 105-Md plasmid is transmissible but otherwise cryptic). B.t. or B. cereus strains which acquired the 88-Md plasmid were seen to produce R1 crystals. Therefore, it was discovered that the 88-Md plasmid is transmissible and encodes R1 crystals, and yields transconjugant strains which are R1 producers. The plasmid arrays of some R1-producing transconjugants are shown in FIG. 3.
TABLE III
TRANSCONJUGANTS HARBORING COLEOPTERAN AND LEPIDOPTERAN-ACTIVE TOXIN PLASMIDS
TRANSCONJUGANT TOXIN PLASMID TOXIN PLASMID
(Source strain) TARGET INSECTS
HD73-26-46 88 (EG2158) CPB
HD73-26-50 88 (EG2158) CPB
44 (HD-263) LEP
HD73-26-54 88 (EG2158) CPB
61 (HD-617) LEP
HD73-26-56 88 (EG2158) CPB
50 (HD-78) LEP
54 (HD-2) LEP
75 (HD-2) LEP
BC569-6-15 88 (EG2158) CPB
68 (HD-536) UNK
HD1-10-1 88 (EG2158) CPB
HD263-8-5 88 (EG2158) CPB
60 (HD-263) LEP
The 88-Md plasmid was put into recipients of three B.t. backgrounds (HD-73, HD-1, and HD-263) and one of B. cereus origin (BC-569). The 88-Md plasmid was shown to coexist with toxin plasmids encoding lepidopteran (P1) toxin crystals, such as the 44-Md toxin plasmid from HD-263, and others (See FIG. 3 and Table III). Transconjugants producing R1 crystals were toxic to CPB, as was EG2158 (Table IV, A and B) and were also toxic to lepidopteran larvae (Table V, A and B).
TABLE IV
A. MORTALITY AND LEAF CONSUMPTION BY FIRST INSTAR COLORADO POTATO BEETLE LARVAE ON POTATO LEAF DISCS TREATED WITH Bacillus thuring iensis
Approx. Leaf Strain Phenotype Number Alive/10 at 24h 48h Consumption (%)
(Control) 10 9 80 HD73-26-46 Rhαnboid+: 88+
