Pesticidal proteins and strains

DNA MOLECULES ENCODING INSECTICIDAL PROTEINS SECRETED FROM VEGETATIVE GROWTH PHASE OF Bacillus spp., USES THEREOF AND TRANSGENIC ORGANISMS COMPRISING THE SAME

The present invention is drawn to methods and compositions for controlling plant and non-plant pests. Particularly, new pesticidal proteins are disclosed which are isolatable from the vegetative growth stage of Bacillus. Bacillus strains, proteins, and genes encoding the proteins are provided. The methods and compositions of the invention may be used in a variety of systems for controlling plant and non-plant pests.

Insect pests are a major factor in the loss of the world's commercially important agricultural crops. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. There is, however, substantial interest in developing effective alternative pesticides.

Microbial pesticides have played an important role as alternatives to chemical pest control. The most extensively used microbial product is based on the bacterium Bacillus thuringiensis (Bt). Bt is a gram-positive spore forming Bacillus which produces an insecticidal crystal protein. (ICP) during sporulation.

Numerous varieties of Bt are known that produce more than 25 different but related ICP's. The majority of ICP's made by Bt are toxic to larvae of certain insects in the orders Lepidoptera, Diptera and Coleoptera. In general, when an ICP is ingested by a susceptible insect the crystal is solubilized and transformed into a toxic moiety by the insect gut proteases. None of the ICP's active against coleopteran larvae such as Colorado potato beetle (Leptinotarsa decemlineata) or Yellow mealworm (Tenebrio molitor) have demonstrated significant effects on members of the genus Diabrotica particularly Diabrotica virgifera virgifera^be western corn rootworm (WCRW) or Diabrotica longicornis barbori, the northern corn rootworm.

Bacillus cereus (Be) is closely related to Bt A major distinguishing characteristic is the absence of a parasporal crystal in Be. Be is a widely distributed bacterium that is commonly found in soil and has been isolated from a variety of foods and drugs. The organism has been implicated in the spoilage of food.

Although Bt has been very useful in controlling insect pests, there is a need to expand the number of potential biological control agents.

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Shivakumar et al. [(1986) Plasmid, Vol. 16, No. 3, p.230] describe the cloning and characterization of two crystal protein genes expressed in B. subtilis during vegetative growth.

Walther et al. [(1986) Applied and Environmental Microbiology, Vol. 5, No. 4, pp.650-653] describe the induction of 100% mortality of the mosquito Aedes aegypti larvae upon infection by Bacillus thuringiensis subsp. israelensis. The larvicidal potential was localized within the cells, and was likely due to a heat-labile hemolysin which was immunologically distinct from the general cytolytic factor released during solubilization of Bacillus thuringiensis subsp. israelensis (Bti) crystals.

Ward et al. [(1986) J. Mol. Biol. Vol. 191, pp.13-22] cloned and characterized the pattern of expression of ®-endotoxin in sporogenic and asporogenic strains of Bacillus thuringiensis var. israelensis. The ®-endotoxin gene is expressed at a low level during vegetative growth in all three strains, but the synthesis of the toxin increases markedly during the third hour of stationary phase for both the sporogenic strain and an asporogenic mutant containing the OJ lesion.

Sekar V. [(1988) Current Microbiology, Vol. 17, pp.347-349] describe the expression of ICP transcript and the corresponding crystal antigen of the coleopteran-toxic Bacillus thuringiensis var. tenebrionis at detectable levels in vegetative cells. The expression of the ICP gene increases dramatically and reaches a plateau in early stationary cells.

WO 90/13651 describes the characterization of novel strains of B. thuringiensis, like JHCC 4835 and JHCC 4353, and their efficacy in the control of various pests. In particular a gene coding for a novel insecticidal endotoxin, of 81 kDa, was \ characterized from the recombinant strain MC1022/pJHll. This novel endotoxin is toxic to both Lepidoptera and Coleoptera.

Thanabalu et al. [(1992) Journal of Bacteriology, vol. 174, no. 15, pp.5051-5056] describe the cloning of a gene encoding a lOOkDa protein, designated mtx (mosquitocidal toxin) gene, from Bacillus sphaericusi SSII-1, and characterization of the Mtx protein. This study was followed by another publication by the same authors [Thanabalu et al. (1993) Journal of Bacteriology, vol. 175, no. 8, pp.23142320], where the Mtx protein was further characterized, and specific roles were assigned to trypsin-derived 27 kDa and 70 kDa Mtx peptides. The N-terminal 27 kDa peptide has homology with ADP-ribosyltransferase toxins, whereas the Cterminal 70 kDa peptide seems to cause morphological changes to certain mosquito cells in culture.

Yoshisue et al. [(1992) Biosci. Biotech. Biochem, Vol. 56(9), pp. 14291433־] investigated the effect of a 20-kDa protein of Bti, which is necessary for the efficient lb expression of the 27-kDa mosquitocidal protein in E. coll, when supplied in trans. A significant increase in the production of the fused CryIVA protein, supplied by a recombinant plasmid, was observed when in the presence of the 20-kDa protein gene.

Koziel et al. [(1993) Biotechnology, V01.ll, pp. 194-200] describe the generation of elite transgenic maize plants expressing a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis. Plants expressing high levels of the insecticidal protein exhibited outstanding resistance to repeated heavy infestations of the European corn borer.

In US 5,262,158, novel isolates of Bacillus thuringiensis are described, which are active against acaride pests. The genes encoding novel toxins from Bacillus thuringiensis strains PS52A1 and PS69D1 were cloned and characterized. In addition, Bacillus thuringiensis isolates were tested for their activity against mites.

WO 94/21795 describes the characterization of pesticidal proteins and auxiliary proteins of Bacillus strains which are produced during vegetative growth. In particular, this publication describes the isolation, purification and characterization of Corn Rootworm active protein from strains AB78, AB81, AB6 and AB88. Other strains of Bacillus sp. were also isolated and characterized.

WO 95/15383 describes the characterization of a mosquitocidal toxin encoded by the B. sphaericus mtx gene.

All parts of the present description related to the following deposited strains: NRRL B-21221, NRRL B-21222_ANRRL B-21223, NRRL B-21224, NRRL B-21225, NRRL B-21226, NRRL B-21227, NRRL B-21228, NRRL B-21229, NRRL B-21230, NRRL B-21058, NRRL B-21059, NRRL B-21060 and NRRL B-21061; and SEQ ID Nos. 118־ bearthe filing date of the Israeli application, i.e., September 21, 1995. These strains and sequences are physically post-dated herein.

All parts of the present description related to the following deposited strains: NRRL B-21422, NRRL B-21423, NRRL B-21438 and NRRL B-21439; and SEQ ID Nos. 19-50 bear the date of the second priority document, i.e., June 5,1995. These strains and sequences are physically post-dated herein.

All parts of the present description related to SEQ ID Nos. 51 and 52 bear their Israeli filing date, i.e., September 27,1995. These sequences are physically post-dated herein.

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Summary of the Invention

In a first aspect,׳ the present . invention provides a .DNA molecule encoding a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp., said DNA molecule being complementary to a- sequence which hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO. 31 and SEQ ID NO: 51 at 65°C in a buffer comprising 7% SDS and 0.5 M sodium phosphate.

In one embodiment, said DNA molecule encodes a protein as defined by SEQ ID NO: 29, SEQ ID NO: 32, and SEQ ID NO: 52, and said DNA molecule has the nucleotide sequence given in SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: ⁵¹- . ' '

In another embodiment, said DNA molecule comprises a nucleotide sequence that has been wholly or partially synthesized using plant preferred codons, having the sequence given in SEQ ID NO: 30 or SEQ ID NO: 51.

In another embodiment, said DNA molecule comprises a nucleotide sequence that has been wholly or partially optimized synthesized using codons preferred by microorganisms.

In a further embodiment, said DNA molecule is obtainable by a process comprising the steps of: (i) obtaining a DNA molecule comprising a nucleotide sequence encoding a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp.; (ii) hybridizing said DNA molecule with a DNA. molecule complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO:51; and (iii) isolating said.hybridized DNA.

, Id 115382/1

In another aspect, the present invention provides an expression cassette comprising a DNA molecule as described in the invention, operably linked to expression sequences including the transcriptional and translational regulatory signals necessary for expression of the associated DNA constructs in a host organism and optionally further regulatory sequences. Preferably, the host organism is a plant.

The present invention also provides a vector molecule comprising an expression cassette as described above.

In a further aspect, the present invention provides a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp. encoded by a DNA molecule as described in the invention, said protein having a molecular weight of about 60 to about 100 kDa,. and the amino acid sequence given in SEQ ID NO: 29 or SEQ ID NO: 32.

In one embodiment, said protein which may be isolated during the vegetative growth phase of Bacillus thuringiensis AB88 is deposited under the Accession Number NRRL B-21225, or Bacillus thuringiensis AB424 deposited under the Accession Number NRRL B-21439.

.In another further aspect, the present invention provides a host organism comprising a DNA molecule according to the invention, an expression cassette comprising said DNA molecule, or a vector molecule comprising said expression cassette, preferably stably incorporated into the genome of the host organism, said host organism being selected from the group consisting of plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes and algae.

In one particular embodiment, said host is a microorganism transformed with an expression cassette or.a vector molecule according to the invention, characterized in that said microorganism is preferably a microorganism that multiplies

I le ' ’ 115382/1 on plants. Preferably, characterized in that the microorganism is Bacillus thuringiensis AB88 deposited under the Accession Number NRRL B-21225, or Bacillus thuringiensis AB424 deposited under the Accession Number NRRL B-21439.

In another aspect, the present invention provides a transgenic plant expressing a vegetative . insecticidal protein according as described in the invention, including parts as well as progeny and seed thereof stably transformed with a DNA molecule or an expression cassette according to the invention. Said plant may further express Bt δ-endotoxm as a second distinct insect control principle. Preerably, said plant is a maize plant.

In one particular embodiment, said plant is a hybrid plant.

In yet another aspect the present invention provides a seed of a plant according to the invention, treated with a seed protecting coating.

In another even further aspect, the present invention provides a method for isolating a DNA molecule according to the invention, said method comprising:(i) obtaining a DNA molecule comprising a nucleotide sequence encoding a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp.; (ii) hybridizing said DNA molecule with a DNA molecule complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO:51; and (iii)isolating hybridized DNA.

The invention further provides a method of increasing insect target range characterized in that an expression cassette as described in the invention is expressed in a plant together with at least . one second insecticidal protein that is different from the vegetative insecticidal protein encoded by said expression cassette.

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115382/.1

In one embodiment of said method, the second insecticidal protein is selected from the group consisting of Bt δendotoxins, protease inhibitors, lectins, a-amylases and peroxidases.

In yet another aspect the present invention provides a method of protecting plants against damage caused by an insect pest comprising transforming said plants with a DNA molecule encoding a vegetative insecticidal protein secreted during the vegetative growth phase of Bacillus spp ., said DNA molecule being complementary to a sequence which hybridizes with a nucleotide selected from the group consisting of SED ID NO:28, SEQ ID NO: 30,.SEQ ID NO: 31 and SEQ ID NO: 51 at 65°C in a buffer comprising 7% SDS and 0.5 M sodium phosphate.

Finally, the present invention provides a method of producing a plant or plant cell expressing a vegetative insecticidal protein comprising transforming said plant or plant cell with an expression cassette or a vector-molecule according to the invention.

Detailed Description of the Invention

Within the present invention compositions and methods for controlling plant pests are provided. In particular, novel pesticidal proteins are provided which are produced during vegetative growth .of Bacillus strains. The proteins are useful as pesticidal agents.

More specifically, the present invention relates to a substantially purified Bacillus strain which produces a pesticidal protein during vegetative growth wherein said Bacillus is not B. sphaericus SSI 1-1. Preferred are a Bacillus cereus strain having Accession No. NRRL B-21058 and Bacillus thuringiensisstrain having Accession No. NRRL B-21060. Also preferred is a Bacillus strain selected from Accession Numbers NRRL B-21224, NRRL B-21225, NRRL B-21226, NRRL B-21227, NRRL B-21228, NRRL B-21229, NRRL B-21230, and NRRL B-21439.

The invention further relates to an insect-specific protein isolatable during the vegetative growth phase of Bacillus spp, but preferably of a Bacillus thuringiensis and

B. cereus strain, and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1. The insect-specific protein of the invention is preferably toxic to Coleoptera or Lepidoptera insects and has a molecular weight of about 30 kDa or greater, preferably of about 60 to about 100 kDa, and more preferably of about 80 kDa.

More particularly, the insect-specific protein of the invention has a spectrum of insecticidal activity that includes an activity against Agrotis and/or Spodoptera species, but preferably a black cutworm [Agrotis ipsilon; BCW] and/or fall armyworm [Spodoptera frugiperda] and/or beet armyworm [Spodoptera exigua ] and/or tobacco budworm and/or.corn earworm [Helicoverpa zea] activity.

The insect-specific protein of the invention can preferably be isolated, for example, from Bacillus cereus having Accession No. NRRL B-21058, or from Bacillus thuringiensis having Accession No. NRRL B-21060.

The insect-specific protein of the invention can also preferably be isolated from a Bacillus spp strain selected from Accession Numbers NRRL B-21224, NRRL B21225, NRRL B-21226, NRRL B-21227, NRRL B-21228, NRRL B-21229, NRRL B21230, and NRRL B-21439.

The present invention especially encompasses an insect-specific protein that has the amino acid sequence selected from the group consisting of SEQ ID NO:5 and

SEQ ID N0:7, including any proteins that are structurally and/or functionally homologous thereto.

Further preferred is an insect-specific protein, wherein said protein has the sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:29SEQ ID NO:32 and SEQ ID^;NO:2, including any proteins that are structurally and/or functionally homologous thereto.

Especially preferred is an insect-specific protein, wherein said protein has the sequence selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:32, including any proteins that are structurally and/or functionally homologous thereto.

A further preferred embodiment of the invention comprises an insect-specific protein of the invention, wherein the sequences representing the secretion signal have been removed or inactivated.

The present invention further encompasses auxiliary proteins which enhance the insect-specific activity of an insect-specific protein. The said auxiliary proteins preferably have a molecular weight of about 50 kDa and can be isolated, for example, from the vegetative growth phase of a Bacillus cereus strain, but especially of Bacillus cereus strain AB78.

A preferred embodiment of the invention relates to an auxiliary protein, wherein the sequences representing the secretion signal have been removed or inactivated.

The present invention further relates to multimeric pesticidal proteins, which comprise more than one polypeptide chain and wherein at least one of the said polypeptide chains represents an insect-specific protein of the invention and at least one of the said polypeptide chains represents an auxiliary protein of the invention, which activates or enhances the pesticidal activity of the said insect-specific protein.

The multimeric pesticidal proteins according to the invention preferably have a molecular weight of about 50 kDa to about 200 kDa.

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The invention especially encompasses a multimeric pesticidal protein, which comprises an insect-specific protein of the invention and an auxiliary protein according to the invention, which activates or enhances the pesticidal activity of the said insectspecific protein.

The present invention further relates to fusion proteins comprising several protein domains including at least an insect-specific protein of the invention and/or an auxiliary protein according to the invention produced by in frame genetic fusions, which, when translated by ribosomes, produce a fusion protein with at least the combined attributes of the insect-specific protein of the invention and/or an auxiliary protein according to the invention and, optionally, of the other components used in the fusion.

A specific embodiment of the invention relates to a fusion protein comprising a ribonuclease S־protein, an insect-specific protein of the invention and an auxiliary protein according to the invention.

A further specific embodiment of the invention relates to a fusion protein comprising an insect-specific protein according to the invention and an auxiliary protein according to the invention having either the insect-specific protein or the auxiliary protein at the N-terminal end of the said fusion protein.

Preferred is a fusion protein, which comprises an insect-specific protein as given in SEQ ID ׳NO:5 and an auxiliary protein as given in SEQ ID NO: 2 resulting in the protein given in SEQ ID NO: 23, including any proteins that are structurally and/or functionally homologous thereto.

Also preferred is a fusion protein, which comprises an insect-specific protein as given in SEQ ID NO:35 and an auxiliary protein as given in SEQ ID NO: 27 resulting in the protein given in SEQ ID NO: 50, including any proteins that are structurally and/or functionally homologous thereto.

The invention further relates to a fusion protein comprising an insect-specific protein of the invention and/or an auxiliary protein according to the invention fused to a signal sequence, preferably a secretion signal sequence or a targeting sequence that directs the transgene product to a specific organelle or cell compartment, which signal sequence is of herterologous origin with respect to the recipient protein.

Especially preferred within this invention is a fusion protein wherein the said, protein has a sequence as given in SEQ ID NO: 43, or in SEQ ID NO: 46, including any proteins 'that are structurally and/or functionally homologous thereto.

As used in the present application, substantial sequence homology means close structural relationship between sequences of amino acids. For example, substantially homologous proteins may be 40% homologous, preferably 50% and most preferably 60% or 80% homologous, or more. Homology also includes a relationship wherein one or several subsequences of amino acids are missing, or subsequences with additional amino acids are interdispersed.

A further aspect of the invention relates to a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein isolatable during the vegetative growth phase of Bacillus spp. and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1. In particular, the present invention relates to a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein wherein the spectrum of insecticidal activity includes an activity against Agrot/s and/or Spodoptera species, but preferably a black cutworm [Agrotis ipsilon; BCW] and/or fall armyworm [Spodoptera frugiperda] and/or beet armyworm [Spodoptera exigua ] and/or tobacco budworm and/or corn earworm [Helicoverpa zea] activity.

Preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ, ID NO: 4, or SEQ ID NO: 6, including any DNA molecules that are structurally and/or functionally homologous thereto.

Also ;preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:31, or SEQ ID NO:1־, including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to a DNA molecule comprising a nucleotide sequence which encodes an auxiliary protein according to the invention which enhances the insect-specific activity of an insect-specific protein.

Preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given SEQ ID NO:19, including any DNA molecules that are structurally and/or functionally homologous thereto.

A further embodiment of the invention relates to a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein isolatable during the vegetative growth phase of Bacillus spp. and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1, which nucleotide sequence has been optimized for expression in a microorganism or a plant.

Preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO:17 or SEQ ID NO:18, including any DNA molecules that are structurally and/or functionally homologous thereto.

Also preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, or

SEQ ID NO:30, including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to a DNA molecule which comprises a nucleotide sequence encoding a multimeric pesticidal protein, which comprises more than one polypeptide chains and wherein at least one of the said polypeptide chains represents an insect-specific protein of the invention and at least one of the said polypeptide chains represents an auxiliary protein according to the invention, which activates or enhances the pesticidal activity of the said insect-specific protein.

Preferred is a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein of the invention and an auxiliary protein according to the invention, which activates or enhances the pesticidal activity of the said insect-specific protein.

Especially preferred is a DNA molecule, wherein said molecule comprises a nucleotide sequence as given in SEQ ID NO:1 or SEQ ID NO:19, including any nucleotide sequences that are structurally and/or functionally homologous thereto. A further embodiment of the invention relates to a DNA molecule which comprises a nucleotide sequence encoding a fusion protein comprising several protein domains including at least an insect-specific protein of the invention and/or an auxiliary protein according to the invention produced by in frame genetic fusions, which, when translated by ribosomes, produce a fusion protein with at least the combined attributes of the irisect-specific protein of the invention and/or an auxiliary protein according to the invention and, optionally, of the other components used in the fusion.

Preferred within the invention is a DNA molecule which comprises a nucleotide sequence encoding a fusion protein comprising an insect-specific protein according to the invention and an auxiliary protein according to the invention having either the insect-specific protein or the auxiliary protein at the N-terminal end of the said fusion protein.; Especially preferred is a DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO:22, including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to a DNA molecule which comprises a nucleotide sequence encoding a fusion protein comprising an insect-specific protein of the invention and/or an auxiliary protein of the invention fused to a signal sequence, preferably a secretion signal sequence or a targeting sequence that directs the transgene product to a specific organelle or cell compartment, which signal sequence is of herterologous origin with respect to the recipient DNA.

The present invention further encompasses a DNA molecule comprising a nucleotide sequence encoding a fusion protein or a mulitmeric protein according to the invention that has been optimized for expression in a microorganism or plant.

Preferred is an optimized DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO:42, SEQ ID NO:45, or SEQ ID NO:49, including any DNA molecules that are structurally and/or functionally homologous thereto.

The invention further relates to an optimized DNA molecule, wherein the sequences encoding the secretion signal have been removed from its 5' end, but especially to an optimized DNA molecule, wherein the said molecule comprises a nucleotide sequence as given in SEQ ID NO: 35 or SEQ ID NO:39, including any DNA molecules that are structurally and/or functionally homologous thereto.

As used in the present application, substantial sequence homology means close structural relationship between sequences of nucleotides. For example, substantially homologous DNA molecules, may be 60% homologous, preferably 80% and most preferably 90% or 95% homologous, or more. Homology also includes a relationship wherein one or several subsequences of nucleotides or amino acids are missing, or subsequences with additional nucleotides or amino acids are interdispersed.

Also comprised by the present invention are DNA molecules which hybridizes to a DNA molecule according to the invention as defined hereinbefore, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said insect-specific protein at least 10 nucleotides in length, under moderately stringent conditions and which molecules have insect-specific activity and also the insect-specific proteins being encoded by the said DNA molecules.

Preferred are DNA molecules, wherein hybridization occurs at 65°C in a buffer comprising 7% SDS and 0.5 M sodium phosphate.

Especially preferred is a DNA molecule comprising a nucleotide sequence which encodes an insect-specific protein according to the invention obtainable by a process comprising (a) obtaining a DNA molecule comprising a nucleotide sequence encoding an insectspecific protein; and (b) hybridizing said DNA molecule with an oligonucleotide probe acording to claim 107 obtained from a DNA molecule comprising a nucleotide sequence as given in SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 31; and (c) isolating said hybridized DNA.

The invention further relates to an insect-specific protein, wherein the said protein is encoded by a DNA molecule according to the invention.

Also encompassed by the invention is an expression cassette comprising a DNA molecule according to the invention operably linked to expression sequences including the transcriptional and translational regulatory signals necessary for expression of the associated DNA constructs in a host organism, preferably a microorganism or a plant, and optionally further regulatory sequences.

The invention further relates to a vector molecule comprising an expression cassette-according to the invention.

The expression cassette and/or the vector molecule according to the invention are preferably part of the plant genome.

A further embodiment of the invention relates to a host organism, preferably a host organism selected from the group consisting of plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes and algae, comprising a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette, preferably stably incorporated into the genome of the host organism.

The invention further relates to a transgenic plant, but preferably a maize plant, including parts as well as progeny and seed thereof comprising a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette, preferably stably incorporated into the plant genome.

Preferred is a transgenic plant including parts as well as progeny and seed thereof which has been stably transformed with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette.

Also preferred is a transgenic plant including parts as well as progeny and seed thereof which expresses an insect-specific protein according to the invention.

The invention further relates to a transgenic plant, preferably a maize plant, according to the invention as defined hereinbefore, which further expresses a second distinct insect control principle, but preferably a Bt δ-endotoxin. The said plant is preferably a hybrid plant.

Parts of transgenic plants are to be understood within the scope of the invention to comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed with a DNA molecule according to the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention.

The invention further relates to plant propagating material of a plant according to the invention, which is treated with a seed protectant coating.

The invention further encompasses a microorganism transformed with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette, wherein the said microorganism is preferably a microorganism that multiply on plants and more preferably a root colonizing bacterium.

A further embodiment of the invention relates to an encapsulated insect-specific protein which comprises a microorganism comprising an insect specific protein according to the invention.

The invention also relates to an entomocidal composition comprising a host organism of the invention, but preferably a purified Bacillus strain, in an insecticidallyeffective amount together with a suitable carrier.

Further comprised by the invention is an entomocidal composition comprising an isolated protein molecule according to the invention, alone or in combination with a host organism of the invention and/or an encapsulated insect-specific protein according to the invention, in an insecticidally-effective amount, together with a suitable carrier.

A further embodiment of the invention relates to a method of obtaining a purified insect-specific protein according to the invention, said method comprising applying a solution comprising said insect-specific protein to a NAD column and eluting bound protein.

Also comprised is a method for identifying insect activity of an insect-specific protein according to the invention, said method comprising:

growing a Bacillus strain in a culture;

obtaining supernatant from said culture;

allowing insect larvae to feed on diet with said supernatant; and, determining mortality.

Another aspect of the invention relates to a method for isolating an insect-specific protein according to the invention, said method comprising:

growing a Bacillus strain in a culture;

obtaining supernatant from said culture; and, isolating said insect-specific protein from said supernatant.

The invention also encompasses a method for isolating a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein exhibiting the insecticidal activity of the proteins according to the invention, said method comprising: obtaining a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein; and hybridizing said DNA molecule with DNA obtained from a Bacillus species; and isolating said hybridized DNA.

The invention further relates to a method of increasing insect target range by using an insect specific protein according to the invention in combination with at least one second insecticidal protein that is different from the insect specific protein according to the invention, but preferably with an insecticidal protein selected from the group consisting of Bt δ-endotoxins, protease inhibitors, lectins, a-amylases and peroxidases.

Preferred is a method for increasing insect target range within a plant by expressing within the said plant a insect specific protein according to the invention in combination with at least one second insecticidal protein that is different from the insect specific protein according to the invention, but preferably with an insecticidal protein selected from the group consisting of Bt δ-endotoxins, protease inhibitors, lectins, a-amylases and peroxidases.

Also comprised is a method of protecting plants against damage caused by an insect pest, but preferably by Spodoptera and/or Agrotis species, and more preferably by an insect pest selected from the group consisting of black cutworm [Agrotis ipsilon; BOW], fall armyworm [Spodoptera frugiperda], beet armyworm [Spodoptera exigua ], tobacco budworm and corn earworm [Helicoverpa zea] comprising applying to the plant or the growing area of the said plant an entomocidal composition or a toxin protein !according to the invention;

The invention further relates to method of protecting plants against damage caused by an insect pest, but preferably by Spodoptera and/or Agrotis species, and more preferably by an insect pest selected from the group consisting of black cutworm [Agrotis ipsilon; BOW], fall armyworm [Spodoptera frugiperda], beet armyworm [Spodoptera exigua ], tobacco budworm and corn earworm [Helicoverpa zea] comprising planting a transgenic plant expressing a insect-specific protein according to the invention within an area where the said insect pest may occur.

The invention also encompasses a method of producing a host organism which comprises stably integrated into its genome a DNA molecule according to the invention and preferably expresses an insect-specific protein according to the invention comprising transforming the said host organism with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette.

A further embodiment of the invention relates to a method of producing a transgenic plant or plant cell which comprises stably integrated into the plant genome a DNA molecule according to the invention and preferably expresses an insectspecific¹ protein according to the invention comprising transforming the said plant and plant cell, respectively, with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette.

The invention also relates to a method of producing an entomocidal composition comprising mixing an isolated Bacillus strain and/or a host organism and/or an isolated protein molecule, and/or an encapsulated protein according to the invention in an insecticidally-effective amount with a suitable carrier.

The invention also encompasses a method of producing transgenic progeny of a transgenic parent plant comprising stably incorporated into the plant genome a DNA molecule comprising a nucleotide sequence encoding an insect-specific protein according to the invention comprising transforming the said parent plant with a DNA molecule according to the invention, an expression cassette comprising the said DNA molecule or a vector molecule comprising the said expression cassette and transferring the pesticidal trait to the progeny of the said transgenic parent plant involving known plant breeding techniques.

Also encompassed by the invention is oligonucleotide probe capable of specifically hybridizing to a nucleotide sequence encoding a insect-specific protein isolatable during the vegetative growth phase of Bacillus spp. and components thereof, wherein said protein is not the mosquitocidal toxin from B. sphaericus SSII-1, wherein said probe comprises a contiguous portion of the coding sequence for the said insectspecific protein at least 10 nucleotides in length and the use of the said oligonucleotide probe for screening of any Bacillus strain or other organisms to determine whether the insect-specific protein is naturally present or whether a particular transformed organism includes the said gene

The present invention recognizes that pesticidal proteins are produced during vegetative growth of Bacillus strains. Having recognized that such a class exists, the present invention embraces all vegetative insecticidal proteins, hereinafter referred to as VIPs, except for the mosquitocidal toxin from B. sphaericus.

The present VIPs are not abundant after sporulation and are particularly expressed during log phase growth before stationary phase. For the purpose of the present invention vegetative growth is defined as that period of time before the onset of sporulation. Genes encoding such VIPs can be isolated, cloned and transformed into various delivery vehicles for use in pest management programs.

For purposes of the present invention, pests include but are not limited to insects, fungi, bacteria, nematodes, mites, ticks, protozoan pathogens, animal-parasitic liver flukes, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera.iHymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.

Tables 1 -10 gives a list of pests associated with major crop plants and pests of human and veterinary importance. Such pests are included within the scope of the present invention.

TABLE 1

Lepidootera (Butterflies and Moth)

Maize

Ostrinia nubilalis, European corn borer

Agrotis ipsilon, black cutworm

Helicoverpa zea, corn earworm

Spodoptera frugiperda, fall armyworm

Diatraea grandiosella, southwestern corn borer Elasmopalpus lignosellus, lesser cornstalk borer

Diatraea saccharalis, sugarcane borer

Sorghum

Chilo partellus, sorghum borer

Spodoptera frugiperda, fall armyworm

Helicoverpa zea, corn earworm

Elasmopalpus lignosellus, lesser cornstalk borer Feltia subterranea, granulate cutworm

Wheat

Pseudaletia unipunctata, army worm

Spodoptera frugiperda, fall armyworm

Elasmopalpus lignosellus, lesser cornstalk borer Agrotis orthogonia, pale western cutworm

Elasmopalpus lignosellus, lesser cornstalk borer

Sunflower

Suleima helianthana, sunflower bud moth

Homoeosoma electellum, sunflower moth

Cotton

Heliothis virescens, cotton boll worm

Helicoverpa zea, cotton bollworm

Spodoptera exigua, beet armyworm

Pectindphora gossypiella, pink bollworm

Rice

Diatraea saccharalis, sugarcane borer

Spodoptera frugiperda, fall armyworm

Helicoverpa zea, corn earworm

Soybean

Pseudoplusia includens, soybean looper

Anticarsia gemmatalis, velvetbean caterpillar

Plathypena scabra, green cloverworm

Ostrinia nubilalis, European corn borer

Agrotis ipsilon, black cutworm

Spodoptera exigua, beet armyworm

Heliothis virescens, cotton boll worm

Helicoverpa zea, cotton bollworm

Barley

Ostrinia nubilalis, European corn borer

Agrotis ipsilon, black cutworm

TABLE 2

Coleoptera (Beetles)

Maize

Diabrotica virgifera virgifera, western corn rootworm

Diabrptica longicornis barberi, northern corn rootworm

Diabrotica undecimpunctata howardi, southern corn rootworm

Melanotus spp., wireworms

Cyclocephala borealis, northern masked chafer (white grub)

Cycldcephala immaculata, southern masked chafer (white grub)

Popillia japonica, Japanese beetle

Chaetocnema pulicaria, corn flea beetle

Sphenophorus maidis, maize billbug

Sorghum

Phyllophaga crinita, white grub

Eleodes, Conoderus, and Aeolus spp., wireworms

Oulema melanopus, cereal leaf beetle

Chaetocnema pulicaria, corn flea beetle

Sphenophorus maidis, maize billbug

Wheat ^;

Oulema melanopus, cereal leaf beetle

Hypera punctata, clover leaf weevil

Diabrotica undecimpunctata howardi, southern corn rootworm

Sunflower

Zygogramma exclamationis, sunflower beetle Bothyrus gibbosus, carrot beetle

Cotton ₍

Anthonomus grandis, boll weevil

Rice

Colaspis brunnea, grape colaspis Lissorhoptrus oryzophilus, rice water weevil

Sitophilus oryzae, rice weevil

Soybean

Epilachna varivestis, Mexican bean beetle

TABLE 3

Homoptera (Whiteflies, Aphids etc.)

Maize

Rhopalosiphum maidis, corn leaf aphid Anuraphis maidiradicis, corn root aphid

Sorghum

Rhopalosiphum maidis, corn leaf aphid

Sipha flava, yellow sugarcane aphid

Wheat '

Russian wheat aphid

Schizaphis graminum, greenbug

Macrosiphum avenae, English grain aphid

Cotton

Aphis gossypii, cotton aphid

Pseudatomoscelis seriatus, cotton fleahopper

Trialeurodes abutilonea, bandedwinged whitefly

Rice

Nephptettix nigropictus, rice leafhopper i

Soybean

Myzus persicae, green peach aphid

Empoasca fabae, potato leafhopper

Barley

Schizaphis graminum, greenbug

Oil Seed Rape

Brevicoryne brassicae, cabbage aphid

TABLE 4

Hemiptera (Bugs)

Maize

Blissus leucopterus leucopterus, chinch bug

Sorghum

Blissus leucopterus leucopterus, chinch bug

Cotton

Lygus lineolaris, tarnished plant bug

Rice

Blissus leucopterus leucopterus, chinch bug

Acrosternum hilare, green stink bug

Soybean

Acrosternum hilare, green stink bug

Barley

Blissus leucopterus leucopterus, chinch bug

Acrosternum hilare, green stink bug Euschistus serves, brown stink bug

TABLE 5

Qrthoptera (Grasshoppers, Crickets, and Cockroaches)

Maize

Melanoplus femurrubrum, redlegged grasshopper

Melanoplus sanguinipes, migratory grasshopper

Wheat

Melanoplus femurrubrum, redlegged grasshopper

Melanoplus differentialis, differential grasshopper

Melanoplus sanguinipes, migratory grasshopper

Cotton

Melanoplus femurrubrum, redlegged grasshopper

Melanoplus differentialis, differential grasshopper

Soybean

Melanoplus femurrubrum, redlegged grasshopper

Melanoplus differentialis, differential grasshopper

Structural/Household

Periplaneta americana, American cockroach

Blattella germanica, German cockroach

Blatta orientalis, oriental cockroach

TABLE 6

Diptera (Flies and Mosquitoes)

Maize

Hylemya platura, seedcorn maggot

Agromyza parvicornis, corn blotch leafminer

Sorghum

Contarinia sorghicola, sorghum midge

Wheat

Mayetiola destructor, Hessian fly

Sitodiplosis mosellana, wheat midge

Meromyza americana, wheat stem maggot

Hylemya coarctata, wheat bulb fly

Sunflower

Neolasioptera murtfeldtiana, sunflower seed midge

Soybean

Hy/emya p/atura, seedcorn maggot

Barley

Hylemya platura, seedcorn maggot

Mayetiola destructor, Hessian fly

Insects attacking humans and animals and disease carriers

Aedes aegypti, yellowfever mosquito

Aedes albopictus, forest day mosquito

Phlebotomus papatasii, sand fly

Musca domestica, house fly

Tabanus atratus, black horse fly

Cochliomyia hominivorax, screwworm fly

TABLE 7

Thvsanoptera (Thrips)

Maize

Anaphothrips obscurus, grass thrips

Wheat

Frankliniella fusca, tobacco thrips

Cotton

Thrips tabaci, onion thrips

Frankliniella fusca, tobacco thrips

Soybean

Sericothrips variabilis, soybean thrips

Thrips tabaci, onion thrips

TABLE 8

Hymenoptera (Sawflies, Ants, Wasps, etc.)

Maize

Solenopsis milesta, thief ant

Wheat

Cephas cinctus, wheat stem sawfly

TABLE 9

Other Orders and Representative Species

Dermaptera (Earwigs)

Forficula auricularia, European earwig

Isoptera (Termites)

Reticulitermes flavipes, eastern subterranean termite

Mallophaga (Chewing Lice)

Cuclotogaster heterographa, chicken head louse

Bovicola bovis, cattle biting louse

Anoplura (Sucking Lice)

Pediculus humanus, head and body louse

Siphonaptera (Fleas)

Ctenocephalides felts, cat flea

TABLE 10

Acari (Mites and Ticks)

Maize

Tetranychus urticae, twospotted spider mite

Sorghum

Tetranychus cinnabarinus, carmine spider mite

Tetranychus urticae, twospotted spider mite

Wheat

Aceria tulipae, wheat curl mite

Cotton

Tetranychus cinnabarinus, carmine spider mite

Tetranychus urticae, twospotted spider mite

Soybean

Tetranychus turkestani, strawberry spider mite

Tetranychus urticae, twospotted spider mite

Barley

Petrobia latens, brown wheat mite

Important human and animal Acari

Demacentor variabilis, American dog tick

Argas persicus, fowl tick

Dermatophagoides farinae, American house dust mite

Dermatophagoides pteronyssinus, European house dust mite

Now that it has been recognized that pesticidal proteins can be isolated from the vegetative growth phase of Bacillus, other strains can be isolated by standard techniques and tested for activity against particular plant and non-plant pests. Generally Bacillus strains can be isolated from any environmental sample, including soil, plant, insect, grain elevator dust, and other sample material, etc., by methods known in the art. See, for example, Travers et al. (1987) Appl. Environ. Microbiol. 53:1263-1266; Saleh etal. (1969) Can J. Microbiol. 15:1101-1104; DeLucca et al. (1981) Can. J. Microbiol. 27:865-870; and Norris, etal. (1981) The genera Bacillus and Sporolactobacillus, In Starr et al. (eds.), The Prokaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria, Vol. II, Springer-Verlog Berlin Heidelberg. After isolation, strains can be tested for pesticidal activity during vegetative growth. In this manner, new pesticidal proteins and strains can be identified.

Such Bacillus microorganisms which find use in the invention include Bacillus cereus and Bacillus thuringiensis, as well as those Bacillus species listed in Table 11.

TABLE 11

List of Bacillus species

Morphological Group 1

B. megaterium

B. cereus*

B. cereus var. mycoides

B. thuringiensis*

B. licheniformis

B. subtilis*

B. pumilus

B. firmus*

B. coagulans

Morphological Group 2

B. polymyxa

B. macerans

B. circulans

B. stearothermophilus

B. alvei*

B. laterosporus*

B. brevis

B. pulvifaciens

B. popilliae*

B. lentimorbus*

B. larvae*

Morphological Group 3

B. sphaericus*

B. pasteurii

Unassigned Strains

Subgroup A

B. apiarus*

B. filicolonicus

B. thiaminolyticus

B. alcalophilus

Subgroup B

B. cirroflagellosus

B. chitinosporus

B. lentus

Subgroup 0

B. badius

B. aneurinolyticus

B. macroides

B. freundenreichii

Subgroup D

B. pantothenticus

B. epiphytus

Subgroup E1

B. aminovorans

B. globisporus

B. insolitus

B. psychrophilus

Subgroup E2

B. psychrosaccharolyticus

B. macquariensis ‘־Those Bacillus strains that have been previously found associated with insects Grouping according to Parry, J.M. etal. (1983) Color Atlas of Bacillus species, Wolfe

Medical Publications, London.

In accordance with the present invention, the pesticidal proteins produced during vegetative growth can be isolated from Bacillus. In one embodiment, insecticidal proteins produced during vegetative growth, can be isolated. Methods for protein isolation are known in the art. Generally, proteins can be purified by conventional chromatography, including gel-filtration, ion-exchange, and immunoaffinity chromatography, by high-performance liquid chromatography, such as reversed-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography, high-performance chromatofocusing and hydrophobic interaction chromatography, etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc. Such methods are known in the art. See for example Current Protocols in Molecular Biology, Vols. 1 and 2, Ausubel et al. (eds.), John Wiley & Sons, NY (1988). Additionally, antibodies can be prepared against substantially pure preparations of the protein. See, for example, Radka et al. (1983) J. Immunol. 128:2804; and Radka et al. (1984) Immunogenetics 19:63. Any combination of methods may be utilized to purify protein having pesticidal properties. As the protocol is being formulated, pesticidal activity is determined after each purification step.

Such purification steps will result in a substantially purified protein fraction. By substantially purified or substantially pure is intended protein which is substantially free of any compound normally associated with the protein in its natural state. Substantially pure preparations of protein can be assessed by the absence of other detectable protein bands following SDS-PAGE as determined visually or by densitometry scanning. Alternatively, the absence of other amino-terminal sequences or N-terminal residues in a purified preparation can indicate the level of purity. Purity can be verified by rechromatography of pure preparations showing the absence of other peaks by ion exchange, reverse phase or capillary electrophoresis. The terms substantially pure or substantially purified are not meant to exclude artificial or synthetic !mixtures of the proteins with other compounds. The terms are also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the protein, and which may be present, for example, due to incomplete purification.

Once purified protein is isolated, the protein, or the polypeptides of which it is comprised, can be characterized and sequenced by standard methods known in the art. For example, the purified protein, or the polypeptides of which it is comprised, may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, lysyl-C endopeptidase, etc. (Oike etal. (1982) J. Biol. Chem. 257:9751-9758; Liu et al. (1983) Int. J. Pept. Protein Res. 21:209-215). The resulting peptides are separated, preferably by HPLC, or by resolution of gels and electroblotting onto PVDF membranes, and subjected to amino acid sequencing. To accomplish this task, the peptides are preferably analyzed by automated sequenators. It is recognized that N-terminal, C-terminal, or internal amino acid sequences can be determined. From the amino acid sequence of the purified protein, a nucleotide sequence can be synthesized which can be used as a probe to aid in the isolation of the gene encoding the pesticidal protein.

It is recognized that the pesticidal proteins may be oligomeric and will vary in molecular weight, number of protomers, component peptides, activity against particular pests, and in other characteristics. However, by the methods set forth herein, proteins active against a variety of pests may be isolated and characterized.

Once׳the purified protein has been isolated and characterized it is recognized that it may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the pesticidal proteins can be prepared by mutations in the DNA. Such variants will possess the desired pesticidal activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

In this^; manner, the present invention encompasses the pesticidal proteins as well as components and fragments thereof. That is, it is recognized that component protomers, polypeptides or fragments of the proteins may be produced which retain pesticidal activity. These fragments include truncated sequences, as well as N-terminal, C-terminal, internal and internally deleted amino acid sequences of the proteins. _־

Most deletions, insertions, and substitutions of the protein sequence are not expected to produce radical changes in the characteristics of the pesticidal protein.

However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.

The proteins or other component polypeptides described herein may be used alone or in combination. That is, several proteins may be used to control different insect pests.

Some proteins are single polypeptide chains while many proteins consist of more than one polypeptide chain, i.e., they are oligomeric. Additionally, some VIPs are pesticidally active as oligomers. In these instances, additional protomers are utilized to enhance the pesticidal activity or to activate pesticidal proteins. Those protomers which enhance or activate are referred to as auxiliary proteins. Auxiliary proteins activate or enhance a pesticidal protein by interacting with the pesticidal protein to form an.oligomeric protein having increased pesticidal activity compared to that observed in the absence of the auxiliary protein.

Auxiliary proteins activate or increase the activity of pesticidal proteins such as the VIP1 protein from AB78. Such auxiliary proteins are exemplified by, but not limited to, the VIP2 protein from AB78. As demonstrated in the Experimental section of the application, auxiliary proteins can activate a number of pesticidal proteins. Thus, in one embodiment of the invention, a plant, Parent 1, can be transformed with an auxiliary protein. This Parent 1 can be crossed with a number of Parent 2 plants transformed with one or more pesticidal proteins whose pesticidal activities are activated by the auxiliary protein.

Amongst the pesticidal proteins of the invention a new class of insect-specific proteins could be surprisingly identified within the scope of the present invention. The said proteins, which are designated throughout this application as VIP3, can be obtained from Bacillus spp strains, but preferably from Bacillus thuringiensis strains and most preferably from Bacillus thuringiensis strains AB88 and AB424. The said VIPs are present mostly in the supernatants of Bacillus cultures amounting to at least 75% of the total in strain AB88. The VIP3 proteins are further characterized by their unique spectrum of insectical acitivity, which includes an activity against Agrotis and/or Spodoptera species, but especially a black cutworm [BCW] and/or fall armyworm and/or beet armyworm and/or tobacco budworm and/or corn earworm activity.

Black cutworm is an agronomically important insect quite resistant to δ-endotoxins. Macintosh et al (1990) J Invertebr Pathol 56,258-266 report that the δ-endotoxins CrylA(b) and CrylA(c) possesses insecticidal properties against BCW with LC₅₀ of more than 80 gg and 18 gg/ml of diet respectively. The vip3A insecticidal proteins according to the invention provide >50% mortality when added in an amount of protein at least 10 to 500, preferably 50 to 350, and more preferably 200 to 300 fold lower than the amount of Cry IA proteins needed to achieve just 50% mortality. Especially preferred within the invention are vip3A insecticidal proteins which provide 100% mortality when added in an amount of protein at least 260 fold lower than the amount of CrylA proteins needed to achieve just 50% mortality.

The vip3 insecticidal proteins according to the invention are present mostly in the supernatants of the cultures and are therefore are to be classified as secreted proteins. They preferably contain in the N-terminal sequence a number of positively charged residues followed by a hydrophobic core region and are not N-terminally processed during export.

As the other pesticidal proteins reported hereto within the scope of the invention, the VIP3 proteins can be detected in growth stages prior to sporulation establishing a further clear distinction from other proteins that belong to the δ-endotoxin family. Preferably, expression of the insect-specific protein starts during mid-log phase and continues during sporulation. Owing to the specific expression pattern in combination with the high stability of the VIP3 proteins, large amounts of the VIP3 proteins can be found in supernatants of sporulating cultures. Especially preferred are the VIP3 proteins identified in SEQ ID NO:29 and SEQ ID NO:32 and the corresponding DNA molecules comprising nucleotide sequences encoding the said proteins, but especially those DNA molecules comprising the nucleotide sequences given in SEQ ID NO:28, SEQ ID NQ:30 and SEQ ID NO:31.

The pesticidal proteins of the invention can be used in combination with Bt endotoxins or other insecticidal proteins to increase insect target range. Furthermore, the use of the VIPs of the present invention in combination with Bt δ-endotoxins or other insecticidal principles of a distinct nature has particular utility for the prevention and/or management of insect resistance. Other insecticidal principles include protease inhibitors (both serine and cysteine types), lectins, a-amylase and peroxidase. In one preferred embodiment, expression of VIPs in a transgenic plant is accompanied by the expression of one or more Bt δ-endotoxins. This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary. Alternatively, a plant, Parent 1, can be genetically engineered for the expression of VIPs. A second plant, Parent 2, can be genetically engineered for the expression of Bt δ-endotoxin. By crossing Parent 1 with Parent 2, progeny plants are obtained which express all the genes introduced into Parents 1 and 2. Particularly preferred Bt δ-endotoxins are those disclosed in EP-A 0618976, herein incorporated by reference.

A substantial number of cytotoxic proteins, though not all, are binary in action. Binary toxins typically consist of two protein domains, one called the A domain and the other called the B domain (see Sourcebook of Bacterial Protein Toxins, J. E. Alouf and J. H. Freer eds.(1991) Academic Press). The A domain possesses a potent cytotoxic activity. The B domain binds an external cell surface receptor before being internalized. Typically, the cytotoxic A domain must be escorted to the cytoplasm by a translocation domain. Often the A and B domains are separate polypeptides or protomers, which are associated by a protein-protein interaction or a di-sulfide bond. However, the toxin can be a single polypeptide which is proteolytically processed within the cell into two domains as in the case for Pseudomonas exotoxin A. In summary binary toxins typically have three important domains, a cytotoxic A domain, a receptor binding B domain and a translocation domain. The A and B domain are often associated by protein-protein interacting domains.

The receptor binding domains of the present invention are useful for delivering any protein, toxin, enzyme, transcription factor, nucleic acid, chemical or any other factor into target insects having a receptor recognized by the receptor binding domain of the binary toxins described in this patent. Similarly, since binary toxins have translocation domains which penetrate phosopholipid bilayer membranes and escort cytotoxins across those membranes, such translocation domains may be useful in escorting any protein, toxin, enzyme, transcription factor, nucleic acid, chemical or any other factor across a phospholipid bilayer such as the plasma membrane or a vesicle membrane. The translocation domain may itself perforate membranes, thus having toxic or insecticidal properties. Further, all binary toxins have cytotoxic domains; such a

- ²⁸־ . !

i cytotoxic domain may be useful as a lethal protein, either alone or when delivered into any target cell(s)> by any means.

Finally, since binary toxins comprised of two polypeptides often form a complex, it is likely that there are protein-protein interacting regions within the components of the binary toxins of the invention. These protein-protein interacting domains may be useful in forming associations between any combination of toxins, enzymes, transcription factors, nucleic acids, antibodies, cell binding moieties, or any other chemicals, factors, proteins or protein domains.

Toxins, enzymes, transcription factors, antibodies, cell binding moieties or other protein domains can be fused to pesticidal or auxiliary proteins by producing in frame genetic fusions which, when translated by ribosomes, would produce a fusion protein with the combined attributes of the; VIP and the other component used in the fusion. Furthermore, if the protein domain fused to the VIP has an affinity for another protein, nucleic acid, carbohydrate, lipid, or other chemical or factor, then a three-component complex can be formed. This complex will have the attributes of all of its components. A similar rationale can be used for producing four or more component complexes. These complexes are useful as insecticidal toxins, pharmaceuticals, laboratory reagents, and diagnostic reagents, etc. Examples where such complexes are currently used are fusion toxins for,potential cancer therapies, reagents in ELISA assays and immunoblot analysis.

One strategy of altering pesticidal or auxiliary proteins is to fuse a 15-amino-acid “Si-tag” to the protein without destroying the insect cell binding domain(s), translocation domains or protein-protein interacting domains of the proteins. The Stag has a high affinity (K_d = 10’⁹ M) for a ribonuclease S-protein, which, when bound to the S-;tag, forms an active ribonuclease (See F. M. Richards and H. W. Wyckoff! (1971) iri ^,'The Enzymes, Vol. IV (Boyer, P.D. ed.). pp. 647-806. Academic Press,

I . ^! : -

New York). The fusion can be made in such a way as to destroy or remove the ! . ^: i . ׳ י. ’ cytotoxic activity of the pesticidal or auxiliary protein, thereby replacing the VIP cytotoxic activity with a new cytotoxic ribonuclease activity. The final toxin would be comprised of the S-protein, a pesticidal protein and an auxiliary protein, where either the pesti'cidal protein or the auxiliary protein is produced as translational fusions with the S-tag. Similar strategies can be used to fuse other potential cytotoxins to pesticidal or auxiliary proteins including (but not limited to) ribosome inactivating proteins, insect hormones, hormone receptors, transcription factors, proteases, phosphatases, Pseudomonas exotoxin A, or any other protein or chemical factor that is lethal’when delivered into cells. Similarly, proteins can be delivered into cells which are not lethal, but might alter cellular biochemistry or physiology.

The spectrum of toxicity toward different species can be altered by fusing domains to pesticidal or auxiliary proteins which recognize cell surface receptors from other species. Such domains might include (but are not limited to) antibodies, transferrin, hormones, or peptide sequences isolated from phage displayed affinity selectable libraries. Also, peptide sequences which are bound to nutrients, vitamins, hormones, or other chemicals that are transported into cells could be used to alter the spectrum of toxicity. Similarly, any other protein or chemical which binds a cell surface receptor or the membrane and could be internalized might be used to alter the spectrum of activity of VIP1 and VIP2.

The pesticidal proteins of the present invention are.those proteins which confer a specific pesticidal property. Such proteins may vary in molecular weight, having component polypeptides at least a molecular weight of 30 kDa or greater, preferably about 50 kDa or greater.

The auxiliary proteins of the invention may vary in molecular weight, having at least a molecular weight of about 15 kDa or greater, preferably about 20 kDa or greater; more preferably, about 30 kDa or greater. The auxiliary proteins themselves may have component polypeptides.

It is possible that the pesticidal protein and the auxiliary protein may be components of a multimeric, pesticidal protein. Such a pesticidal protein which includes the auxiliary proteins as one or more of its component polypeptides may vary in molecular weight, having at least a molecular weight of 50 kDa up to at least200 ׳ kDa, preferably about 100 kDa to 150 kDa.

An auxiliary protein may be used in combination with the pesticidal proteins of the invention to enhance activity or to activate the pesticidal protein. To determine whether the auxiliary protein will affect activity, the pesticidal protein can be expressed alone and in combination with the auxiliary protein and the respective activities compared in feeding assays for pesticidal activity.

It may be beneficial to screen strains for potential pesticidal activity by testing activity of the strain alone and in combination with the auxiliary protein. In some instances an auxiliary protein in combination with the native proteins of the strains yields pesticidal activity where none is seen in the absence of an auxiliary protein.

The auxiliary protein can be modified, as described above, by various methods known in the art. Therefore, for purposes of the invention, the term Vegetative Insecticidal Protein (VIP) encompasses those proteins produced during vegetative growth which alone or in combination can be used for pesticidal activity. This includes pesticidal proteins, auxiliary proteins and those proteins which demonstrate activity only in the presence of the auxiliary protein or the polypeptide components of these proteins.

It is recognized that there are alternative methods available to obtain the nucleotide and amino acid sequences of the present proteins. For example, to obtain the nucleotide sequence encoding the pesticidal protein, cosmid clones, which express the pesticidal protein, can be isolated from a genomic library. From larger active cosmid clones, smaller subclones can be made and tested for activity. In this manner, clones Vvhich express an active pesticidal protein can be sequenced to determine the nucleotide sequence of the gene. Then, an amino acid sequence can be deduced for the protein. For general molecular methods, see, for example, Molecular Cloning, A Laboratory Manual, Second Edition, Vols. 1-3, Sambrook et al. (eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and the references cited therein.

The present invention also encompasses nucleotide sequences from organisms other than Bacillus, where the nucleotide sequences are isolatable by hybridization with the Bacillus nucleotide sequences of the invention. Proteins encoded by such nucleotide sequences can be tested for pesticidal activity. The invention also: encompasses the proteins encoded by the nucleotide sequences. Furthermore, the invention encompasses proteins obtained from organisms other than Bacillus wherein the protein cross-reacts with antibodies raised against the proteins of the invention. Again the isolated proteins can be assayed for pesticidal activity by the methods disclosed herein or others well-known in the art.

Once the nucleotide sequences encoding the pesticidal proteins of the invention have behn isolated, they can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms and plants.

The pesticidal genes of the invention can be optimized for enhanced expression in plants. See, for example EP-A 0618976; EP-A 0359472; EP-A 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl Acad. Sei. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17:477-498. In this manner, the genes can be synthesized utilizing plant preferred codons. That is the preferred codon for a particular host is the single codon which most frequently encodes that amino acid in that host The maize preferred codon, for example, for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is found in Murray etal. (1989), Nucleic Acids Research 17:477498, the disclosure of which is incorporated herein by reference. Synthetic genes can also be made based on the distribution of codons a particular host uses for a particular amino acid.

In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.

In like manner, the nucleotide sequences can be optimized for expression in any microorganism. For Bacillus preferred codon usage, see, for example US Patent No. 5,024,837 and Johansen etal. (1988) Gene 65:293-304.

Methodologies for the construction of plant expression cassettes as well as the introduction of foreign DNA into plants are described in the art. Such expression cassettes may include promoters, terminators, enhancers, leader sequences, introns and other regulatory sequences operably linked to the pesticidal protein coding sequence. It is further recognized that promoters or terminators of the VIP genes can be used hn expression cassettes.

Generally, for the introduction of foreign DNA into plants Ti plasmid vectors have been utilized for the delivery of foreign DNA as well as direct DNA uptake, liposomes, electroporation, micro-injection, and the use of microprojectiles. Such methods had been published in the art. See, for example, Guerche et al., (1987) Plant Science 52:111-1.16; Neuhause etal., (1987) Theor, Appl. Genet. 75:30-36: Klein etal., (1987) Nature 327: 70-73; Howell etal., (1980) Science 208:1265: Horsch etal., (1985) Science 227:1229-1231; DeBlock et al., (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, ln‘c. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). See also US patent application serial no.

08/008,374 herein incorporated by reference. See also, EP-A 0193259 and EP-A

0451878. It is understood that the method of transformation will depend upon the plant cell to be transformed.

It is further recognized that the components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. See, for example Perlak et al.

(1991) Proc. Natl. Acad. Sei. USA 88:3324-3328; Murray et al., (1989) Nucleic Acids Research 17:477-498; and WO 91/16432.

The construct may also include any other necessary regulators such as terminators, (Guerineau et al., (1991), Mol. Gen. Genet. 226:141-144; Proudfoot, (1991).,Cell, 64:671-674; Sanfacon etal., (1991). Genes Dev. 5:141-149: Mogen et al., (1990). Plant Cell. 2:1261-1272; Munroe etal., (1990). Gene. 91:151-158; Ballas etaletal., (1989). Nucleic Acids Res. 17:7891-7903; Joshi etal., (1987). Nucleic Acid Res., 15:9627-9639); plant translational consensus sequences (Joshi, C.P., (1987).׳Nucleic Acids Research, 15:6643-6653), introns (Luehrsen and Walbot,.

(1991). Mol. Gen. Genet., 225:81-93) and the like, operably linked to the nucleotide sequence. It may be beneficial to include 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation.

Translational leaders are known in the art and include:

Picornavirus leaders, for example, EMCV leader (encephalomyocarditis 5' noncoding region) (Elroy-Stein, 0., Fuerst, T.R., and Moss, B. (1989) PNAS USA 86:6126-6130);

Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison etal., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and

Human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G., and Sarnow, P1991) ״), Nature, 353:90-94;

Untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A., and Gehrke, L1987) ״), Nature. 325:622-625;

Tobacco mosaic virus leader (TMV), (Gallie, D.R. et al., (1989), Molecular Biology of RNA, pages 237-256; and

Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S.A. etal., (1991), Virology, 81:382-385. See also, Della-Cioppa etal., (1987), Plant Physiology, 84:965-968.

A plant terminator may be utilized in the expression cassette. See, Rosenberg et al., (1987), Gene. 56:125; Guerineau etal., (1991). Mol. Gen. Genet., 226:141-144; Proudfoot, (1991), Cell, 64:671674־; Sanfacon etal., (1991). Genes Dev. 5:141-149; Mogen eta/., (1990), Plant Cell. 2:1261-1272; Munroe eta/., (1990), Gene. 91:151158־; Ballas etal., (1989). Nucleic Acids Res. 17:7891-7903; Joshi etal., (1987). Nucleic Acid Res. 15:9627-9639.

For tissue specific expression, the nucleotide sequences of the invention can be operably linked to tissue specific promoters. See, for example, EP-A 0618976, herein incorporated by reference.

Further comprised within the scope of the present invention are transgenic plants, in particular transgenic fertile plants transformed by means of the aforedescribed processes and their asexual and/or sexual progeny, which comprise and preferably also express the pesticidal protein according to the invention. Especially preferred are hybrid plants.

The transgenic plant according to the invention may be a dicotyledonous or a monocotyledonous plant. Preferred are monocotyledonous plants of the Graminaceae family involving Lolium, Zea, Triticum, Triticale, Sorghum, Saccharum, Bromus, Oryzae, Avena, Hordeum, Secale and Setaria plants.

Especially preferred are transgenic maize, wheat, barley, sorghum, rye, oats, turf grasses and rice.

Among the dicotyledonous plants soybean, cotton, tobacco, sugar beet, oilseed rape, and sunflower are especially preferred herein.

The expression 'progeny' is understood to embrace both, asexually and sexually generated progeny of transgenic plants. This definition is also meant to include all mutants, and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initially transformed parent plant, together with all crossing and fusion products of the transformed plant material.

Another object of the invention concerns the proliferation material of transgenic plants.

The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually or asexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, pollen, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.

Parts of plants, such as for example flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed by means of the process of the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention.

Before the plant propagation material [fruit, tuber, grains, seed], but expecially seed is sold as a commerical product, it is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.

In order to treat the seed, the protectant coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to.plants are possible, eg treatment directed at the. buds or the fruit.

The plant seed according to the invention comprising a DNA molecule comprising a nucleotide sequence encoding a pesticidal protein according to the invention may be treated with a seed protectant coating comprising a seed treatment compound, such as, for example, captan, carboxin, thiram (TMTD®), methalaxyl (Apron®) and pirimiphos-methyl (Actellic®) and others that are commonly used in seed treatment. Preferred within the scope of the invention are seed protectant coatings comprising an ehtomocidal composition according to the invention alone or in combination with one ׳ of the a seed protectant coating customarily used in seed treatment.

It is thus a further object of the present invention to provide plant propagation material for cultivated plants, but especially plant seed that is treated with a seed protectant coating as defined hereinbefore.

It is recognized that the genes encoding the pesticidal proteins can be used to transform insect pathogenic organisms. Such organisms include Baculoviruses, fungi, protozoa, bacteria and nematodes.

The Bacillus strains of the invention may be used for protecting agricultural crops and products from pests. Alternatively, a gene encoding the pesticide may be introduced via a suitable vector into a microbial host, and said host applied to the environment or plants or animals. Microorganism hosts may be selected which are known ,to occupy the phytosphere (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wildtype microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes׳, fungi, particularly yeast, e g., Saccharomyces, Cryptococcus, Kluyverpmyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest.are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacterxyli and Azotobacter vinlandir, and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

A number of ways are available for introducing a gene expressing the pesticidal protein into the microorganism host under conditions which allow for stable maintenance and expression of the gene. For example, expression cassettes can be constructed which include the DNA constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the DNA constructs, and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include but are not limited to promoter, transcriptional initiation start site, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, US Patent 5,039,523; US Patent No. 4,853,331; EPO

0480762A2; Sambrook et al. supra; Molecular Cloning, a Laboratory Manual, Maniatis etal. (eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Advanced

Bacterial Genetics, Davis et al. (eds.) Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY (1980); and the references cited therein.

Suitable host cells, where the pesticide-containing cells will be treated to prolong the activity of the toxin in the cell when the then treated cell is applied to the environment of the target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus', Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such a Saccharomyces and Schizosaccharromyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces. and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.׳, or such other organisms as Escherichia, LactoBacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeurginosa, Pseudomonas fluorescens,

Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.

VIP genes can be introduced into micro-organisms that multiply on plants (epiphytes) to deliver VIP proteins to potential target pests. Epiphytes can be grampositive or gram-negative bacteria for example.

Root colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a Bacillus cereus strain which colonizes roots could be isolated from roots of a plant (for example see J. Handelsman, S. Raffel, E. Mester; L. Wunderlich and C. Grau, Appl. Environ. Microbiol. 56:713-718, (1990)). VIP1 and/or V.IP2 and/or VIP3 could be introduced into a root colonizing Bacillus cereus by standard methods known in the art.

Specifically, VIP1 and/or VIP2 derived from Bacillus cereus strain AB78 can be introduced into a root colonizing Bacillus cereus by means of conjugation using standard methods (J. Gonzalez, B. Brown and B. Carlton, Proc. Natl. Acad. Sci. 79:6951-6955, (1982)).

Also, VIP1 and/or VIP2 and/or VIP3 or other VIPs of the invention can be introduced into the root colonizing Bacillus by means of electro-transformation. Specifically, VIPs can be cloned into a shuttle vector, for example, pHT3101 (D. Lereclus et al., FEMS Microbiol. Letts. 60:211-218 (1989)) as described in Example

10. The shuttle vector pHT3101 containing the coding sequence for the particular VIP can then be transformed into the root colonizing Bacillus by means of electroporation (D. Lereclus et al. 1989, FEMS Microbiol. Letts. 60:211 -218).

Expression systems can be designed so that VIP proteins are secreted outside the cytoplasm of gram negative bacteria, E. coli, for example. Advantages of having VIP proteins secreted are (1) it avoids potential toxic effects of VIP proteins expressed within the cytoplasm and (2) it can increase the level of VIP protein expressed and (3) can aid in efficient purification of VIP protein.

VIP proteins can be made to be secreted in E. coli, for example, by fusing an appropriate E. co//signal peptide to the amino-terminal end of the VIP signal peptide or replacing the VIP signal peptide with the E. co//signal peptide. Signal peptides recognized by E. colican be found in proteins already known to be secreted in E. coli, for example the OmpA protein (J. Ghrayeb, H. Kimura, M. Takahara, Y. Masui and M. Inouye, EMBO J., 3:24371984) 2442־)). OmpA is a major protein of the E. co//outer membrane and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide (G. Duffaud, P. March and M. Inouye, Methods in Enzymology, 153:492 (1987)).

Specifically, unique BamHI restriction sites can be introduced at the aminoterminal and carboxy-terminal ends of the VIP coding sequences using standard methods known in the art. These BamHI fragments can be cloned, in frame, into the vector pIN-lll-ompA 1, A2 or A3 (J. Ghrayeb, H. Kimura, M. Takahara, H. Hsiung, Y. Masui and M. Inouye, EMBO J. 3:24371984) 2442־)) thereby creating 0mpA:VIP fusion gene which is secreted into the periplasmic space. The other restriction sites in the polylinker of pIN-lll-ompA can be eliminated by standard methods known in the art so that the VIP amino-terminal amino acid coding sequence is directly after the ompA signal peptide cleavage site. Thus, the secreted VIP sequence in E. coliwoM then be identical to the native VIP sequence.

When the VIP native signal peptide is not needed for proper folding of the mature protein, such signal sequences can be removed and replaced with the ompA signal sequence. Unique BamHI restriction sites can be introduced at the amino-termini of the proprotein coding sequences directly after the signal peptide coding sequences of VIP and at the carboxy-termini of VIP coding sequence. These BamHI fragments can then be cloned into the pIN-lll-ompA vectors as described above.

General methods for employing the strains of the invention in pesticide control or in engineering other organisms as pesticidal agents are known in the art. See, for example US Patent No. 5,039,523 and EP 0480762A2.

VIPs can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bacillus thuringiensis strains have been used as insecticidal sprays. In the case of a VIP(s) which is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the VIP protein(s) into the growth medium during the fermentation process. The VIPs are retained within the cell and the cells are then processed to yield the encapsulated VIPs. Any suitable microorganism can be used for this purpose. Psuedomonas has been used to express

Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide. (H. Gaertner etal. 1993, In Advanced

Engineered Pesticides, L. Kim ed.)

Various strains of Bacillus thuringiensis are used in this manner. Such Bt strains produce endotoxin protein(s) as well as VIPs. Alternatively, such strains can produce only VIPs. A sporulation deficient strain of Bacillus subtilis has been shown to produce high levels of the Cry 111A endotoxin from Bacillus thuringiensis (Agaisse, H. and Lereclus, D., “Expression in Bacillus subtilis of the Bacillus thuringiensis CrylllA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spoOA mutant”, J. Bacteriol., 176:4734-4741 (1994)). A similar spoOA mutant can be prepared in Bacillus thuringiensis and used to produce encapsulated VIPs which are not secreted into the medium but are retained within the cell.

To have VIPs maintained within the Bacillus cell the signal peptide can be disarmed so that it no longer functions as a secretion signal. Specifically, the putative signal peptide for VIP1 encompasses the first 31 amino acids of the protein with the putative consensus cleavage site, Ala-X-Ala, at the C-terminal portion of this sequence (G. von Heijne , J. Mol. Biol. 184:99-105 (1989)) and the putative signal peptide for VIP2 encompasses the first 40 amino acids of the protein with the putative cleavage site after Ala40. The cleavage sites in either VIP1 or VIP2 can be mutated with methods known in the art to replace the cleavage site consensus sequence with alternative amino acids that are not recognized by the signal peptidases.

Alternatively, the signal peptides of VIP1, V1P2 and/or other VIPs of the invention can be eliminated from the sequence thereby making them unrecognizable as secretion proteins in Bacillus. Specifically, a methionine start site can be engineered in front of the proprotein sequence in VIP1, starting at Asp32, or the proprotein sequence in VIP2, starting at Glu41 using methods known in the art.

VIP genes can be introduced into micro-organisms that mutiply on plants (epiphytes) to deliver VIP proteins to potential target pests. Epiphytes can be grampositive or gram-negative bacteria for example.

The Bacillus strains of the invention or the microorganisms which have been genetically altered to contain the pesticidal gene and protein may be used for

-‘40- :.

protecting agricultural crops and products from pests. In one aspect of the invention, whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).

Alternatively, the pesticides are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment Of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.

The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, mollusicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

Preferred methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention which contains at least one of the insect-specific proteins produced by the bacterial strains of the present invention are leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest

The׳ present invention thus further provides an entomocidal composition comprising as an active ingrdient at least one of the novel insect-specific proteins according to the invention and/or a recombinant microorganism containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insectspecific proteins in recombinant form, but especially a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis, containing at least one one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form, or a derivative or mutant thereof, together with an agricultural adjuvant such as a carrier, diluent, surfactant or application-promoting adjuvant. The composition may also contain a further biologically active compound. The said compound can be both a fertilizer or micronutrient donor or other preparations that influence plant growth. It can also be a selective herbicide, insecticide, fungicide, bactericide, nematicide, molluscide or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers

The composition may comprise from .0.1 to 99% by weight of the active ingredient, from 1 to 99.9% by weight of a solid or liquid adjuvant, and from 0 to 25% by weight of a surfactant. The acitve ingredient comprising at least one of the novel insect-specific proteins according to the invention or a recombinant microorganism containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insectspecific proteins in recombinant form, but especially a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific; proteins in recombinant form, or a derivative or mutant thereof, or the composition containing the said acitve ingredient, may be administered to the plants or crops to be protected together with certain other insecticides or chemicals (1993 Crop Protection Chemicals Reference, Chemical and Pharmaceutical Press, Canada) without loss of potency. It is compatible with most other commonly used agricultural spray materials but should not be used in extremely alkaline spray solutions. It may be administered as a dust, a suspension, a wettable powder or in any other material form suitable for agricultural application.

The invention further provides methods for for controlling or inhibiting of insect pests by applying an active ingredient comprising at least one of the novel insectspecific proteins according to the invention or a recombinant microorganism containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form or a composition comprising the said active ingredient to (a) an environment in which the insect pest may occur, (b) a plant or plant part in order to protect said plant or plant part from damage caused by an insect pest, or (c) seed in order to protect a plant which develops from said seed from damage caused by an insect pest.

A preferred method of application in the area of plant protection is application to the foliage of the plants (foliar application), with the number of applications and the rate of application depending on the plant to be protected and the risk of infestation by the pest in question. However, the active ingredient may also penetrate the plants through the roots (systemic action) if the locus of the plants is impregnated with a liquid formulation or if the active ingredient is incorporated in solid form into the locus of the plants, for example into the soil, e.g. in granular form (soil application). In paddy rice crops, such granules may be applied in metered amounts to the flooded rice field.

The compositions according to the invention are also suitable for protecting plant propagating material, e.g. seed, such as fruit, tubers or grains, or plant cuttings, from insect pests. The propagation material can be treated with the formulation before planting: seed, for example, can be dressed before being sown. The acitve ingredient of the invention can also be applied to grains (coating), either by impregnating the grains with a liquid formulation or by coating them with a solid formulation. The formulation can also be applied to the planting site when the propagating material is being planted, for example to the seed furrow during sowing. The invention relates also to those methods of treating plant propagation material and to the plant propagation material thus treated.

The compositions according to the invention comprising as an active ingredient a recombinant microorganism containing at least one of the novel toxin genes in recombinant form, but especially a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form, or a derivative or mutant thereof may be applied in any method known for treatment of seed-or soil with bacterial strains. For example, see US Patent

N0.4,863,866. The strains are effective for biocontrol even if the microorganism is not living. Preferred is, however, the application of the living microorganism.

Target crops to be protected within the scope of the present invention comprise,

e.g., the following species of plants:

cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beet (sugar beet and fodder beet), forage grasses (orchardgrass, fescue, and the like), drupes, pomes and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries), leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants (cucumber, marrows, melons) fiber plants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus, cabbages and other Brassicae, onions, tomatoes, potatoes, paprika), lauraceae (avocados, carrots, cinnamon, camphor), deciduous trees and conifers (e.g. , linden-trees, yew-trees, oak-trees, alders, poplars, birch-trees, firs, larches, pines), or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants, as well as ornamentals (including composites).

A recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain, containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form is normally applied in the form of entomocidal compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with further biologically active compounds. These compounds may be both fertilizers or micronutrient donors or other preparations that influence plant growth. They may also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several ׳of these preparations, if desired together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation.

The active ingredient according to the invention may be used in unmodified form or together with any suitable agriculturally acceptable carrier. Such carriers are adjuvants conventionally employed in the art of agricultural formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances. Like the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objective and the prevailing circumstances. Advantageous rates of application are normally from about 50 g to about 5 kg of active ingredient (a.i.) per hectare (ha, approximately 2.471 acres), preferably from about 100 g to about 2kg a.i./ha. Important rates of application are about 200 g to about 1kg a.i./ha and 200g to 500g

a.i./ha.

For seed dressing advantageous application rates are 0.5 g to 1000 g a.i.per 100 kg seed, preferably 3 g to 100 g a.i. per 100 kg seed or 10 g to 50 g a.i.per 100 kg seed.

Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. The formulations, i.e. the entomocidal compositions, preparations or mixtures containing the recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form as an active ingredient or combinations thereof with other active ingredients, and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, e.g., by homogeneously mixing and/or grinding the active ingredients with extenders, e.g., solvents, solid carriers, and in some cases surface-active compounds (surfactants).

Suitable solvents are: aromatic hydrocarbons, preferably the fractions containing 8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethylsulfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils such as epoxidised coconut oil or soybean oil; or water.

The solid carriers used, e.g., for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite;

and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used,

e.g. especially dolomite or pulverized plant residues.

Depending on the nature of the active ingredients to be formulated, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term surfactants will also be understood as comprising mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds. Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C™ -C₂₂), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained, e.g. from coconut oil or tallow oil. Further suitable surfactants are also the fatty acid methyltaurin salts as well as modified and unmodified phospholipids.

More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates. The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts or unsubstituted dr substituted ammonium salts and generally contain a C_B -C₂₂ alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. the sodium or calcium salt of lignosulfonic acid, of dodecylsulfate, or of a fixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing about 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

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

Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit. Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan, such as polyoxyethylene sorbitan trioleate, are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which contain, as N־substituent, at least one C₈ -C₂₂ alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or hydroxyl-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g., stearyltrimethylammonium chloride or benzyldi-(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described, e.g., in McCutcheon’s Detergents and Emulsifiers Annual, MC Publishing Corp. Ridgewood, N.J., 1979; Dr. Helmut Stache, Tensid Taschenbuch (Handbook of Surfactants), Carl Hanser Verlag, Munich/Vienna.

Another particularly preferred characteristic of an entomocidal composition of the present invention is the persistence of the active ingredient when applied to plants and soil. Possible causes for loss of activity include inactivation by ultra-violet light, heat, leaf exudates and pH. For example, at high pH, particularly in the presence of reductant, δ-endotoxin crystals are solubilized and thus become more accessible to proteolytic inactivation. High leaf pH might also be important, particularly where the leaf surface can be in the range of pH 8-10. Formulation of an entomocidal composition of the present invention can address these problems by either including additives to help prevent loss of the active ingredient or encapsulating the material in such a way that the active ingredient is protected from inactivation. Encapsulation

-Μ can be accomplished chemically (McGuire and Shasha, J Econ Entomol 85:14251433,1992) or biologically (Barnes and Cummings, 1986; EP-A 0 192 319). Chemical encapsulation involves a process in which the active ingredient is coated with a polymer while biological encapsulation involves the expression of the δ-endotoxin genes in a microbe. For biological encapsulation, the intact microbe containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form is used as the active ingredient in the formulation. The addition of UV protectants might effectively reduce irradiation damage. Inactivation due to heat could also be controlled by including an appropriate additive.

Preferred within the present application are formulations comprising living microorganisms as active ingredient either in form of the vegetative cell or more preferable in form of spores, if available. Suitable formulations may consist, for example, of polymer gels which are crosslinked with polyvalent cations and comprise these microorganisms. This is described, for example, by D.R. Fravel et al. in Phytopathology, Vol. 75, No. 7, 774-777,1985 for alginate as the polymer material. It is also known from this publication that carrier materials can be co-used. These formulations are as a rule prepared by mixing solutions of naturally occurring or synthetic gel-forming polymers, for example alginates, and aqueous salt solutions of polyvalent metal ions such that individual droplets form, it being possible for the microorganisms to be suspended in one of the two or in both reaction solutions. Gel formation starts with the mixing in drop form. Subsequent drying of these gel particles is possible. This process is called ionotropic gelling. Depending on the degree of drying, compact and hard particles of polymers which are structurally crosslinked via polyvalent cations and comprise the microorganisms and a carrier present predominantly uniformly distributed are formed. The size of the particles can be up to 5 mm.

Compositions based on partly crosslinked polysaccharides which, in addition to a microorganism, for example, can also comprise finely divided silicic acid as the carrier material, crosslinking taking place, for example, via Ca⁺⁺ ions, are described in EP-A1 -0 097 571. The compositions have a water activity of not more than 0.3. W.J. Cornick et al. describe in a review article [New Directions in Biological Control: Alternatives for Suppressing Agricultural Pests and Diseases, pages 345-372, Alan R.

Liss, Inc. (1990)] various formulation systems, granules with vermiculite as the carrier and compact alginate beads prepared by the ionotropic gelling process being mentioned. Such compositions are also disclosed by D.R.Fravel in Pesticide Formulations and Application Systems: 11th Volume, ASTM STP 1112 American Society for Testing and Materials, Philadelphia, 1992, pages 173 to 179 and can be used to formulate the recombinant microorganisms according to the invention.

The entomocidal compositions of the invention usually contain from about 0.1 to about ,99%, preferably about 0.1 to about 95%, and most preferably from about 3 to about90%־ of the active ingredient, from about 1 to about 99.9%, preferably from about 1 to about 99%, and most preferably from about 5 to about 95% of a solid or liquid Adjuvant, and from about 0 to about 25%, preferably about 0.1 to about 25%, and most preferably from about 0.1 to about 20% of a surfactant.

In a preferred embodiment of the invention the entomocidal compositions usually contain 0.1 to 99%, preferably 0.1 to 95%, of a recombinant Bacillus spp strain, such as Bacillus cereus or Bacillus thuringiensis strain containing at least one DNA molecule comprising a nucleotide sequence encoding the novel insect-specific proteins in recombinant form, or combination thereof with other active ingredients, 1 to 99.9% of a solid or liquid adjuvant, and 0 to 25%, preferably 0.1 to 20%, of a surfactant.

Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations of substantially lower concentration. The entomocidal compositions may also contain further ingredients, such as stabilizers, antifoams, viscosity regulators, binders, tackifiers as well as fertilizers or other active ingredients in order to obtain special effects.

In one embodiment of the invention a Bacillus cereus microorganism has been isolated which is capable of killing Diabrotica virgifera virgifera, and Diabrotica longicornis barberi. The novel B. cereus strain AB78 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, IL 61604, USA and given Accession No. NRRL B-21058.

A fraction protein has been substantially purified from the B. cereus strain. This purification of the protein has been verified by SDS-PAGE and biological activity. The protein has a molecular weight of about 60 to about 100 kDa, particularly about 70 to about 90 kDa, more particularly about 80 kDa, hereinafter VIP.

Amino-terminal sequencing has revealed the N-terminal amino-acid sequence to Uc י ^NH2-Lys-Arg-Glu-ll_e._Asp.G|_uA_Sp-Thr-Asp-Thr-_Asx-Gly-_Asp-Ser-llo-Pro(SEQ ID NO:8) where Asx represents either Asp or As״. The entire amino acid sequence is given in SEQ ID NO:7. The DNA sequence which encodes the amino acid sequence of SEQ ID NO:7 is disclosed in SEQ ID NO6־

A״ oligonuleotide probe for the region of the gene encoding amino acids 3-9 of the NH₂-ter״״,1״s has been generated. The probe was synthesized based on the codon ־״age of a Sac,־״/׳ _{tMngiensis (Bt) 5}._end₁₀״xin _{gene Τή}θ the oligonucleotide probe used lor Southern hybridizations was as follows:

5־ GAA ATT GATCAA GAT AON GAT-3’ (SEQIDNO-9) where N represents any base.

In addition, the DNA probe for the Be AB78 VIP1 gene described herein, permits e screening of any Baallus strain or other organisms to determine whether the VIP1 gene (or related gene) _is ״aturatly θ organism includes the VIP1 gene.

The invention now being.generatly described, the same will be better understood y reference to the following detailed examples that are provided for the purpose of . ustration and are not to be considered limiting of the invention unless so specified

A standard nomenclature has been developed based on the sequence identity oi *he proteins encompassed by the present i״״e״,_ion. _{The gene}he detailed examples which fellow and their relationship to the names used in t e first priority document are shown below.

<td> Gene/Protein</td><td> Gene / Description of Protein</td>

<td> Name under</td><td> Protein</td>

<td> Standard</td><td> Name in</td>

<td> Nomenclature</td><td> Parent</td>

<td> VIP1A(a)</td><td> VIP 1 VIP1 from strain AB78 as disclosed in SEQ ID NO:5.</td>

<td> VIP2A(a)</td><td> VIP2 VIP2 from strain AB78 as disclosed in SEQ ID NO:2.</td>

<td> VIPIA(b)</td><td> VIP1 VIP1 from Bacillus thuringiensisvar. homolog tenebrionis as disclosed in SEQ ID NO:21.</td>

<td> VIP2A(b)</td><td> VIP2 VIP2 from Bacillus thuringiensisvar. homolog tenebrionis as disclosed in SEQ ID NQ:20.</td>

<td> VIP3A(a)</td><td> VIP from strain AB88 as disclosed in SEQ ID NO:28 of the present application</td>

<td> VIP3A(b)</td><td> VIP from strain AB424 as disclosed in SEQ ID NO:31 of the present application</td>

EXPERIMENTAL

Formulation Examples

The active ingredient used in the following formulation examples are Bacillus cereus strain AB78 having Accession No. NRRL B-21058; Bacillus thuringiensis strains having Accession Nos. NRRL B-21060, NRRL B-21224, NRRL B-21225, NRRL B21226, NRRL B-21227, and NRRL B-21439; and Bacillus spp strains having Accession Nos NRRL B-21228, NRRL B-21229, and NRRL B-21230. All the mentioned strains are natural isolates comprising the insect-specific proteins according to the invention.

Alternatively, the isolated insect-specific proteins are used as the active ingredient alone or in combination with the above-mentioned Bacillus strains.

A1. Wettable powders

<td></td><td> a)</td><td> b)</td><td> c)</td>

<td> Bacillus thuringiensis spores</td><td> 25%</td><td> 50%</td><td> 75%</td>

<td> sodium lignosufonate</td><td> 5%</td><td> 5%</td><td> —</td>

<td> sodium laurylsulfate</td><td> 3%</td><td> —</td><td> 5%</td>

<td> sodium diisobutylnaphthalenesulfonate</td><td> .</td><td> 6%</td><td> 10%</td>

<td> octylphenol polyethylene glycol ether</td><td> —</td><td> 2%</td><td> —</td>

<td> (7-8 moles of ethylene oxid) highly dispersed silicid acid</td><td> 5%</td><td> 10%</td><td> 10%</td>

<td> kaolin</td><td> 62%</td><td> 27%</td><td> --</td>

The spores are thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations.

A2. Emulsifiable concentrate

Bacillus thuringiensis spores 10% octylphenol polyethylene glycol ether (4:5 moles ethylene oxide) 3% clacium;dodecylbenzensulfonate 3% castor oil polyglycol ether (36 moles of ethylene oxide) 4% cyclohexanone 30% xylene mixture 50%

Emulsions of any required concentration can be obtained from this concentrate by dilution with water.

A3. Dusts

a) b)

Bacillus thuringiensis spores 5% 8% talcum 95% kaolin - 92%

Ready for use dusts are obtained by mixing the active ingredient with the carriers and grinding the mixture in a suitable mill.

A4. Extruder Granulate

Bacillus thuringiensis spores 10% sodium lignosulfonate 2% carboxymethylcellulose 1% kaolin 87%

The active ingredient or combination is mixed and ground with the adjuvants and the mixture is subsequently moistened with water. The mixture is extruded, granulated and the dried in a stream of air.

A5. Coated Granule

Bacillus thuringiensis spores 3% polyethylene glycol (mol wt 200) 3% kaolin 94%

The active ingredient or combination is uniformly applied in a mixer to the kaolin moistened with polyethylene glycol. Non-dusty coated granulates are obtained in this manner.

A6. Suspension Concentrate

Bacillus thuringiensis spores 40% ethyleneglycol 10% nonylphenol polyethylene glycol ether (15 moles of ethylene oxide) 6% sodium lignosulfonate 10% carboxymethylcellulose 1%

37% aqueous formaldehyde solution 0.2% silicone oil in the form of a 75% aqueous solution 0.8% water 32%

The active ingredient or combination is intimately mixed with the adjuvants giving a suspension concentrate from which suspensions of any desired concentration can be obtained by dilution with water.

EXAMPLE 1. AB78 ISOLATION AND CHARACTERIZATION

Bacillus cereus strain AB78 was isolated as a plate contaminant in the laboratory on T3 media (per liter: 3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate (pH 6.8), and 0.005 g MnCI₂; Travers, R.S. 1983). During log phase growth, AB78 gave significant activity against western corn rootworm. Antibiotic activity against gram-positive Bacillus spp. was also demonstrated (Table 12);

TABLE 12

Antibiotic activity of AB78 culture supernatant

Zone of inhibition(cm)

Bacteria tested AB78 Streptomycin

E. coli 0.0 3.0

<td> B. megaterium</td><td> 1.1</td><td> 2.2</td>

<td> B. mycoides</td><td> 1.3</td><td> 2.1</td>

<td> B. cereus CB</td><td> 1.0</td><td> 2.0</td>

<td> B. cereus 11950</td><td> 1.3</td><td> 2.1</td>

<td> B. cereus 14579</td><td> 1.0</td><td> 2.4</td>

<td> B. Cereus AB78</td><td> 0.0</td><td> 2.2</td>

<td> Btvar. israelensis</td><td> 1.1</td><td> 2.2</td>

<td> Btvar. tenebrionis</td><td> 0.9</td><td> 2.3</td>

Morphological characteristics of AB78 are as follows:

Vegetative rods straight, 3.1-5.0 mm long and 0.5-2.0 mm wide. Cells with rounded ends, single in short chains. Single subterminal, cylindrical-oval, endospore formed per cell. No parasporal crystal formed. Colonies opaque, erose, lobate and flat. No pigments produced. Cells motile. Flagella present.

Growth characteristics of AB78 are as follows:

Facultative anaerobe with optimum growth temperature of 21-30°C. Will grow at 15, 20, 25,30 and 37°C. Will not grow above 40°C. Grows in 5-7% NaCl.

Tabte 13 provides the biochemical profile of AB78.

TABLE 13

Biochemical characteristics of B. cereus strain AB78.

<td> Acid from L-arabinose</td><td> Methylene blue reoxidized</td><td> +</td>

<td> Gas from L-arabinose</td><td> Nitrate reduced</td><td> +</td>

<td> Acid from D-xylose</td><td> NO₃ reduced to NO₂</td><td> +</td>

<td> Gas from D-xylose -</td><td> VP</td><td> -ו-</td>

<td> Acid from D-glucose +</td><td> H₂O₂ decomposed</td><td> -».</td>

<td> Gas from D-glucose</td><td> Indole</td><td> -</td>

<td> Acid from lactose</td><td> Tyrosine decomposed</td><td> +</td>

<td> Gas from lactose</td><td> Dihydroxiacetone</td><td> -</td>

<td> Acid from sucrose</td><td> Litmus milk acid</td><td> -</td>

<td> Gas from sucrose</td><td> Litmus milk coagulated</td><td> -</td>

<td> Acid from D-mannitol</td><td> Litmus milk alkaline</td><td> -</td>

<td> Gas from D-mannitol</td><td> Litmus milk peptonized</td><td> -</td>

<td> Proprionate utilization +</td><td> Litmus milk reduced</td><td> -</td>

<td> Citrate utilization +</td><td> Casein hydrolyzed</td><td> +</td>

<td> Hippurate hydrolysis w</td><td> Starch hydrolyzed</td><td> +</td>

<td> Methylene blue reduced + Lecithinase produced w</td><td> Gelatin liquidified</td><td> +</td>

w= weak reaction

EXAMPLE 2. BACTERIAL CULTURE

A subculture of Be strain AB78 was used to inoculate the following medium, known as TB broth:

<td> Tryptone Yeast Extract Glycerol kh₂po₄k₂hpo₄pH 7.4</td><td> 12 g/l 24 g/l 4 ml/l 2.1 g/l 14.7 g/l</td>

The potassium phosphate was added to the autoclaved broth after cooling.

Flasks were incubated at 30°C on a rotary shaker at 250 rpm for 24 h-36 h, which represents an early to mid-log growth phase.

The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

During vegetative growth, usually 24-36 h. after starting the culture, which represents an early to mid-dog growth phase, AB78 bacteria were centrifuged from the culture supernatant. The culture supernatant containing the active protein was used in bioassays.

EXAMPLE 3. INSECT BIOASSAYS

B. cereus strain AB78 was tested against various insects as described below.

Western, Northern and Southern corn rootworm, Diabrotica virgifera virgifera, D. longcornis barberi and D. undecempunctata howardi, respectively: dilutions were made of AB78 culture supernatant grown 24-36 h., mixed with molten artificial diet (Marrone et al. (1985) J. of Economic Entomology 78:290-293) and allowed to solidify. Solidified diet was cut and placed in dishes. Neonate larvae were placed on the diet and held at 30 C. Mortality was recorded after 6 days.

£ ¢0//clone bioassav: E. colicells were grown overnight in broth containing 100 jig/ml ampicillin at 37°C. Ten ml culture was sonicated 3X for 20 sec each. 500 μΙ of sonicated culture was added to molten western corn rootworm diet.

Colorado potato beetle, Leptinotarsa decemlineata: dilutions in Triton X-100 (to give final concentration of 0.1% TX-100) were made of AB78 culture supernatant grown 24-36 h. Five cm² potato leaf pieces were dipped into these dilutions, air dried, and placed on moistened filter paper in plastic dishes. Neonate larvae were placed on the leaf pieces and held at 30°C. Mortality was recorded after 3-5 days.

Yellow mealworm, Tenebrio molitor dilutions were made of AB78 culture supernatant grown 24-36 h., mixed with molten artificial diet (Bioserv #F9240) and allowed to solidify. Solidified diet was cut and placed in plastic dishes. Neonate larvae were placed on the diet and held at 30°C. Mortality was recorded after 6-8 days.

European corn borer, black cutworm, tobacco budworm, tobacco hornworm and beet armyworm; Ostrinia nubilalis, Agrotis ipsilon, Heliothis virescens, Manduca sexta and Spodoptera exigua, respectively: dilutions, in TX-100 (to give final concentration of 0.1% TX-100), were made of AB78 culture supernatant grown 24-36 hrs. 100 μί was pipetted onto the surface of 18 cm of solidified artificial diet (Bioserv #F9240) and allowed to air dry. Neonate larvae were then placed onto the surface of the diet and held at 30°C. Mortality was recorded after 3-6 days.

Northern house mosquito, Culex p/p/ens:-dilutions were made of AB78 culture supernatant grown 24-36 h. 100 μΙ was pipetted into 10 ml water in a 30 ml plastic cup. Third instar larvae were added to the water and held at room temperature. Mortality was recorded after 24-48 hours. The spectrum of entomocidal activity of AB78 is given in Table 14.

TABLE 14

Activity of AB78 culture supernatant against various insect species

Insect species tested to date ________ Order___________Activity

<td></td><td></td>

<td> Western corn rootworm (Diabrotica virgifera virgifera) Northern corn rootworm</td><td> Col +++</td>

<td> (Diabrotica longicornis barbed) Southern corn rootworm (Diabrotica undecimpunctata howardi) Colorado potato beetle (Leptinotarsa decemlineata) Yellow mealworm</td><td> Col +++ Col Col</td>

<td> (Tenebrio molitoi)</td><td> Col</td>

<td colspan="2"> European corn borer</td>

<td> (Ostrinia nubilalis)</td><td> Lep</td>

<td> Tobacco budworm (Heliothis virescens) Tobacco hornworm</td><td> Lep</td>

<td> (Manduca sexta)</td><td> Lep</td>

<td> Beet armyworm (Spodoptera exigua) Black cutworm</td><td> Lep</td>

<td> (Agrotis ipsilon)</td><td> Lep</td>

<td> Northern house mosquito (Culex pipiens)</td><td> Dip</td>

The newly discovered B. cereus strain AB78 showed a significantly different spectrum of insecticidal activity as compared to known coleopteran active δ-endqtoxins from Bt. In particular, AB78 showed more selective activity against beetles than known coleopteran-active Bt strains in that it was specifically active against Diabrotica spp. More specifically, it was most active against D. virgifera virgifera and D. longicornis barberi but not D. undecimpunctata howardi.

A number of Bacillus strains were bioassayed for activity during vegetative growth (Table 15) against western corn rootworm. The results demonstrate that AB78 is unique in that activity against western corn rootworm is not a general phenomenon.

TABLE 15

Activity of culture supernatants from various Bacillus spp. against western corn rootworm

<td> Bacillus strain</td><td> Percent WCRW mortality</td>

<td> B. cereus AB78 (Bat.1)</td><td> 100</td>

<td> B: cereus AB78 (Bat.2)</td><td> 100</td>

<td> B. cereus (Carolina Bio.)</td><td> 12</td>

<td> B. cereus MCC 11950</td><td> 12</td>

<td> B. cereus ATCC 14579</td><td> 8</td>

<td> B. mycoides (Carolina Bio.)</td><td> 30</td>

<td> B. popilliae</td><td> 28</td>

<td> B. thuringiensis HD135</td><td> 41</td>

<td> B. thuringiensis H D191</td><td> 9</td>

<td> B. thuringiensis GC91</td><td> 4</td>

<td> B. thuringiensis isrealensis</td><td> 24</td>

<td> Water Control</td><td> 4</td>

Specific activity of AB78 against western corn rootworm is provided in Table 16.

TABLE 16

Activity of AB78 culture supernatant against neonate western corn rootworm

Culture supernatant Percent concentration (ul/ml)________WCRW mortality

<td> 100</td><td> 100</td>

<td> 25</td><td> 87</td>

<td> 10</td><td> 80</td>

<td> 5</td><td> 40</td>

<td> 2.5</td><td> 20</td>

<td> 1</td><td> 6</td>

<td> 0 .</td><td> 0</td>

The LC50 was calculated to be 6.2 μΙ of culture supernatant per ml of western corn rootworm diet.

The cell pellet was also bioassayed and had no activity against WCRW. Thus, the presence of activity only in the supernatant indicates that this VIP is an exotoxin.

EXAMPLE 4 ISOLATION AND PURIFICATION OF CORN ROOTWORM

ACTIVE PROTEINS FROM AB78.

Culture media free of cells and debris was made to 70% saturation by the addition of solid ammonium sulfate (472 g/L). Dissolution was at room temperature followed by cooling in an ice bath and centrifugation at 10,000 X g for thirty minutes to pellet the precipitated proteins. The supernatant was discarded and the pellet was dissolved i I in 1/10 the original volume of 20 mM TRIS-HCI at pH 7.5. The dissolved pellet was desalted either by dialysis in 20 mM TRIS-HCI pH 7.5, or passing through a desalting column;

.The desalted material was titrated to pH 3.5 using 20 mM sodium citrate pH 2.5. Following a thirty minute room temperature incubation the solution was centrifuged at

3000 X g for ten minutes. The supernatant at this stage contained the greatest amount of active protein.

Following neutralization of the pH to 7.0 the supernatant was applied to a Mono-Q, anion exchange, column equilibrated with 20 mM TRIS pH 7.5 at a flow rate of 300 mL/min. The column was developed with a stepwise and linear gradient employing 400 mM NaCl in 20 mM TRIS pH 7.5.

Bioassay of the column fractions and SDS-PAGE analysis were used to confirm the active fractions. SDS-PAGE analysis identified the biologically active protein as having components of a molecular weight in the range of about 80 kDa and 50 kDa.

EXAMPLE 5. SEQUENCE ANALYSIS OF THE CORN ROOTWORM ACTIVE PROTEIN

The 80 kDa component isolated by SDS-PAGE was transferred to PVDF membrane and was subjected to amino-terminal sequencing as performed by I repetitive Edman cycles on an ABI470 pulsed-liquid sequencer. Transfer was carried out in 10 mM CAPS buffer with 10% methanol pH 11.0 as follows:

Incubation of the gel following electrophoresis was done in transfer buffer for five minutes. ProBlott PVDF membrane was wetted with 100% MeOH briefly then equilibrated in transfer buffer. The sandwich was arranged between foam sponges and filter paper squares with the configuration of cathode-gel-membrane-anode.

Transfer was performed at 70 V constant voltage for 1 hour.

Following transfer, the membrane was rinsed with water and stained for two minuteis with 0.25% Coomassie Blue R-250 in 50% MeOH.

Destaining was done with several rinses with 50% MeOH 40% water 10% acetic acid.

Following destaining the membrane was air dried prior to excision of the bands for sequence analysis. A BlottCartridge and appropriate cycles were utilized to achieve maximum efficiency and yield. Data analysis was performed using model 610 Sequence Analysis software for identifying and quantifying the PTH-amino acid derivatives for each sequential cycle.

The N-terminal sequence was determined to be:

NH2-Lys-Arg־Glu-lle-Asp-Glu-Asp-Thr-Asp-Thr-Asx-Gly-Asp-Ser-lle־Pro1 1 5 3 82 /2 (SEQ ID N0:8) where Asx represents Asp er As״. The complete amino acid sequence for the 80 kDa component is disclosed in SEQ ID NO-7 The DNA sequence which encodes SEQ ID NO:7 is disclosed in SEQ ID NO-6

EXAMPLE 6 CONSTRUCTION OF DNA PRORF

An oligonucleotide probe lor the region of the gene encoding amino 3cids 3-9 of he N-termmal sequence (Example 5) was generated. Th־ probe was synthesized based on the codon usage of a _{8acfc lhumgiens1s (Bt)}nucleotide sequence

5.- GAA ATT GAT CAA GAT ACN GAT -3' (SEQ ID NO:9) was used as a probe in Southern hybridizations. The oligonucleotide using standard procedures and equipment.

was synthesized

EXAMPLE 7. ISOEI FCTRIC PDINTpETERMINATION OF THE CORM .ROOTWORM ACTIVE PROTEIN

Purified protein from step 5 of the publication process was anaiyzed on a 3-9 pl ,־־electnc focus,ng gel using the Phastgel eiectrophoresis system (Pharmacia) Standard operate procedures for the unit were followed for both the separation and ver staining development procedures. The pl was approximated at about 4 9

EXAMPLE 8. PCR DATA ON AB78

PCR analysis (See, for example US Patent No. 5,506,099; and, ^Car°zz1 era/. (1991) AbqL Environ, M!crobipL57(11)3057-3061־ h_Pr_P^Γ6^^βΓθ^ηί:6^^{was use}d^t0 verify that the β. cereus strain AB^TSd^dno?00013^30^ ׳nsecticidsl crystal protein genes 018. thun_{ngiensis or e}. _{י ?)}

TABLE 17

Sac/7/us insecticidal crystal protein gene primers tested by PCR against AB78

DNA.

<td colspan="2"> Primers Tested Product Produced</td>

<td> 2 sets specific for Cry 111A</td><td> Negative</td>

<td> CrylllB</td><td> Negative</td>

<td> 2 sets specific for CrylA</td><td> Negative</td>

<td> CrylA(a)</td><td> Negative</td>

<td> CrylA(b) specific</td><td> Negative</td>

<td> CrylB</td><td> Negative</td>

<td> CryIC specific</td><td> Negative</td>

<td> Cry IE specific</td><td> Negative</td>

<td> 2 sets specific for S. sphaericus</td><td> Negative</td>

<td> 2 sets specific for CryIV</td><td> Negative</td>

<td> Bacillus control (PI-PLC) ,</td><td> Positive</td>

EXAMPLE 9. COSMID CLONING OF TOTAL DNA FROM B. CEREUSSTRMN

AB78

The VIP1 A(a) gene was cloned from total DNA prepared from strain AB78 as follows:

Isolation of AB78 DNA was as follows:

1. Grow bacteria in 10 ml L־broth overnight. (Use 50 ml sterile centrifuge tube)

2. Add 25 ml of fresh L-broth and ampicillin (30 gg/ml).

3. Grow cells 2-6 h. at 30°C with shaking.

4. Spin cells in a 50 ml polypropylene orange cap tube in IEC benchtop clinical centrifuge at 3/4 speed.

5. Resuspend cell pellet in 10 ml TES (TES = 50 mM TRIS pH 8.0,100 mM EDTA, 15 mM NaCI).

6. Add 30 mg lysozyme and incubate 2 hrs at 37°C.

7. Add 200 μΙ 20% SDS and 400 μΙ Proteinase K stock (20 mg/ml). Incubate at 37°C.

8. Add 200 μΙ fresh Proteinase K. Incubate 1 hr. at 55°C. Add 5 ml TES to make 15 ml final volume.

9. Phenol extract twice (10 ml phenol, spin at room temperature at 3/4 speed in an IEC benchtop clinical centrifuge). Transfer supernatant (upper phase) to a clean tube using a wide bore pipette.

10. Extract once with 1:1 vol. phenokchloroform/isoamyl alcohol (24:1 ratio).

11. Precipitate DNA with an equal volume of cold isopropanol; Centrifuge to pellet DNA.

12. Resuspend pellet in 5 ml TE.

13. Precipitate DNA with 0.5 ml 3M NaOAc pH 5.2 and 11 ml 95% ethanol. Place at-20°C for 2 h.

14. Hook DNA from tube with a plastic loop, transfer to a microfuge tube, spin, pipette off excess ethanol, dry in vacuo.

15. , Resuspend in 0.5 ml TE. Incubate 90 min. at 65°C to help get. DNA back into solution.

16. Determine concentration using standard procedures.

Cosmid Cloning of AB78

All procedures, unless indicated otherwise, were performed according to Stratagene Protocol, Supercos 1 Instruction Manual, Cat. No. 251301.

Generally, the steps were as follows:

A. Sau 3A partial digestion of the AB78 DNA.

B. Preparation of vector DNA

C. Ligation and packaging of DNA

D. Tittering the cosmid library

1. Start a culture of HB101 cells by placing 50 ml of an overnight culture in mis of TB with 0.2% maltose. Incubate 3.5 hrs. at 37°C.

2. Spin out cells and resuspend in 0.5 ml 10 mM MgSO4.

¹ 3. Add together:

100 !cells

100 I diluted packaging mixture

100 110 mM MgSO4

ITB

4. Adsorb at room temperature for 30 minutes with no shaking.

5. Add 1 ml TB and mix gently. Incubate 30 minutes at 37°C.

6. Plate 200 I onto L־amp plates. Incubate at 37°C overnight.

At least 400 cosmid clones were selected at random and screened for activity against western corn rootworm as described in Example 3. DNA from 5 active clones and 5 non-active clones were used in Southern hybridizations. Results demonstrated that hybridization using the above described oligonucleotide probe correlated with western corn rootworm activity (Table 18).

Cosmid clones P3-12 and P5-4 have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession Nos. NRRL B-21061 and NRRL B-21059 respectively.

TABLE 18

Activity of AB78 cosmid clones against western corn rootworm.

Mean

Clone percent mortality (N=4)

Clones which hybridize with probe

P1-7347

PT-8364

P2-269

P3-1285

P&amp;497

Clones which do not hybridize with probe

P1-2

P3t8

P3-912

P3-18Ο

Ρ4-69

EXAMPLE 10. IDENTIFICATION OF A 6 KB REGION ACTIVE AGAINST WESTERN CORN ROOTWORM.

DNA from P3-12 was partially digested with restriction enzyme Sau 3A, and ligated into the E. coli vector pUC19 and transformed into E. coli. A DNA probe specific for the 80 kDa VIP1 A(a) protein was synthesized by PCR amplification of a portion of P3-12 DNA. Oligonucleotides MK113 and MK117, which hybridize to portions of VIP1 A(a), were synthesized using the partial amino acid sequence of the 80 kDa protein. Plasmid subclones were identified by colony hybridization to the PCR-generated probe, and tested for activity against western corn rootworm. One such clone, PL2, hybridized to the PCR-generated fragment, and was active against western com rootworm in the assay previously described.

A 6 kb Cla I restriction fragment from pL2 was cloned into the Sma I site of the E. co/&lt;Bac///t/s shuttle vector pHT 3101 (Lereclus, D. etal., FEMS Microbiology Letters 60:211 -218 (1989)) to yield pCIB6201. This construct confers anti-western corn rootworm activity upon both Bacillus and E.colistrains, in either orientation. pCIB6022 contains this same 6 kb Cla I fragment in pBluescript SK(+) (Stratagene), produces equivalent VIP1A(a) protein (by western blot), and is also active against western corn rootworm.

The nucleotide sequence of pCIB6022 was determined by the dideoxy termination method of Sanger etal., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977), using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase® Terminator Double-Stranded DNA Sequencing Kit and analyzed on an ABI 373 automatic sequencer. The sequence is given in SEQ ID NO:1. The 6 kb fragment encodes both VIP1 A(a) and VIP2A(a), as indicated by the open reading frames described in SEQ ID NO:1. The sequence encoding VIP2A(a) is further disclosed in SEQ ID NO:4. The relationship between VIP1 A(a) and VIP2A(a) within the 6 kb fragment found in pCIB6022 is depicted in Table 19. pCIB6022 was deposited with the Agricultural Research Service, Patent Culture Collection) (NRRL),

Northern Regional Research Center, 1815 North University Street, Peoria, Illinois

61604, USA, and given the Accession No. NRRL B-21222.

EXAMPLE 11. FUNCTIONAL DISSECTION OF THE VIP1 A(a) DNA REGION.

To confirm that the VIP1 A(a) open reading frame (ORF) is necessary for insecticidal activity a translational frameshift mutation was created in the gene. The restriction enzyme Bgl II recognizes a unique site located 857 bp into the coding region of VIP1 A(a). pCIB6201 was digested with Bgl II, and the single-stranded ends filled-ΐη with DNA polymerase (Klenow fragment) and dNTPS. The plasmid was religated and transformed into E. coli. The resulting plasmid, pCIB6203, contains a four nucleotide insertion in the coding region of VIP1A(a). pCIB6203 does not confer WCRW insecticidal activity, confirming that VIP1 A(a) is an essential component of western corn rootworm activity.

To further define the region necessary to encode VIP1 A(a), subclones of the VIP1 A(a) and VIP2A(a) (auxiliary protein) region were constructed and tested for their ability to complement the mutation in pCIB6203. pCIB6023 contains the 3.7kb Xba IEcoRV fragment in pBluescript SK(+) (Stratagene). Western blot analysis indicates that pCIB6023 produces VIP1 A(a) protein of equal size and quantity as clones PL2 and pCIB6022. pCIB6023 contains the entire gene encoding the 80 kD protein. pCIB6023 was deposited with the Agricultural Research Service, Patent Culture &lt; Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given the Accession No. NRRL B-21223N. pCIB6206 contains the 4.3 kb Xba l-Cla I fragment from pCIB6022 in pBluescript SK(+) (Stratagene). pCIB6206 was also deposited with the Agricultural Research Service, Patent Culture Collection, (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given the Accession No. NRRL B21321.

pCIB6023, pCIB6206, and pCIB6203 do not produce detectable western corn rootworm activity when tested individually. However, a mixture of cells containing pCIB6203 (VIP1 A(a)-mutated, plus VIP2A(a)) and cells containing pCIB6023 (only

2/ 5382 ח

VIP.1 A(a)) ־hows high activity _{against westem com rootworm}

To further define the limit־ of VIP2_A(a), we constructed pCIB6024 which cont»־ ^{theeatlrey0 V,P2A}(^a)׳b^ut ¹acks most of the VIP1_A(a) coding region pC!B6024was constnjcted by gel purifying the 2.2 kb Cla l-Sca I restriction fragment from pCIB6022 I ing in the single-stranded ends with DNA polymerase (Klpnnw f dNTPs. _and 1i_gatin_g this _lragn1en, _{int0 pB|usscSK(+)} enzyme Eco RV. Cells containing pCIB6024 exhibit no activity _against western com rootworm. However, a mixture of celts containing _{pCIB6 24 and ells}^^{n a,n,n9} PC^{|B6023 sh}°ws high activity against western corn rootworm .{See TaWe

Thus, PCIB6023 and p_CIB6206 must produce a functional _V|_P1A(a)prod״״, while PCIB6203 and pCIB₆024 must produce a functionai V.pX _eneproduct These results suggest a requirement for a gene product(־) from the W2A(a) region. ,״ combination with VIPf _A(a), _{t0 confer maxima|}rootworm activity. (See Figure 1). ״־״»״> com

־

In Figure 1, boxed regions represent the extent of VIPiA(a) and VIP2A(at Whi! >

P״ »״־, VIP1 encoding rhe 80 _kDa pept,d־ ob״־־ed in 2L 0^^ ”

I

EXAMPLE 12. AB78 ANTIBODY PRODUCTION

Antibody production was initiated in 2 Lewis rats to allow for both the possibility of moving to production of hybridoma cell lines and also to produce enough serum for limited screening of genomic DNA library. Another factor was the very limited amount of antigen available and the fact that it could only be produced to purity by PAGE and subsequent electrotransfer to nitrocellulose.

Due to the limited availability of antigen on nitrocellulose, the nitrocellulose was emulsified in DMSO and injected into the hind footpads of the animals to elicit B-cell production in the popliteal lymph nodes just upstream. A strong reacting serum was produced as judged by western blot analysis with the first production bleed. Several subsequent injections and bleeds produced enough serum to accomplish all of the screening required.

Hybridoma production with one of the rats was then initiated. The popliteal lymph node was excised, macerated, and the resulting cells fused with mouse myeloma P3x63Ag8.653. Subsequent cell screening was accomplished as described below. Four initial wells were selected which gave the highest emulsified antigen reaction to be moved to limited dilution cloning. An additional 10 wells were chosen for expansion and cryoperservation.

Procedure to Emulsify AB78 on nitrocellulose in DMSO for ELISA screening:

After electrotransfer of AB78 samples run on PAGE to nitrocellulose, the reversible strain Ponceau S is used to visualize all protein transferred. The band corresponding to AB78 toxin, previously identified and N-terminal sequenced, was identified and excised from nitrocellulose. Each band is approximately 1 mm x 5 mm in size to minimize the amount of nitrocellulose emulsified. A single band is placed in a microfuge tube with 250 μΙ of DMSO and macerated using a plastic pestle (Kontes, Vineland, NJ). To aid in emulsification, the DMSO mixture is heated for 2-3 minutes at 37 C-45 C. Some further maceration might be necessary following heating; however, all of the nitrocellulose should be emulsified. Once the AB78 sample is emulsified, it is placed on ice. In preparation for microtiter plate coating with the emulsified antigen, the sample must be diluted in borate buffered saline as follows: 1:5,1:10,1:15,1:20, 1:30, 1:50, 1:100, and 0. The coating antigen must be prepared fresh immediately prior to use.

ELISA protocol:

1. Coat with AB78/DMSO in BBS. Incubate overnight at 4°C.

2. Wash plate 3X with 1X ELISA wash buffer.

3. Block (1% BSA &amp; 0.05% Tween 20 in PBS) for 30 minutes at Room Temperature.

4. Wash plate 3X with 1X ELISA wash buffer.

5. Add rat serum. Incubate 1.5 hours at 37°C.

6. Wash plate 3X with 1X ELISA wash buffer.

7. Add goat anti-rat at a concentration of 2 gg/ml in ELISA diluent. Incubate 1 hr. at 37°C.

8. Wash plate 3X with 1X ELISA wash buffer.

9. Add rabbit anti-goat alkaline phosphatase at 2 gg/ml in ELISA diluent. Incubate 1 hr. at 37°C.

10. Wash 3X with 1X ELISA wash buffer.

11. Add Substrate. Incubate 30 minutes at room temperature.

12. Stop with 3N NaOH after 30 minutes.

Preparation of VIP2A(a) Antisera

A partially purified AB78 culture supernatant was separated by discontinuous SDS PAGE (Novex) following manufacturer’s instructions. Separated proteins were electrophoresed to nitrocellulose (S&amp;S #21640) as described by Towbin etal., (1979). The nitrocellulose was stained with Ponceau S and the VIP2A(a) band identified. The VIP2A(a) band was excised and emulsified in DMSO immediately prior to injection. A rabbit was initially immunized with emulsified VIP2A(a) mixed approximately 1:1 with Freund's Complete adjuvant by intramuscular injection at four different sites. Subsequent immunizations occurred at four week intervals and were identical to the first, except for the use of Freund’ Incomplete adjuvant. The first serum harvested following immunization reacted with VIP2A(a) protein. Western blot analysis of AB78 culture supernatant using this antisera identifies predominately full length VIP2A(a) protein.

ד

EXAMPLE 13. ACTIVATION OF INSECTICIDAL ACTIVITY OF ΝΟΝ-ACTIVE BT STRAINS WITH AB78 VIP CLONES.

Adding pCIB6203 together with a 24 h culture (early to mid-log phase) supernatant from Bt strain GC91 produces 100% mortality in Diabrotica virgifera virgifera. Neither pCIB6203 nor GC91 is active on Diabrotica virgifera virgifera by itself. Data are shown below:

Test material Percent Diabrotica mortality ______

GC9116 pCIB6203 + GC91100

Control0

EXAMPLE 14. ISOLATION AND BIOLOGICAL ACTIVITY OF B. CEREUS AB81.

A second B. cereus strain, designated AB81, was isolated from grain bin dust samples by standard methodologies. A subculture of AB81 was grown and prepared for bioassay as described in Example 2. Biological activity was evaluated as described in Example 3. The results are as follows:

Insect species tested

Percent

Mortality

Ostrinia nubilalis0

Agrotis ipsilon0

Diabrotica virgifera virgifera55

EXAMPLE 15. ISOLATION AND BIOLOGICAL ACTIVITY OF

B. THURINGIENSIS AB6.

A B. thuringiensis strain, designated AB6, was isolated from grain bin dust samples by standard methods known in the art. A subculture of AB6 was grown and prepared for bioassay as described in Example 2. Half of the sample was autoclaved 15 minutes to test for the presence of β-exotoxin.

Biological activity was evaluated as described in Example 3. The results are as follows:

<td> Insect species tested</td><td> Percent Mortality</td>

<td> Ostrinia nubilalis</td><td> 0</td>

<td> Agrotis ipsilon Agrotis ipsilon (autoclaved sample) Diabrotica virgifera virgifera</td><td> 100 0 0</td>

The reduction of insecticidal acitivity of the culture supernatant to insignificant levels by autoclaving indicates that the active principle is not β-exotoxin.

Strain AB6 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given Accession No. NRRL B-21060.

EXAMPLE 16. ISOLATION AND BIOLOGICAL CHARACTERIZATION OF

B. THURINGIENSIS AB88.

A Bt strain, designated AB88, was isolated from grain bin dust samples by standard methodologies. A subculture of AB88 was grown and prepared for bioassay as described ih Example 2. Half of the sample was autoclaved 15 minutes to test for the presence of β-exotoxin. Biological activity was evaluated against a number of insect species as described in Example 3. The results are as follows:

<td rowspan="2"> Insect species tested</td><td rowspan="2"> Order</td><td colspan="2"> Percent mortality of culture supernatant Non-</td>

<td> autoclaved</td><td> Autoclav ed</td>

<td> Agrotis ipsilon</td><td> Lepidoptera</td><td> 100</td><td> 5</td>

<td> Ostrinia nubilalis Spodoptera</td><td> Lepidoptera</td><td> 100</td><td> 0</td>

<td> frugiperda</td><td> Lepidoptera</td><td> 100</td><td> 4</td>

<td> Helicoverpa zea</td><td> Lepidoptera</td><td> 100</td><td> 12</td>

<td> Heliothis virescens Leptinotarsa</td><td> Lepidoptera</td><td> 100</td><td> 12</td>

<td> decemlineata Diabrotica</td><td> Coleoptera</td><td> 0</td><td> 0</td>

<td> virgifera virgifera</td><td> Coleoptera</td><td> 0</td><td> 5</td>

The reduction of insecticidal acitivity of the culture supernatant to insignificant levels by autoclaving indicates that the active principle is not β-exotoxin.

Delta-endotoxin crystals were purified from strain AB88 by standard methodologies. No activity from pure crystals was observed when bioassayed against Agrotis ipsilon.

EXAMPLE 17 PURIFICATION OF VIPS FROM STRAIN AB88:

Bacterial liquid culture was grown overnight [for 12h] at 30°C in TB media. Cells were centrifuged at 5000 x g for 20 minutes and the supernatant retained. Proteins present in the supernatant were precipitated with ammonium sulfate (70% saturation), centrifuged [at 5000 x g for 15 minutes] and the pellet retained. The pellet was resuspended in the original volume of 20 mM Tris pH 7.5 and dialyzed overnight against the same buffer at 4°C. AB88 dialysate was more turbid than comparable material from AB78. The dialysate was titrated to pH 4.5 using 20 mM sodium citrate (pH 2:5) and, after 30 min incubation at room temperature, the solution was centrifuged at 3000 x g for 10 min. The protein pellet was redissolved in 20 mM BisTris-Propane pH 9.0.

AB88 proteins have been separated by several different methods following clarification including isoelectric focusing (Rotofor, BioRad, Hercules, CA), precipitation at pH 4.5, ion-exchange chromotography, size exclusion chromatography and ultrafiltration.

Proteins were separated on a Poros HQ/N anion exchange column (PerSeptive Biosystems, Cambridge, MA) using a linear gradient from 0 to 500 mM NaCl in 20 mM Bis-Tris-Propane pH 9.0 at a flow rate of 4 ml/min. The insecticidal protein eluted at 250 mM NaCl.

European corn borer (ECB)-active protein remained in the pellet obtained by pH

4.5 precipitation of dialysate. When preparative IEF was done on the dialysate using pH 3-10 ampholytes, ECB insecticidal activity was found in all fractions with pH of 7 or greater. SDS-PAGE analysis of these fractions showed protein bands of MW ~60 kDa and ~80 kDa. The 60 kDa and 80 kDa bands were separated by anion exchange HPLC on a Poros-Q column (PerSeptive Biosystems, Cambridge, MA). N־terminal sequence was obtained from two fractions containing proteins of slightly differing MW, but both of approximately 60 kDa in size. The sequences obtained were similar to each other and to some δ-endotoxins.

anion exchange fraction 23 (smaller): xEPFVSAxxxQxxx (SEQIDNO:10) anion exchange fraction 28 (larger): xEYENVEPFVSAx (SEQ ID NO:11)

When the ECB-active pH 4.5 pellet was further separated by anion exchange on a Poros-Q column, activity was found only in fractions containing a major band of ~60 kDa.

Black cutworm-active protein also remained in the pellet when AB88 dialysate was brought down to pH 4.5. In preparative IEF using pH 3-10 ampholytes, activity was not found in the ECB-activelEF fractions; instead, it was highest in a fraction of pH 4.5-5.0. Its major components have molecular weights of ~35 and ~80 kDa.

The pH 4.5 pellet was separated by anion exchange HPLC to yield fractions containing only the 35 kDa material and fractions containing both 35 kDa and 80 kDa bands.

EXAMPLE 18. CHARACTERIZATION OF AB88 VIP.

Fractions containing the various lepidopteran active vegetative proteins were generated as described in Example 17. Fractions with insecticidal acitivity were separated in 8 to 16% SDS-polyacrylamide gels and transferred to PVDF membranes [LeGendre et al, (1989) in: A Practical Guide to Protein and Peptide Purification for Microsequencing, ed Matsudaria PT (Academic Press Inc, New Yorkl]. Biological analysis of fractions demonstrated that different VIPs were responsible for the different lepidopteran species activity.

The Agrotis ipsilon activity is due to an 80 kDa and/or a 35 kDa protein, either delivered singly or in combination. These proteins are not related to any δ-endotoxins from Bt as evidenced by the lack of sequence homology of known Bt δ-endotoxin sequences. The vip3A(a) insecticidal protein from strain AB88 is present mostly (at least 75% of the total) in supernatants of AB88 cultures.

Also, these proteins are not found in the AB88 δ-endotoxin crystal. N-terminal sequences of the major δ-endotoxin proteins were compared with the N-terminal sequences of the 80 kDa and 35 kDa VIP and revealed no sequence homology. The N-terminal sequence of the vip3A(a) insecticidal protein posses a number of positively charged residues (from Asn2 to Asn7) followed by a hydrophobic core region (from Thr8 to Ile34). Unlike most of the known secretion proteins, the vip3A(a) insecticidal protein from strain AB88 is not N-terminally processed during export.

A summary of the results follows:

<td> AgrotisWP N-terminal sequences</td><td> N-terminal sequence of major δ-endotoxin proteins</td>

<td></td><td> 130 kDa MDNNPNINE (SEQ ID NO:14)</td>

<td> 80 kDa MNKNNTKLPTRALP (SEQ ID NO:12)</td><td> 80 kDa MDNNPNINE (SEQ ID NO:15)</td>

<td> 35 kDa ALSENTGKDGGYIVP (SEQ ID NO:13)</td><td> 60 kDa MNVLNSGRTTI (SEQ ID NO:16)</td>

<td colspan="2"> The Ostrinia nubilalis activity is due to a 60 kDa VIP and the Spodoptera frugiperda activity is due to a VIP of unknown size. Bacillus thuringiensis strain AB88 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center^ 1815 North University Street, Peoria, Illinois 61604, USA and given the</td>

Accession No. N R R L B-21225.

EXAMPLE 18A. ISOLATION AND BIOLOGICAL ACTIVITY OF 8.

THURINGIENSIS AB424

A B. thuringiensis strain, designated AB424, was isolated from a moss covered pine cone sample by standard methods known in the art. A subculture of AB424 was grown and prepared for bioassay as described in Example 2.

Biological activity was evaluated as described in Example 3. The results are as follows:

<td> Insect species tested</td><td> Percent mortality</td>

<td> Ostrinia nubilalis</td><td> 100</td>

<td> Agrotis ipsilon</td><td> 100</td>

<td> Diabrotica virgifera</td><td> 0</td>

<td> virgifera</td><td></td>

Strain AB424 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA, and given Accession No. NRRL B-21439.

EXAMPLE 18B. CLONING OF THE VIP3A(a) and VIP3A(b) GENES WHICH ENCODE PROTEINS ACTIVE AGAINST BLACK CUTWORM.

Total DNA from isolates AB88 and AB424 was isolated [Ausubel etal (1988), in: Current Protocols in Molecular Biology (John Wiley &amp; Sons, NY)] and digested with the restriction enzymes Xbal [library of 4.0 to 5.0 Kb size-fractionated Xbal fragments of B thuringiensis AB88 DNA] and EcoR\ [library of 4.5 to 6.0 Kb size-fractionated EcoRI fragments B thuringiensis AB424 DNA] respectively, ligated into pBluescript vector previously linearized with the same enzymes and dephosphorylated, and transformed into E. coli DH5a strain. Recombinant clones were blotted onto nitrocellulose filters which were subsequently probed with a ^3?P labeled 33-bases long oligonucleotide corresponding to the 11-N terminal amino acids of the 80 kDa protein active against Agrotis ipsilon (black cutworm). Hybridization was carried out at 42°C in 2 x SSC/0.1% SDS (1 x SSC = 0.15 m NaCI/0.015 M sodium citrate, pH 7.4) for 5 min and twice at 50°C in 1 x SSC/0.1 SDS for 10 min. Four out of 400 recombinant clones were positive. Insect bioassays of the positive recombinants exhibited toxicity to black cutworm larvae comparable to that of AB88 or AB424 supernantants.

Plasmid pCIB7104 contains a 4.5 Kb Xbal fragment of AB88 DNA. Subclones were constructed to define the coding region of the insecticidal protein.

E co//pCIB7105 was constructed by cloning the 3.5 Kb Xbal-Acclfragment of pCIB7104 into pBluescript.

Plasmid pCIB7106 contained a 5.0 Kb EcoRIfragment of AB424 DNA. This fragment was further digested with Hindi to render a 2.8 kb EcoRI-Hindl insert (pCIB7107), which still encoded a functional insecticidal protein.

The nucleotide sequence of pCIB7104, a positive recombinant clone from AB88, and of pCIB7107, a positive recombinant clone from AB424, was determined by the dideoxy termination method of Sanger etal., Proc. Natl. Acad. Sci. USA, 74:54635467 (1977), using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase® Terminator Double-Stranded DNA Sequencing Kit and analysed on an ABI373 automatic sequencer.

The clone pCIB7104 contains the VIP3A(a) gene whose coding region is disclosed in SEQ ID NO:28 and the encoded protein sequence is disclosed in SEQ ID NO:29. A synthetic version of the coding region designed to be highly expressed in maize is given in SEQ ID NO:30. Any number of synthetic genes can be designed based on the amino acid sequence given in SEQ ID NO:29.

The clone pCIB7107 contains the VIP3A(b) gene whose coding region is disclosed in SEQ ID NO:31 and the encoded protein is disclosed in SEQ ID NO:32. Both pCIB7104 and pCIB7107 have been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession Nos. NRRL B-21422 and B21423, respectively.

The VIP3A(a) gene contains an open reading frame (ORF) that extends form nucleotide 732 to 3105. This ORF encodes a peptide of 791 amino acids corresponding to a molecular mass of 88,500 daltons. A Shine-Dalgarno (SD) sequence is located 6 bases before the first methionine and its sequence identifies a strong SD for Eac///us.

The VIP3A(b) gene is 98% identical to VIP3A(a).

When blost of total DNA isolated from AB88 B thuringiensis cells were probed with a 33.base fragment that spans the N-terminal region of the VIP3A-insecticidal protein, single bands could be observed in different restriction digests. This result was confirmed by using larger probes spanning the coding region of the gene. A search of the Gen Bank data base revealed no homology to known proteins.

EXAMPLE 18C. EXPRESSION OF THE VIP3A INSECTICIDAL PROTEINS

The time course for expression of the VIP3A(a) insecticidal protein was analyzed by western blot. Samples from Bacillus thuringiensis Ab88 clutures were taken throughout ist growth curve and sporulation. The VIP3A(a) insecticidal protein can be detected in the supernatants of AB88 cultures during logarithmic phase, as early as 15 h after initiating the culture. It reached its maximum level during early stages of stationary phase and remained at high levels during and after sporulation. Similar results were obtained when supernatants of AB424 Bacillus cereus cultures were used. The levels of VIP3A(a) insecticidal protein reflected the expression of the VIP3A(a) gene as determined by Northern blot. The initiation of the sporulation was determined by direct microscopic observations and by analyzing the presence of δendotoxins in cell pellets. Cry-I type prtoeins could be detected late in the stationary phase , during and after sporulation.

EXAMPLE 18D. IDENTIFICATION OF NOVEL VIP3-LIKE GENES BY HYBRIDIZATION

To identify Bacillus containing genes related to the VIP3A(a) from isolate AB88, a collection of Bacillus isolates was screened by hybridization. Cultures of 463 Bacillus strains were grown in microtiter wells until sporulation. A 96-pin colony stampel was used to transfer the cultures to 150 mm plates containing L-agar. Inoculated plates were kept at 30°C for 10 hours, then at 4°C overnight. Colonies were blotted onto nylon filters and probed with a 1.2Kb Hzndlll VIP3A(a) derived fragment. Hybridization was performed overnight at 62°C using hybridization conditions of Maniatis et al. Molecular Cloning: A Laboratory Manual (1982). Filters were washed with 2xSSC/0.1% SDS at 62°C and exposed to X-ray film.

Of the 463 Bacillus strains screened, 60 contain VIP3-like genes that could detected by hybridization. Further characterization of some of them (AB6 and AB426) showed that their supernatants contain a BCW insecticidal protein similar to the Vip3 protein that are active against black cutworm.

EXAMPLE 18E. CHARACTERIZATION OF A B. thuringiensis SJRMH M2194 CONTAINING A CRYPTIC VIP3-LIKE GENE

A B. thuringiensis strain, designated M2194, was shown to contain VIP3-like gene(s) by colony hybridization as described in Example 18C. The M2194 VIP3 like gene is considered cryptic since no expression can be detected throughout the bacterial growth phases either by immunoblot analysis using polyclonal antibodies raised against the VIP3A(a) protein isolated from AB88 or by bioassay as described in Example 3.

Antiserum against purified VIP3A(a) insecticidal protein was produced in rabbits. Nictrocellulםseדbound protein (50 μ9) was dissolved in DMSO and emulsified with Freund's complete adjuvant (Difco). Two rabbits were given subcutaneous injections each month for three month. They were bled 10 days after the second and third injection and the serum was recovered from the blood sample [Harlow et al (1988) in : Antibodies: A Laboratory Manual (Cold Spring Harbor Lab Press, Plainview, NY)].

The M2194 VIP3-like gene was cloned into pKS by following the protocol described in Example 9, which created pCIB7108. E. co//containing pCIB7108 which comprises the M2194 VIP3 gene were active against black cutworm demonstrating that the gene encodes a functional protein with insecticidal activity. The plasmid pCIB7108 has been deposited with the Agricultural Research Service Patent Culture Collection (NRRL) and given Accession No. NRRL B-21438.

EXAMPLE 18F. INSECTICIDAL ACITIVITY OF VIP3A PROTEINS

The activity spectrum of VIP3A insecticidal proteins was qualitatively determined in insect bioassays in which recombinant E coli carrying the VIP‘A genes were fed to larvae. In these assays, cells carrying the VIP3A(a) and VIP3A(b) genes were insecticidal to Agrotis ipsilon, Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens and Helicoverpa zea. Under the same expermimental conditions, bacterial extracts containing VIP3A proteins did not show any activity against Ostrinia nubilalis.

Effect of VIP*A insecticidal proteins on Agrotis ipsilon larvae

<td> Treatment______________________ TB medium</td><td> (%) Mortality_____ 5</td>

<td> AB88 Supernatant</td><td> too</td>

<td> Ab424; Supernatant</td><td> 100</td>

<td> Buffer</td><td> 7</td>

<td> . E coli pKS</td><td> 10</td>

<td> Ec0//pCIB7104 (AB88)</td><td> 100</td>

<td> E co//pCIB7105 (AB88)</td><td> 100</td>

<td> E co//pCIB7106 (AB424)</td><td> 100</td>

<td> E co//pCIB7107 (AB424)</td><td> 100</td>

<td> Effect of VIP3A insecticidal proteins</td><td> on lepidopteran insect larvae</td>

<td> Treatment Insect</td><td> (%) Mortality</td>

<td> E co//pKS BCW</td><td> 10</td>

<td> FAW</td><td> 5</td>

<td> BAW</td><td> 10</td>

<td> TBW</td><td> 8</td>

<td> CEW</td><td> 10</td>

<td> ECB</td><td> 5</td>

<td> Ecoli pCIB7105</td><td></td>

<td> E co//pCIB7107 BCW</td><td> 100</td>

<td> FAW</td><td> 100</td>

<td> BAW</td><td> 100</td>

<td> ; TBW</td><td> 100</td>

<td> CEW</td><td> 50</td>

<td> ECB</td><td> 10</td>

BCW = Black Cut Worm; FAW = Fall Army Worm; BAW = Best Army Worm; TBW = Tobacco Bud Worm; CEW = Corn Ear Worm; ECB = European Corn Borer

EXAMPLE 19. ISOLATION AND BIOLOGICAL ACTIVITY OF OTHER

BACILLUS SP.

Other Bacillus species have been isolated which produce proteins with insecticidal activity during vegetative growth. These strains were isolated from environmental samples by standard methodologies. Isolates were prepared for bioassay and assayed as described in Examples 2 and 3 respectively. Isolates which produced insecticidal proteins during vegetative growth with activity against Agrotis ipsilon in the bioassay are tabulated below. No correlation was observed between the presence of a δ-endotoxin crystal and vegetative insecticidal protein production.

<td colspan="3"> Presence of δ-</td>

<td> Bacillus isolate</td><td> endotoxin crystal</td><td> Percent mortality</td>

<td> AB6</td><td> -ו-</td><td> 100</td>

<td> AB53</td><td> -</td><td> 80</td>

<td> AB88</td><td> -ו-</td><td> 100</td>

<td> AB195</td><td> -</td><td> 60</td>

<td> AB211</td><td> -</td><td> 70</td>

<td> AB217</td><td> -</td><td> 83</td>

<td> AB272</td><td> -</td><td> 80</td>

<td> AB279</td><td> -</td><td> 70</td>

<td> AB289</td><td> +</td><td> 100</td>

<td> AB292</td><td> ו-</td><td> 80</td>

<td> AB294</td><td> -</td><td> 100</td>

<td> AB300</td><td> -</td><td> 80</td>

<td> AB359</td><td> -</td><td> 100</td>

Isolates AB289, AB294 and AB359 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria II 61604, USA and given the Accession Numbers NRRL B-21227, NRRL B-21229, and NRRL B-21226 respectively.

Bacillus isolates which produce insecticidal proteins during vegetative growth with activity against Diabrotica virgifera virgifera are tabulated below.

<td colspan="3"> Presence of δ-</td>

<td> ; Bacillus isolate</td><td> endotoxin crystal</td><td> Percent mortality</td>

<td> AB52</td><td> -</td><td> 50</td>

<td> AB59</td><td> -</td><td> 71</td>

<td> AB68</td><td> -ו-</td><td> 60</td>

<td> AB78</td><td> -</td><td> 100</td>

<td> AB 122</td><td> -</td><td> 57</td>

<td> AB218</td><td> - (</td><td> 64</td>

<td> AB256</td><td> -</td><td> 64</td>

Isolates AB59 and AB256 have been deposited in the,Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Illinois 61604, USA, and given the Accession Numbers NRRL B-21228 and NRRL B-21230, respectively.

EXAMPLE 20. IDENTIFICATION OF NOVEL VIP1/VIP2 LIKE GENES BY HYBRIDIZATION

To identify strains containing genes related to those found in the

VIP1 A(a)/VIP2A(a) region of AB78, a collection of Bacillus strains was screened by hybridization. Independent cultures of 463 Bacillus strains were grown in wells of 96 well microtiter dishes (five plates total) until the cultures spbrulated. Of the strains tested, 288 were categorized as Bacillus thuringiensis, and 175 were categorized as other Bacillus species based on the presence or absence of δ-endotoxin crystals. For each microtiter dish, a 96-pin colony stamper was used to transfer approximately 10 μΙ of spore culture to two 150 mm plates containing L-agar. Inoculated plates were grown 4-8 hours at 30 °C, then chilled to 4 °C. Colonies were transferred to nylon filters, and the cells lysed by standard methods known in the art. The filters were hybridized to a DNA probe generated from DNA fragments containing both VIP1 A(a) and VIP2A(a) DNA sequences. Hybridization was performed overnight at 65 °C using the hybridization conditions of Church and Gilbert (Church, G.M., and W. Gilbert,

PNAS, 81:1991-1995 (1984)). Filters were washed with 2x SSC containing 0.1% SDS at 65 °C and exposed to X-Ray film.

Of the 463 Bacillus strains screened, 55 strains were identified that hybridized to the VIF1 ״ A(a)/VIP2A(a) probe. DNA was isolated from 22 of these strains, and analyzed using a Southern blot with VIP1 A(a)/VIP2A(a) DNA as probes. These strains were grouped into 8 classes based on their Southern blot pattern. Each class differed in Southern blot pattern from AB78. One class had a pattern identical to that of the VIP1 A(a)/VIP2A(a) homologs from Bacillus thuringiensis var tenebrionis (see below); Each of the 22 strains was tested for activity against western corn rootworm (WCRW). Three strains, AB433, AB434, and AB435 were found to be active on WCRW. Western blot analysis using VIP2A(a) antisera revealed that strains AB6, AB433', AB434, AB435, AB444, and AB445 produce a protein(s) of equivalent size to VIP2A(a).

Notable among the strains identified was Bacillus thuringiensis strain AB6, (NRRL B-21060) which produced a VIP active against black cutworm (Agrotis ipsilon) as described in Example 15. Western blot analysis with polyclonal antisera to VIP2A(a) and polyclonal antisera to VIP1 A(a) suggests that AB6 produces proteins similar to VIP2A(a) and VIP1 A(a). Thus, AB6 may contain VIPs similar to VIP1A(a) and VIP2A(a), but with a different spectrum of insecticidal activity.

EXAMPLE 21. CLONING OF A VIP1 A(a)/VIP2A(a) HOMOLOG FROM

BACILLUS THURINGIENSISVAR, TENEBRIONIS.

Several previously characterized Bacillus strains were tested for presence of DNA similar to VIP1 A(a)/VIP2A(a) by Southern blot analysis. DNA from Bacillus strains AB78, AB88, GC91, HD-1 and ATCC 10876 was analyzed for presence of VIP1 A(a)/VIP2A(a) like sequences. DNA from Bt strains GC91 and HD-1, and the Be strain ATCC 10876 did not hybridize to VIP2A(a)/VIP1A(a) DNA, indicating they lack DNA sequences similar to VIP1 A(a)/VIP2A(a) genes. Similarly, DNA from the insecticidal strain AB88 (Example 16) did not hybridize to VIP1 A(a)/VIP2A(a) DNA region, suggesting that the VIP activity produced by this strain does not result from VIP1 A(a)/VIP2A(a) homologs. In contrast, Bacillus thuringiensis var. tenebrionis (Btt) contained sequences that hybridized to the VIP1 A(a)/VIP2A(a) region. Further analysis confirmed that Btt contains VIP1 A(a)/VIP2A(a) like sequences.

To characterize the Btt homologs of VIP2A(a) and VIP1A(a), the genes encoding these proteins were cloned. Southern blot analysis identified a 9.5 kb Eco Rl restriction fragment likely to contain the coding regions for the homologs. Genomic DNA was digested with Eco Rl, and DNA fragments of approximately 9.5 kb in length were gel-purified. This DNA was ligated into pBluescript SK(+) digested with Eco Rl, and transformed into E. coli to generate a plasmid library. Approximately 10,000 colonies were screened by colony hybridization for the presence of VIP2A(a) homologous sequences. Twenty eight positive colonies were identified. All twenty eight clones are identical, and contain VIP1 A(a)/VIP2A(a) homologs. Clone pCIB7100 has been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria Illinois 61604, USA, and given the Accession Number B-21322. Several subclones were constructed from pCIB7100. A 3.8 kb Xba I fragment from pCIB7100 was cloned into pBluescript SK(+) to yield pCIB7101. A 1.8 kb Hind III fragment and a 1.4 kb Hind III fragment from pCIB7100 were cloned into pBluescript SK(+) to yield pCIB7102 and pCIB7103, respectively. Subclones pCIB7101, pCIB7102 and pCIB7i 03 have been deposited in the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria ’Illinois 61604, USA, and given the Accession Numbers B-21323, B-21324 and B-21325 respectively.

The;DNA sequence of the region of pCIB7100 containing the VIP2A(a)/VIP1 A(a) homologs was determined by the dideoxy chain termination method (Sanger etal., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467). Reactions were performed using PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kits and PRISM Sequenase® Terminator Double-Stranded DNA Sequencing Kits, and analyzed on an ABI model 373 automated sequencer. Custom oligonucleotides were used as primers to determine the DNA sequence in certain regions. The DNA sequence of this region is shown in SEQ ID NO:19.

The 4 kb region shown in SEQ ID NO:19 contains two open readings frames (ORFs), which encode proteins with a high degree of similarity to VIP1 A(a) and VIP2A(a) proteins from strain AB78. The amino acid sequence of the VIP2A(a)

־ homolog, designaled as VIP2A(b) using the standardized nomenclature, is found at SEQ ID NO:20 and the amino acid sequence of the VIPt A(a) homolog, designated as VIP1 A(b> using the standardized nomenclature, is disclosed at SEQ ID NO-21 The VIP2A(b) protein exhibits 91% amino acid identity to VIP2A(a) from AB78. An alignment of the amino acid sequences of the two VIPS proteins is provided in Table 19. The VIPlA(b) protein exhibits 77 % amino acid identity to VIP1 A(a) from AB78. An alignment of these two VIP1 proteins is provided in Table 20. The alignment shown in Table 20discloses the similarity between VIP1 A(b) and VIPt A(a) from AB78. This alignment reveals that the amino terminal regions of the two VIP1 proteins share higher amino acid identity in the amino terminal region than in the carboxy terminal region. In fact, the amino terminal two thirds (up to aa 618 of the VIP1 A(b) sequence shown in Table 20) of the two proteins exhibit 91% ״entity, _whilethe carboxy-termmal third (from aa 619-833 of VIP1A(b)) exhibit only 35% identity

Western blot analysis indicated that Bacillus thuringiensis var. tenebrionis (Bit) produces both VIPt A(a) like and VIP2A(a) like proteins. However, these proteins do not appear to have activity against western corn rootworm. Bioassay for activity against western com rootworm was performed using either a 24 h culture supernatant from Btt or E. coliclone pCIB7100 (which contains the entire region of the VIP1 AfayVIP2A(a) homologs). No activity against western com rootworm was detected in either case.

Given the similarity between the VIP2 proteins from Btt and AB78, the ability of ™«ר^B10״^substitu,e⁰׳^{, v,p2A(a)} י״™׳ ^{ab78 was ,es,ed}.^c* ־״».״״־ PCIB 206 (Wh,־h produces AB78 VIPt A(a) but not VIP2A(a) protein) were mixed with culture supernatant, and tested for activity against western corn rootworm While neither Btt culture supernatant nor cells containing pCIB6206 had activity on WCRW the mixture of Btt and pCIB6₂06 gave high activity against WCRW. Furthermore ' additional b.oassay showed that the Btt clone pCIB7100, which contains the Btt ' VIP1A(b)/VIP2A(b) genes in E coli, also confers activity against WCRW when mixed ΓΓνί⁶״'^ThUS’ ¹θ ^V'^P2A(b) '™י“ ^Pr°^{dUCed by B״ iS} '“** equivalent to the VIR2A(a) protein produced by AB78.

Thus, the ability to identify new strains with insecticidal activity by using VIP ONA as hybridization probes has been demonstrated. Furthermore, Musstrains that contain VIP1A(a)/VIP2A(a) like sequences, produce VIPt A(a)/v1P2A(a) like protein, yet demonstrate tox.city toward different insect pests. Similar _{methods can iden}״_f many more members of the VIP1/VIP2 family. _Fufl״_ermore. _5״θ _q( can. identify homologs of other varieties of VIPs (for example, the VIPs from AB88)

TABLE 19

Alignment of VfP₂ Amino Acid Sequences from BaciHus thunngiens.s ״ar. tenebnoms (VIP2A(b)) vs. AB78 (VIP2A(a)) ״־ ‘^MtKKjrwSKTIQVVIRTVLLSTVySITl.IMIVVIKADQLNIlISQSK 50 _ID p״,.״

AB71 ־ _{M SEQ m N0:2} ^^^tlLKIPDNAEDEKEDKGKAKEWGKEKGEEWRPPATEKGEMNHFLDN _M0 ..............-1111 I. | , f |, ,, ^^^^KXTDKVEDEKWKEKAKHOtEKEKEHKLTATEKGKMNNELDN ' I I1111:111III.11H11HH

151 ™™־LTEG^^^ ^IHIi::i:iiiiIiIiIIii_HII '1.1111111111::1111111,,, '^;'^IiVIIi'^Vt'״eSGKGS^,n׳PTKAGVILNHSEVBtn,1p_MGy_MVHVDp_Vg^_v^ ^^'''^OVEG'rLKKSLDEKNDInAEAIISiOlKIyEDWAKNLTASQBEAIX) ₃₀₍₎, ^:^:llllllll 11:111:11.11,,1,1 _3M

350

...¹¹¹¹^1111111'״IIIIIIIIIIHIHIIIIIIIUIII ¹“^(,'^ΜΡ^^ί’^ϊ0Ι5ΟΡΕΡ3_Ι.ΚΟΓΕΕ0ιτ^βφ||_)1ίΟγ^τ₃^$^^^^^_ί,ρ '351

KIILRUJVPKGSTGAYLSMGGFSSEKEIIiOKDSKYaiDKiUEVIIKGV «0 .... י........Hmm ^KIILRL°^VPKGST®n3MGGFKEKEILWKDSKYHIDIOTEVlIKGV 450

451 KRYWDATLLTN 462 I I II I I I | | | | |

451 KRYWDATLLTN 462

TABLE 20

A״gnm״־_t of v!P1 Amino Acid S^־»־״ _{from Baclllus}fe/7edr/on/s (VIPiA(b)) vs.AB78(VIP1A(a)) ⁵θ SEQ ID NO:21

SEQ ID NO :5 ^M'⁽'^״^״A^'^/vTOlLLAPMELNGHVHAVNADSKINQIS־ITQ|iMQQKa4D '¹ininii 1111.iium.num

Btt

Ab78 ⁵¹ n «״ ¹¹ 11:111 י ¹¹י ¹¹¹¹¹¹־ f 1 n iininiimi

»1־1

111 1 ^{1 11111111111}'¹¹¹¹¹¹1^l:11¹1111111111 η n ״,

1־1

-LRNPEFNKKE

Hill mil ^ί>^^^Ε^50ΤΚΓΝΙΟ₅ΚΤΓΚΕυ<0ΓΚ4.[)52_Η2502_ν2 ,^,11,11111111Hn1nn1nn.ini ־90151 PIKIEYQSDTKFNIDSKTFKELKLFKIDSQNQPQQVQQDELRNPEFNKKE 200

198 SQEFLAKASKTNLFKQKMKRDIDEDTDTDGDSIPDLWEENGYTIQNKVAV 247

III HI 1:1 l-l I HI III 1:1 II HI III III III III III I III:: II

201 SQEFLAKPSKINLFTQKMKREIDEDTDTDGDSIPDLWEENGYTIQNRIAV 250

248 KWDDSLASKGYTKFVSNPLDSHTVGDPYTDYEKAARDLDLSNAKETFNPL. 297

III 11 III 11 HI I III II: IIIIII11IIIIIIIIIIIII11 HI IIII

251 KWDDSLASKGYTKFVSNPLESHTVGDPYTDYEKAARDLDLSNAKETFNPL 300

298 VAAFPSVNVSMEKVILSPNENLSNSVESHSSTNWSYTNTEGASIEAGGGP 347 mill I Illi I Illi II II Hill II Illi 111111111111:1 II II

301 VAAFPSVNVSMEKVILSPNENLSNSVESHSSTNWSYTNTEGASVEAGIGP 350

348 LGLSFGVSVTYQHSETVAQEWGTSTGNTSQFNTASAGYLNANVRYNNVGT 397

I: IIIIII. II HI III HI III III IIIIII IIIII III IIII III 11

351 KGISFGVSVNYQHSETVAQEWGTSTGNTSQFNTASAGYLNANVRYNNVGT 400

398 GAIYDVKPTTSFVLNNNTIATITAKSNSTALRISPGDSYPEIGENAIAIT 447 ^£ II I III I I I 11 I I I I I: I I I I I I 11 11 I I I 1.1111:111. 1:1:1m

401 GAIYDVKPTTSFVLNNDTIATITAKSNSTALNISPGESYPKKGQNGIAIT 450

448 SMDDFNSHPITLNKQQVNQLINNKPIMLETDQTDGVYKIRDTHGNIVTGG 497

11111111111111 -11: -1:1111:1111:11111111:1111111111

4.51 SWDFNSHPITIJ^QVDIUINNKPMI^TNQTDGVYKIKDTHGNIVTGG 500

498 EWNGVTQQIKAKTASIIVDDGKQVAEKRVAAKDYGHPEDKTPPLTLKDTL 547 > 11 mm 11mm 1111. 111 r 1111111:: 111111.11111.1

501 EWNGVIQQIKAKTASIIVDDGERVAEKRVAAKDYENPEDKTPSLTLKDAL 550

548 KLSYPDEIKETNGLLYYDDKPIYESSVMTYLDENTAKEVKKQINDTTGKF 597 ' III I III II I .: I III I.: HI HI III III HI II III . I I: III III I

551 KLSYPDEIKEIEGLLYYKNKPIYESSVMTYLDENTAKEWKQLNDTTGKF 600 . . . ,598 W/NHLYDVKLTPKMNFTIKMASLYDGAENNHNSLGTWYLTYNVAGGNTG 647 I 11.1 I I I I I I I I I I 1. 11 I:. I I I .II. I. I I: I. I I I . I I I. I . .. 601 KDVSHLYDVKLTPKMNVTIKLSILYDNAESNDNSIGKWTNTNIVSGGNNG 650 '648 KRQYRSAHSCTWALSSEAKKKLNQNAlTYYLSMYMFTtoSTTEPTIEVAGE 697 1:11-1.:. 1::.1.:1.111.1 :11:1:111.:.1:.1.:.11

651 KKQYSSNNPDZ\NLTI1ITDAQEKLNKNRDYYISLYMKSEKNTQCEITIDGE 700

698 ^AITSKKVKEWQNYQRVDILVKNSEWMDKIYIRGNGTTNVYGDDVT 747 :11.I.I.:1.:11.1:11:.I .11:.:.1:.1:..:: 11:.

701 IYPITTKTVNVNKDNYKRLDIIAHNIKSNPISSLHIKTNDEITLFWDDIS 750

748 IPEVSAINPASLSDEEIQEIFKDSTIEYGNPSFVADAVTFK..... 788

I.: 1. 1.1. 1.1.11.: 1:. .ן.::.::...:.

751 ITDVASIKPENLTDSEIKQIYSRYGIKLEDGILIDKKGGIHYGEFINEAS 800

789 . NIKPLQNYVKEYEIYHK....SHRYEKKTVFDIMGVHYEYSIAREQ 830

II.IIII I|.|.: . |. .1..::. .:.:::. ..

801 FNIEPLQNYVTKYKVTYSSELGQNVSDTLESDKIYKDGTIKFDFTKYSKN 850

831 KKA 833

851 EQG 853

EXAMPLE 22. FUSION OF VIP PROTEINS TO MAKE A SINGLE POLYPEPTIDE

VIP proteins may occur in nature as single polypeptides, or as two or more interacting polypeptides. When an active VIP is comprised of two or more interacting protein chains, these protein chains can be produced as a single polypeptide chain from a gene resulting from the fusion of the two (or more) VIP coding regions. The genes encoding the two chains are fused by merging the coding regions of the genes to produce a single open reading frame encoding both' VIP polypeptides. The composite polypeptides can be fused to produce the smaller polypeptide as the NH₂terminus of the fusion protein, or they can be fused to produce the larger of the polypeptides as the NH₂ terminus of the fusion protein. A linker region can optionally be used between the two polypeptide domains. Such linkers are known in the art. This linker pan optionally be designed to contain protease cleavage sites such that once the single fused polypeptide is ingested by the target insect it is cleaved in the linker region to liberate the two polypeptide components of the active VIP molecule.

VIP1 A(a) and VIP2A(a) from B. cereus strain AB78 are fused to make a single polypeptide by fusing their coding regions. The resulting DNA comprises a sequence given in SEQ ID NO:22 with the encoded protein given in SEQ ID NO:23. In like manner, other fusion proteins may be produced.

The fusion of the genes encoding VIP1 A(a) and VIP2A(a) is accomplished using standard techniques of molecular biology. The nucleotides deleted between the VIP1A(a) and VIP2A(a) coding regions are deleted using known mutagenesis techniques or, alternatively, the coding regions are fused using PCR techniques.

The fused VIP polypeptides can be expressed in other organisms using a synthetic gene, or partially synthetic gene, optimized for expression in the alternative host. For instance, to express the fused VIP polypeptide from above in maize, one makes a synthetic gene using the maize preferred codons for each amino acid, see for example EP-A 0618976, herein incorporated by reference. Synthetic DNA sequences created according to these methods are disclosed in SEQ ID NO:17 (maize optimized version of the 100 kDa VIP1A(a) coding sequence), SEQ ID NO:18 (maize optimized version of the 80 kDa VIP1 A(a) coding sequence) and SEQ ID NO:24 (maize optimized version of the VIP2A(a) coding sequence).

Synthetic VIP1 and VIP2 genes optimized for expression in maize can be fused using PCR techniques, or the synthetic genes can be designed to be fused at a common restriction site. Alternatively, the synthetic fusion gene can be designed to encode a single polypeptide comprised of both VIP1 and VIP2 domains.

Addition of a peptide linker between the VIP1 and VIP2 domains of the fusion protein can be accomplished by PCR mutagenesis, use of a synthetic DNA linker encoding the linker peptide, or other methods known in the art.

The fused VIP polypeptides can be comprised of one or more binding domains. If more than one binding domain is used in the fusion, multiple target pests are controlled using such a fusion. The other binding domains can be obtained by using all or part of other VIPs; Bacillus thuringiensis endotoxins, or parts thereof; or other proteins capable of binding to the target pest or appropriate biding domains derived from such binding proteins.

One example of a fusion construction comprising a maize optimized DNA sequence encoding a single polypeptide chain fusion having VIP2A(a) at the Nterminal end and VIP1 A(a) at the C-terminal end is provided by pCIB5531. A DNA sequence encoding a linker with the peptide sequence PSTPPTPSPSTPPTPS (SEQ ID NO:47) has been inserted between the two coding regions. The sequence encoding this linker and relevant cloning sites is 5¹- CGC GGG OCT TCT ACT CCC CCA ACT CCC TCT CCT AGC ACG CCT CCG ACA CCT AGC GAT ATC GGA TC C -3' (SEQ ID NO:48). Oligonucleotides were synthesized to represent both the upper and lower strands and cloned into a pUC vector following hybridization and phosphorylation using standard procedures. The stop codon in VIP2A(a) was removed using PCR and replaced by the Bglll restriction site with a Smal site. A translation fusion was made by ligating the Bam HI / Pstl fragment of the VIP2A(a) gene from pCIB5522 (see Example 24), a PCR fragment containing the Pstl-end fragment of the VIP2A(a) gene (identical to that used to construct pCIB5522), a synthetic linker having ends that would ligate with a blunt site at the 5’ end and with BamHI at the 3’ end and the modified synthetic VIP1 A(a) gene from pCIB5526 described below (See SEQ ID NO:35). The fusion was obtained by a four way ligation that resulted in a plasmid containing the VIP2A(a) gene without a translation stop codon, with a linker and the VIP1 A(a) coding region without the Bacillus secretion signal. The DNA sequence for this construction is disclosed in SEQ ID NO:49, which encodes the fusion protein disclosed in SEQ ID NQ:50. A single polypeptide fusion where VIP1 A(a) is at the N-terminal end and VIP2A(a) is at the C-terminal end can be made in a similar fashion. Furthermore, either one or both genes can be linked in a translation fusion with or without a linker at either the 5’ or the 3’ end to other molecules like toxin encoding genes or reporter genes.

EXAMPLE 23. TARGETING OF VIP2 TO PLANT ORGANELLES

Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino-terminal end of various proteins. This signal is cleaved during chloroplast import, yielding the mature protein (e.g. Comai etal. J. Biol. Chem. 263:15104-15109 (1988)). These signal sequences can be fused to heterologous gene products such as VIP2 to effect the import of those products into the chloroplast (van den Broeck etal. Nature 313:358363 (1985)). DNA encoding for appropriate signal sequences can be isolated from the 5’ end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized.

Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger etal. Plant Molec. Biol. 13:411-418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products such as VIP2 to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Similarly, targeting to cellular protein bodies has been described by Rogers etal. (Proc. Natl. Acad. Sci. USA 82:6512-6516 (1985)).

By the fusion of the appropriate targeting sequences described above to coding seqbences of interest such as VIP2 it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino-terminal ATG of the transgene. The signal sequence selected should include the known cleavage site and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the start codon ATG, or alternatively replacement of some amino acids within the coding sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by (Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier, pp 10811091 (1982); Wasmann etal. Mol. Gen. Genet. 205:446-453 (1986)). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.

The above described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell targeting goal under the transcriptional regulation of a promoter which has an expression pattern different to that of the promoter from which the targeting signal derives.

A DNA sequence encoding a secretion signal is present in the native Bacillus VIP2 gene. This signal is not present in the mature protein which has the N־terminal sequence of LKITDKVEDF (amino acid residues 57 to 66 of SEQ ID NO:2). It is possible to engineer VIP2 to be secreted out of the plant cell or to be targeted to subcellular organelles such as the endoplasmic reticulum, vacuole, mitochondria or plastids including chloroplasts. Hybrid proteins made by fusion of a secretion signal peptide to a marker gene have been successfully targeted into the secretion pathway. (Itirriaga G. etal., The Plant Cell. 1:381-390 (1989), Oenecke et al., The Plant Cell. 2:511990) 59־). Amino-terminal sequences have been identified that are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler &amp; Ho, Plant Cell 2: 769-783 (1990)).

The presence of additional signals are required for the protein to be retained in the endoplasmic reticulum or the vacuole. The peptide sequence KDEL/HDEL at the carboxy-terminal of a protein is required for its retention in the endoplasmic reticulum (reviewed by Pelham, Annual Review Cell Biol., 5:1-23 (1989). The signals for retention of proteins in the vacuole have also been characterized. Vacuolar targeting signals may be present either at the amino-terminal portion, (Holwerda et al., The Plant Cell. 4:307-318 (1992), Nakamura etal., Plant Physiol., 101:1-5 (1993)), carboxy- terminal portion, or in the internal sequence of the targeted protein. (Tague etal., The Plant Cell, 4:307-318 (1992), Saalbach eta/. The Plant Cell. 3:695-708 (1991)). Additionally, amino-terminal sequences in conjunction with carboxy-terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14:357-368 (1990)). Similarly, proteins may be targeted to the mitochondria or plastids using specific carboxy terminal signal peptide fusions (Heijne et al., Eur. J, Biochem. 180:535-545 (1989), Archer and Keegstra, Plant Molecular Biology^ 23:1105-1115 (1993)).

־

In order to target VIP2, either tor secretion or to the various subcellufar _organete atze optimized DNA sequence encoding a known signal peptide(s) may be ' designed to be at the 5’ or the 3־ end of tho ' ^y the cell a DNA &lt;,. ^{aS T}° ^{secrete VIP2} ־ '״״' cell. DNA sequence encoding the eukaryotic secretion signal peptide

I^GWSWIFLFLLSGAAGVHCL (SEQ ID NO:25) from WO 96/00783 or any other described in the literature (hirriaga efaL JMantCehj ;381.390 ( 989), Denecke, ef aZ, ThePlantc1990) 2:51-59 .״־)) may be added to the 5' end of ״־her the complete VtP2 gene sequence or to the sequence truncated to encode t e mature protem or the gene truncated to nucleotide 286 cr encoding a protein to start a ammo acid residue 94 (methionine). To target VIP2to be retained in the ב“-^{a dna sequence ,he er s!9nai} ־*׳>*EL. add,hon to the secretion signal, can be added to the 3’ end of the gene For vacuolar targeting a DNA sequence encoding the signal peptide 31^(^ 99^0 ⁽$^Εθ‘D N°²⁰^ ׳f^werda et a/., The_Plant_Cell, 4:3078 (1992)) can be des.g״ed to be adjacent to the secretion signal or a sequence encoding a carboxyl signat peptide as described by DombrowsH a,at. The Plant

1993) 87-596־:־) or a functional variation may be inserted at the 3^_egene^Similarly, VIP2 can be designed to be targeted to either the mitochondria or the P astids, including the chloroplasts, by inserting sequences in the VIP2 sequence escnbed that would encode the required targeting _s1gnals. _The־ignal present in ViP2 may be retained or removed Irom the Una. construe,™ ne example 01 a construction which incorporates a eukaryotic secretion signal ״sed to a coding sequence lor a VIP is provided by pCIB5528. Oli_g״״־_clectides corresponding to both the upper and lower strand Of sequences encoding the secretion signal peptide of SEQ ID NO:25 was synthesized and hp&lt;־ th ggatccacc atg ggc tgg agc tgg atc ttc ctc X ^{sequence 5}'

GTG CAC TGC C^^AC™ ^C“ θ&lt;*

־. end of the secretion signal reseat J ends > sites BamHI and Pen τη ,. ^־Wends ״־־«Pending to restriction ^bamH and Pstl. The oligonucleotide was hybridized and nhne _hligated into PCIB5527 (construction described in Example 23A1 η η n coding sequence is disciosed in SEQ ID NO:42 which encodes the pZ “

-פ? in SEQ ID NO:43. This encoded protein comprises the eukaryotic secretion signal חו place of the Bacillus secretion signal.

One example of a construction which incorporates a vacuolar targetting signal fused to a coding sequence for a VIP is provided by pCIB5533. Oligonucleotides corresponding to both the upper and lower strand of sequences encoding the vacuolar targetting peptide of SEQ ID NO:3 was synthesized and has the sequence 5'-CCG CGG GCG TGC ACT GCC TCA GCA GCA GCA GCT TCG CCG ACA GCA ACC CCA TCC GCG TGA CCG ACC GCG CCG CCA GCA CCC TGC AG-3' (SEQ ID NO:44). When hybridized, the 5' end of the vacuolar targetting signal resembled “sticky-ends״ corresponding to restriction sites SacII and Pstl. The oligonucleotide was hybridized and phosphorylated and ligated into pCIB5528 (construction described above) which had been digested with SacII / Pstl using standard procedures. The resulting maize optimized coding sequence is disclosed in SEQ ID NO:45 which encodes the protein disclosed in SEQ ID NO:46. This encoded protein comprises the vacuolar targetting peptide in addition to the eukaryotic secretion signal.

The VIP1 gene can also be designed to be secreted or targeted to subcellular organelles by similar procedures.

EXAMPLE 23A. REMOVAL OF BACILLUS SECRETION SIGNAL FROM

VIP1 A(a) AND VIP2A(a)

VIP1 A(a) and VIP2A(a) are secreted during the growth of strain AB78. The nature of peptide sequences that act as secretion signals has been described in the literature (Simonen and Palva, Microbiological reviews, pg. 109-137 (1993)). Following the information in the above publication, the putative secretion signal was identified in both genes. In VIP1 A(a) this signal is composed of amino acids 1-33 {See SEQ ID NO:5). Processing of the secretion signal probably occurs after the serine at amino acid 33. The secretion signal in VIP2A(a) was identified as amino acids 1-49 {See SEQ ID NO:2). N-terminal peptide analysis of the secreted mature VIP2A(a) protein revealed the N-terminal sequence LKITDKVEDFKEDK. This sequence is found beginning at amino acid 57 in SEQ ID NO:2. The genes encoding these proteins have been modified by removal of the Bacillus secretion signals.

A maize optimized VIP1 A(a) coding region was constructed which had the sequences encoding the first 33 amino acids, i.e., the secretion signal, removed from its 5' end. This modification was obtained by PCR using an forward primer that contained the sequence 5'-GGA TCC ACC ATG AAG ACC AAC CAG ATC AGC-3' (SEQ ID NO:33), which hybridizes with the maize optimized gene (SEQ ID NO:26) at nucleotide position 100, and added a BamHI restriction site and a eukaryotic translation start site consensus including a start codon. The reverse primer that contained the sequence 5'־AAG CTT CAG CTC CTT G-3' (SEQ ID NO:34) hybridizes on the complementary strand at nucelotide position 507. A 527 bp amplification product was obtained containing the restriction sites BamHI at the 5* end and Hindlll site at the 3' end. The amplification product was cloned into a T- vector (described in Example 24, below) and sequenced to ensure the correct DNA sequence. The BamHI / Hindlll fragment was then obtained by restriction digest and used to replace the BamHI/Hindlll fragment of the maize optimized VIP1A(a) gene cloned in the rootpreferred promoter cassette. The construct obtained was designated pCIB5526. The maize optimized coding region for VIP1A(a) with the Bacillus secretion signal removed is disclosed as SEQ ID NO;35 and the encoded protein is disclosed as SEQ ID NO :36.

The gene encoding the processed form of VIP2A(a), i.e., a coding region with the secretion signal removed, was constructed by a procedure similar to that described for that used to construct the processed form of VIP1 A(a), above. The modification was obtained by PGR using the forward primer 5'-GGA TCC ACC ATG CTG CAG AAC CTG AAG ATC AC -3' (SEQ ID NO:37). This primer hybridizes at nucleotide position 150 of the maize optimized VIP2A(a) gene (SEQ ID NO:27). A silent mutation has been inserted at nucleotide position 15 of this primer to obtain a Pstl restriction site. The reverse primer has the sequence 5'־AAG CTT CCA CTC CTT CTC-3' (SEQ ID NO:38). A 259 bp product was obtained with Hindlll restriction site at the 3' end. The amplification product was cloned into a T- vector, sequenced and ligated to a BamHI /Hindi 11'digested root-preferred promoter cassette containing the maize optimized VIP2A(a). The construct obtained was designated pCIB5527. The maize optimized coding region for VIP2A(a) with the Bacillus secretion signal removed is disclosed as SEQ ID NO:39 and the encoded protein is disclosed as SEQ ID NO:40.

EXAMPLE 24 CONSTRUCTION AND CLONING OF THE VIP1 A(a) AND VIP2A(a) MAIZE OPTIMIZED GENES

Design: The maize optimized genes were designed by reverse translation of the native VIPlA(a) and VIP2A(a) protein sequences using codons that are used most often in maize (Murray etal., Nucleic Acid Research, 17:477-498 (1989)). To facilitate cloning, the DNA sequence was further modified to incorporate unique restriction sites at intervals of every 200-360 nucleotides. VIP1 A(a) was designed to be cloned in 11 such fragments and VIP2A(a) was cloned in 5 fragments. Following cloning of the individual fragments, adjacent fragments were joined using the restriction sites common to both fragments, to obtain the complete gene. To clone each fragment, oligonucleotides (50-85 nucleotides) were designed to represent both the upper and the lower strand of the DNA. The upper oligo of the first oligo pair was designed to have a 15 bp single stranded region at the 3’ end which was homologous to a similar single stranded region of the lower strand of the next oligo pair to direct the orientation and sequence of the various oligo pairs within a given fragment. The oligos are also designed such that when the all the oligos representing a fragment are hybridized, the ends have single stranded regions corresponding to the particular restriction site to be formed. The structure of each oligomer was examined for stable secondary structures such as hairpin loops using the OLIGO program from NBI Inc. Whenever neccesary, nucleotides were changed to decrease the stability of the secondary structure without changing the amino acid sequence of the protein. A plant ribosomal binding site consensus sequence, TAAACAATG (Joshi etal., Nucleic Acid Res., 15:6643-6653 (1987)) or eukaryotic ribosomal binding site concensus sequence CCACCATG (Kozak, Nucleic Acid Research, 12:857-872 (1984)) was inserted at the translational start codon of the gene.

I׳ -

Cloning: Oligos were synthesized by IDT Inc., and were supplied as lyophilized powders. They were resuspended at a concentration of 200 μΜ. To 30 μΙ of each oligo formamide was added a final concentration of 25-50% and the sample was boiled for two minutes before separation on a premade 10% polyacryamide / urea gel obtained from Novex. After electrophoresis, the oligo was detected by UV shadowing by placing the gel on a TLC plate containing a fluorescent indicator and exposing it to UV light. The region containing DNA of the correct size was excised and extracted from the polyacryamide by an overnight incubation of the minced gel fragment in a buffer containing 0.4 M LiCI, 0.1 mM EDTA. The DNA was separated from the gel residue by centrifugation through a Millipore UFMC filter. The extracted DNA was ethanol precipitated by the addition of 2 volumes of absolute alcohol. After centrifugation, the precipitate was resuspended in dH₂0 at a concentration of 2.5 μΜ. Fragments were cloned either by hybridization of the oligos and ligation with the appropriate vector or by amplification of the hybridized fragment using a equimolar mixture of all the oligos for a particular fragment as a template and end-specific PCR primers.

Cloning by hybridization and ligation: Homologous double stranded oligo pairs were obtained by mixing 5 μΙ of the upper and of the lower oligo for each oligo pair with buffer containing 1X polynucleotide kinase (PNK) buffer (70 mM Tris-HCI (pH 7.6), 10 mM MgCI₂,5 mM dithiothreitol (DTT)), 50 mM KCI, and 5 % formamide in a final volume of 50 μΙ. The oligos were boiled for 10 minutes and slow cooled to 37° C or room temperature. 10 μΙ was removed for analysis on a 4% agarose in a TAE buffer system (Metaphore®; FMC). Each hybridized oligo pair was kinased by the addition of ATP at a final concentration of 1 mM, BSA at a final concentration of 100 pg per ml and 200 units of polynucleotide kinase and 1 μΙ of ־!OX PNK buffer in a volume of 10 μΙ. Following hybridization and phosphorylation, the reaction was incubated at 37° C for 2 hours to overnight. 10 μΙ of each of the oligo pairs for a particular fragment, were mixed in a final volume of 50 μΙ. The oligo pairs were hybridized by heating at 80° C for 10 minutes and slow cooling to 37° C. 2 μΙ of oligos was mixed with about 100 ng of an appropriate vector and ligated using a buffer containing 50 mM Tris-HCI (pH 7.8), 10 mM MgCI₂, 10 mM DTT, 1 mM ATP. The reaction was incubated at room temp, for 2 hours to overnight and transformed into DH5a strain of E.coli, plated on L- plates containing ampicillin at a concentration of 100 pg/ml using standard procedures. Positive clones were further characterized and confirmed by PCR miniscreen described in detail in EP-A 0618976 using the universal primers “Reverse and M13 “-20 “ as primers. Positive clones were identified by digestion of DNA with appropriate enzymes followed by sequencing. Recombinants that had the expected DNA sequence were then selected for further work.

PCR Amplification and cloning into T- vector:

PCR amplification was carried out by using a mixture of all the oligomers that represented the upper and the lower strand of a particular fragment (final concentration 5 mM each) as template, specific end primers for the particular fragment (final concentration 2 μΜ) 200 μΜ of each dATP, dTTP, dCTP and dGTP, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCI₂,0.01% gelatin and 5 units of Taq polymerase in a final reaction volume of 50 μΙ, The amplification reaction was carried out in a Perkin Elmer thermocycler 9600 by incubation at 95° C for 1 min (1 cycle), followed by 20 cycles of 95 °C for 45 sec., 50 °C for 45 sec., 72 °C for 30 sec. Finally the reaction was incubated for 5 min at 72°C before analyzing the product. 10 μΙ of the reaction was analyzed on a 2.5% Nusieve (FMC) agarose gel in a TAE buffer system. The correct size fragment was gel purified and used for cloning into a PCR cloning vector or T־vector. T-vector construction was as described by Marchuk et al., Nucleic Acid Research, 19:1154 (1991). pBIuescriptsk+ (Stratagene®, Ca.) was used as the parent vector. Transformation and identification of the correct clone was carried out as described above.

Fragments 1,3, 4, 5, 6, 8, and 9 of VIP1 A(a) and fragments 2 and 4 of VIP2A(a) were obtained by cloning of PCR amplification products; whereas, fragments 2, 7,10 and 11 of VIP1 A(a) and fragments 1,3, and 5 of VIP2A(a) were obtained by hybridization/ ligation.

Once fragments with the desired sequence were obtained, the complete gene was assembled by cloning together adjacent fragments. The complete gene was resequenced and tested for activity against WCRW before moving it into plant expression vectors containing the root preferred promoter (disclosed in U.S. patent application serial no. 08/017,209, herein incorporated by reference) and the rice actin promoter.

One such plant expression vector is pCIB5521. The maize optimized VIP1 A(a) coding region (SEQ ID NO:26) was cloned in a plant expression vector containing the root preferred promoter at the 5' of the gene with the PEP Carboxylase intron #9 followed by the 35S terminator at the 3' end. The plasmid also contains sequences for ampicillin resistance from the plasmid pUC19. Another plant expression vector is pCIB5522, which contains the maize optimized VIP2A(a) coding region (SEQ ID

NO:27) fused to the root preferred promoter at the 5' of the gene with the PEP

Carboxylase intron #9 followed by the 35S terminator at the 3' end.

EXAMPLE 25. NAD AFFINITY CHROMATOGRAPHY

A purification strategy was used based on the affinity of VIP2 for the substrate NAD. The supernatant from the pH 3.5 sodium citrate buffer treatment described in Example 4 was dialyzed in 20 mM TRIS pH 7.5 overnight. The neutralized supernatant was added to an equal volume of washed NAD agarose and incubated with gentle rocking at 4° C overnight. The resin and protein solution were added to a 10 ml disposable polypropylene column and the protein solution allowed to flow out. The column was washed with 5 column volumes of 20 mM TRIS pH 7.5 then washed with 2-5 column volumes of 20 mM TRIS pH 7.5,100 mM NaCl, followed by 2-5 column volumes of 20 mM TRIS 7.5. The VIP proteins were eluted in 20 mM TRIS pH

7.5 supplemented with 5 mM NAD. Approximately 3 column volumes of the effluent were collected and concentrated in a Centricon -10. Yield is typically about 7-15 gg of protein per ml of resin.

When the purified proteins were analyzed by SDS-PAGE followed by silver staining, two polypeptides were visible, one with Mr of approximately 80,000 and one with Mr of approximately 45,000. N-terminal sequencing revealed that the Mr 80,000 protein corresponded to a proteolytically processed form of VIP1 A(A) and the Mr 45,000 form corresponded to a proteolytically processed form of VIP2A(a). The copurification of V|P 1 A(a) with VIP2A(a) indicates that the two proteins probably form a complex and have protein-protein interacting regions. VIP1A(a) and VIP2A(a) proteins purified in this manner were biologically active against western com rootworm.

EXAMPLE 26. EXPRESSION OF MAIZE OPTIMIZED VIP1 A(a) AND VIP2A(a)

E. coli strains containing different plasmids comprising VIP genes were assayed for expression of VIPs. E. colistrains harboring the individual plasmids were grown overnight in L-broth and expressed protein was extracted from the culture as described in Example 3, above. Protein expression was assayed by Western Blot analysis using antibodies developed using standard methods known in the art, similar to those described in Example 12, above. Also, insecticidal activity of the expressed proteins were tested against Western corn rootworm, according to the method in

Example 3, above. The results of the E. coli expression assays are described below.

Expression of VIPs in E. coli

<td> Extract of E. coli Strain Harboring Indicated Plasmid</td><td> Assay No. 1 % Mo</td><td> Assay No. 2 !rtality</td><td> Protein Detected</td>

<td> Control</td><td> 0</td><td> 0</td><td> no</td>

<td> pCIB5521 (maize optimized VIP1A(a))</td><td> 47</td><td> 27</td><td> yes</td>

<td> pCIB5522 (maize optimized VIP2A(a))</td><td> 7</td><td> 7</td><td> yes</td>

<td> pCIB6024 (native VIP2A(a))</td><td> 13</td><td> 13</td><td> yes</td>

<td> PCIB6206 (native VIP1A(a))</td><td> 27</td><td> 40</td><td> yes</td>

<td> Extracts pCIB5521 + pC IB5522 combined</td><td> 87</td><td> 47</td><td></td>

<td> Extracts pCIB5521 + pCIB6024 combined</td><td> 93</td><td> 100</td><td></td>

<td> Extracts pCIB5522 + pCIB6206 combined</td><td> 100</td><td> 100</td><td></td>

<td> Extracts pCIB6024 + pCIB6206 combined</td><td> 100</td><td> 100</td><td></td>

The DNA from these plasmids was used to transiently express the VIPs in a maize protoplast expression system. Protoplasts were isolated from maize 2717 Line 6 suspension cultures by digestion of the cell walls using Cellulase RS and Macerase R10 in appropriate buffer. Protoplasts were recovered by sieving and centrifugation. Protoplasts were transformed by a standard direct gene transfer method using approximately 75 g plasmid DNA and PEG-40. Treated protoplasts were incubated overnight in the dark at room temperature. Analysis of VIP expression was accomplished חס protoplast explants by Western blot analysis and insecticidal activity against Western corn rootworm as described above for the expression in E. coli. The results of the maize protoplast expression assays are described below.

Expression of VIPs in Plant Protoplasts

<td> Extract Tested</td><td> Assay No. 1 % Mo</td><td> Assay No. 2 rtality</td><td> Protein Detected</td>

<td> No DNA control</td><td> 27</td><td> 10</td><td> no</td>

<td> pCIB5521 (p) (maize optimized VIP1A(a))</td><td> 20 (0)</td><td> 30</td><td> yes</td>

<td> pCIB5522 (p) (maize</td><td> 20 (0)</td><td> 20</td><td> yes</td>

<td> optmizied VIP2A(a)) Extracts pCI B5521 (p) +</td><td> 87(82)</td><td> 90</td><td></td>

<td> pCIB5522 (p) combined Extracts pCI B5521 (p) + pCIB5522 (e) combined</td><td> 100</td><td> -</td><td></td>

<td> Extracts pC IB5522 (p) + pCIB5521 (e) combined</td><td> 53 (36)</td><td> -</td><td></td>

<td> Extracts pCIB5521 (p) + pCIB6024 (e) combined</td><td> 100</td><td> -</td><td></td>

<td> Extracts pCIB5522 (p) + pCIB6206 (e) combined</td><td> 100</td><td> -</td><td></td>

<td> pCIB6024(e) (native</td><td> 0</td><td> -</td><td> yes</td>

<td> VIP2A(a)) pCIB6206(e) (native</td><td> 20</td><td> -</td><td> yes</td>

<td> VIP1A(a)) pCI B5521 + pCIB 5522</td><td> 100</td><td> 100</td><td> yes</td>

(plasmids delivered by cotransformation) (p) = extract of protoplast culture transformed with indicated plasmid (e) = extract of E. co//strain harboring indicated plasmid

The expression data obtained with both E. coli and maize protoplasts show that the maize optimized VIP1A(a) and VIP2A(a) genes make the same protein as the native VIP1A(a) and VIP2A(a) genes, respectively, and that the proteins encoded by the maize optimized genes are functionally equivalent to the proteins encoded by the native genes.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The following deposits have been made at Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA:

<td colspan="3"> Strain designation</td><td colspan="2"> Deposition Number Deposition Date</td>

<td>⁵ג 5.׳2</td><td> 1. »</td><td> E. coli PL2</td><td> NRRL B-21221</td><td> March 09,1994</td>

<td></td><td> 2.</td><td> E. C0HPL2.</td><td> NRRL B-21221N</td><td> --------------— ' - * September 02,1994</td>

<td> 2/.9-55</td><td> 3. 1-</td><td> E co//pC IB6022</td><td> NRRL B 21222</td><td> March 09,1994</td>

<td> 11.3.35</td><td> , 4.</td><td> E. co//'pCIB6023</td><td> NRRL B-21223</td><td> ---------------:---------------j March 09, 1994</td>

<td></td><td> 5.</td><td> E. coli pCIB6023</td><td> NRRL B-21223N</td><td> . 1——׳——---—׳—“ September 02,1994</td>

<td></td><td> 6.</td><td> Bacillus thuringiensis</td><td></td><td></td>

<td></td><td></td><td> HD73-78VIP</td><td> NRRL B 21224</td><td> March 09,1994</td>

<td></td><td> 7.</td><td> Bacillus thuringiensis AB88</td><td> NRRLB-21225</td><td> March 09,1994</td>

<td></td><td>⁸-</td><td> Bacillus thuringiensis AB359</td><td> NRRL B-21226</td><td> March 09,1994</td>

<td></td><td> 9.</td><td> Bacillus thuringiensis AB289</td><td> NRRL B-21227</td><td> March 09, 1994</td>

<td></td><td> 10.</td><td> Bacillus sp. AB59 .</td><td> NRRL B-21228</td><td> March 09,1994</td>

<td></td><td> 11.</td><td> . Bacillus sp. AB294</td><td> NRRL B-21229</td><td> March 09,1994</td>

<td></td><td> .12.</td><td> Bacillus sp. AB256</td><td> NRRL B-21230</td><td> March 09,1994</td>

<td></td><td> 13.</td><td> E. coli P5-4</td><td> NRRLB-21059</td><td> March 18,1993</td>

<td></td><td> 14</td><td> - E. coli P3-12</td><td> NRRL B-21061</td><td> March 18,1993</td>

<td></td><td> 15.</td><td> Bacillus cereus AB78</td><td> NRRLB-21058</td><td> March 18,1993</td>

<td></td><td> 16.</td><td> • Bacillus thuringiensis AB6</td><td> NRRL B-21060</td><td> March. 18,1993</td>

<td></td><td> 17.</td><td> E. co//pC IB6202</td><td> NRRLB-21321</td><td> September 02,1994</td>

<td></td><td> 18.</td><td> E. co//pCIB7100</td><td> NRRLB-21322</td><td> September 02,1994</td>

<td></td><td> 19.</td><td> E colipCIB7101</td><td> NRRL B-21323</td><td> September 02,1994</td>

<td></td><td> 20.</td><td> E co//pC IB7102</td><td> NRRL B-21324</td><td> September 02,1994</td>

<td></td><td> 21.</td><td> E co/ί pC IB7103</td><td> NRRL B-21325</td><td> September 02,1994</td>

<td></td><td> 22.</td><td> E co//pClB7104</td><td> NRRL 8:21422</td><td> March 24,1995</td>

<td> 505</td><td> 23.</td><td> E colipCIB7107</td><td> NRRL B-21423</td><td> March 24,1995</td>

<td></td><td> 24,</td><td> E. coli pCIB7108</td><td> NRRL B-21438</td><td> May 05, 1995</td>

<td> .3r</td><td> 25.</td><td> Bacillus thuringiensis AB424</td><td> NRRL B-21439</td><td> May. 05, 1995</td>

2/ 82 3 5 1 1 ־^07,־

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Passages in the description which are out of ambit of the appended claims do not constitute part of the claimed invention.

115382/ , SEQUENCE LISTING (1) GENERAL INFORMATION:

(A) NAME: CIBA-GEIGY AG .

(B) STREET: Klybeckstr. 14! ' ' (C) CITY: Basel (E) COUNTRY: Switzerland (F) POSTAL CODE (ZIP); 4002 (G) TELEPHONE: +41 61 69 11 n (H) TELEFAX: + 41 61 69g 79 76 (D TELEX: 962 991 ^(i״ T ־^ ?״ ״_{ins Md Strains} (iii) NUMBER OF SEQUENCES: 52 (iv) COMPUTER READABLE FORM.

(A) MEDIUM TYPE: Floppy disk &lt;B) COMPUTER: IBM PC^Pco״pX_le (C) OPERATING SYSTEM: PC-DOS/MS-DGS ' (D) SOFIWARE: Datentln ״ .

«crease #1.0, Version #1.30b (2) INFORMATION FOR SEQ ID NO: 1;

(i) SEQUENCE CHARACTERISTICS.' ' ' (A) . LENGTH: 6049 base pairs . (B) TYPE; nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY;.linear . (ii) MOLECULE TYPE: DNA. (genomic) (vi) ORIGINAL SOURCE:

(A) ORGANISM; Bacillus cereus (B) STRAIN: AB78 (C) INDIVIDUAL ISOLATE: NRRL B-21058 (ix) FEATURE:

(A) ΝΛΜΕ/ΚΕΥ: CDS (B) . LOCATION: 1082.24 67 (D) OTHER INFORMATION: /product״ ־־VIP2A(a)־ (ix) feature:

(A) ΝΑΜΕ/ΚΕΥ: misc feature (B) IOCATION: 24757.5126 (D) OTHER INFORMATION. /not־e. kd VIPiA(a) protein. _This^{9 Sequence for} the 100

NO: 4 and translated sepam^.^{9 U} 1 ־EQ ID ¹¹5382/3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO;1:

ATCGATACAA TGTTGTTTTA CTTAGACCGG TAGTCTCTGT AATTTGTTTA ATGCTATATT 60 CTTTACTTTG ATACATTTTA ATAGCCATTT CAACCTTATC AGTATGTTTT TCTGGTCTT־C120

CTCCTTTTTT TCCACGAGCT CTAGCTGCGT TTAATCCTGT TTTGGTACGT TCGCTAATAA180

TATCTCTTTC TAATTCTGCA ATACTTGCCA TCATTCGAAA GAAGAATTTC CCCATAGCAT240

TAGAGGTATC AATGTTGTCA TGMTAGAAA TAAAATCTAC ACCTAGCTCT TTGAATTTTT300

CACTTAACTC AATTAGGTGT TTTGTAGAGC GAGAAATTCG ATCAAGTTTG TAAACAACTA360

TCTTATCGCC TTTACGTAAT ACTTTTAGCA ACTCTTCGAG TTGAGGGCGC TCTTTTTTTA420

TTCCTGTTAT TTTCTCCTGA TATAGCCTTT CTACACCATA TTGTTGCAAA GCATCTATTT. .480

GCATATCGAG ATTTTGTTCT TCTGTGCTGA CACGAGCATA ACCAAAAATC AAATTGGTTT c^ch’tcctat .ctaaatatat ctattaaaat agcaccaaaa accttattaa attaaaataa'

GGAACTTTGT TTTTGGATAT GGATTTTGGT ACTCAATATG GATGAGTTTT TAACGCTTTT660

GTTAAAAAAC AAACAAGTGC CATAAACGGT CGTTTTTGGG ATGACATAAT AAATAATCTG720

TTTGATTAAC CTAACCTTGT ATCOTACAG CCC^GTmA TTTGTACTTC AACTGACTGA780

ATATGAAAAC AACATGAAGG TTTCATAAAA ΊΤΓΑΤΑΤΑΤΤ TTCCATAACG GATGCTCTAT840

CTTTAGGTTA TAGTTAAATT ATAAGAAAAA AACAAACGGA GGGAGTGAAA AAAAGCATCT900

TCTCTATAAT TTTACAGGCT CTTTAATAAG AAGGGGGGAG ATTAGATAAT AAATATGAAT 960 ' ־ ATCTATCTAT AATTGTHGC TTGTACAATA ACTTATCTAA CTTTCATATA CAACAACAAA1020

ACAGACTAAA TCCAGATTGT ATATTCATTT TCAGTTGTTC CTTTATAAAA TAATTTCATA1080

A ATG .AAA AGA ATG GAG GGA AAG TTG ΊΤΓ ATG GTG TCA AAA AAA TTA

Met ,Lys Arg Met Glu Gly Lys Leu Phe Met Vai Ser Lys Lys Leu

15 .10 י־ :. ⁵ .

CAA

Gin

GTA GTT ACT AAA ACT GTA TTG. CTT AGT ACA GTT TTC .TCT ATA TCT Vai Val Thr Lys Thr Vai Leu Leu Ser Thr Vai Phe Ser He Ser ²⁵30

1126

1174

TTA TTA AAT AAT GAA GTG. ATA AAA GCT GAA CAA TTA AAT

Leu Leu Asn . Asn Glu Val He Lys Ala Glu Gin Leu. Asn ³⁵ . 40

CAA AGT AAA TAT ACT AAC TTG CAA AAT CTA AAA ATC ACT

Gin. Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr . 55 ₆₀

GAG GAT TTT AAA GAA GAT AAG GAA AAA GCG AAA GAA TGG

ATA AAT TCT He Asn Ser 45

GAC AAG GTA Asp Lys Val

GGG AAA GAA

1222

1270

1318

<img file="IL115382A_D0001.tif" />

Glu Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu ⁶⁵ 7075

AAA GAA AAA GAG TCG AAA CTA ACT GCT ACT GAA AAA GGA AAA ATGAAT

Lys Glu Lys Glu Trp Lys Leu Thr Ala Thr Glu Lys Gly Lys MetAsn ⁸⁰ . 85 go95

AAT TTT TTA GAT AAT AAA AAT GAT ATA AAG ACA AAT TAT AAA GAAATT

Asn Phe Leu .Asp. Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glulie ¹⁰⁰ 105no

ACT TTT TCT ATG GCA GGC TCA TTT GAA GAT GAA ATA AAA GAT TTAAAA

Thr Phe Ser Met Ala Gly Ser Phe Glu Asp Glu He Lys Asp LeuLys

115 . .120 .

GAA ATT GAT AAG. ATG TTT GAT AAA ACC AAT CTA TCA AAT TCT ATTATC

Glu He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser. Asn Ser lielie ¹³⁰ 135. no

ACC TAT AAA AAT GTG GAA CCG ACA ACA ATT GGA TTT AAT AAA TCT TTA Thr Tyr Lys Asn Vai Glu Pro Thr Thr He Gly Phe Asn Lys Ser Leu 145 150 . 155 . . .

ACA GAA GGT AAT ACG ATT. AAT , TCT GAT GCA ATG .GCA CAG .TTT. AAA GAA Thr Glu Gly Asn Thr lie Asn Ser Asp Ala Met Ala Gin Phe Lys Glu ^L6U 165 170 . ₁₇₅

CAA TTT TTA GAT AGG GAT ATT AAG TTT GAT AGT TAT CTA GAT ACG CAT Gin Phe Leu Asp Arg Asp He Lys Phe Asp Ser Tyr Leu Asp Thr His

190 185 .־. 180

TTA ACT GCT CAA CAA GTT TCC AGT AAA GAA AGA GTT ATT TTG AAG GTT Leu Thr . Ala Gin Gin Vai Ser Ser Lys Glu Arg Vai lie Leu Lys Vai

205 200 י.'.. ¹⁹⁵ '

ACG GTT CCG AGT GGG AAA GGT TCT ACT ACT CCA ACA AAA GCA GGT GTC . Thr. Vai Pro Ser Gly Lys Gly Ser. Thr Thr Pro Thr Lys Ala GlyVai' ²¹⁰ '215,220

ATT TTA AAT AAT AGT GAA TAC AAA ATG CTC ATT GAT AAT GGG TATATG ^{12e ASn ASn Ser Glu} Ty^r,.Lys Met Leu lie Asp Asn Gly ׳TyrMet ²²5 . 230235

GTC CAT GTA GAT AAG GTA TCA AAA CTG GTG AAA AAA GGG GTG GAG TCC

Vai Hrs Vai Asp Lys Vai Ser Lys Vai Vai Lys Lys Gly Vai Glu Cys

TTA CAA ATT GAA GGG ACT TTA AAA AAG AGT CTT GAC TTT AAA AAT GAT

Leu Gin He Glu Gly Thr Leu Lys Lys Ser Leu Asp Phe Lys Asn Asp

260' ., 265 ' . . 270

ATA AAT GCT GAA GCG CAT AGC TGG GGT ATG AAG AAT TAT GAA GAG TGG

He Asn Ala Glu Ala His Ser Trp Gly Met Lys Asn Tyr Glu Glu Trp ²⁷⁵ 280 285

V

1366

1414

1462

1510

1558

1606

1654

1702 .

1750

1798

1846

1894

1942

GCT

Ala

AAA GAT TTA ACC GAT Lys Asp Leu Thr Asp ' 290

AGG CAA

Arg

Gin

GAT TAT AAA

Asp Tyr Lys

GAA

Glu

TCG

Ser

ATC lie 310

AGT

Ser

GGA

Gly

AAT' GAA AAA Asn

Glu

Lys

TTA

Leu

GGC

Gly

GGG

Gly Lys

ATG

Met

AAG

AAA

Lys

CCG

CAA AGG

Gin Arg

AAT AAT

Asn Asn

CTA . GAT GCT

Leu

CCA

Pro. lie

ATA

Asp Ala

CCG

Pro

GAA

GAA

Glu

TAT

Tyr

CAA ATA

Gin He

AAT

Glu Asn

ATT lie 345

GCT

Ala

TTA

TTA

Leu

AGA

GAA TTT

Pro Glu

AAA

LysAsp Phe

GAT

TTT

GAA

Glu

TAT

Phe

GAA

GAT GGG TAT GCT Asp Gly Tyr Ala 300

AAT CAA GGC GGA Leu Arg Asn 315

Gin Gly Gly

1990

2038

AAA AAT

Lys

Asn

ATT

He

TCT GAT GCT

Ser Asp Ala

ACT

Thr Vai Tyr Arg Trp Cys • .350

GTG

TAT

AGA TGG TGT .2086

2134

GGT

Gly Tyr Gin lie

CAA ATT

AGT

Ser

GAT CCG TTA CCT TCT TTA Asp Pro

Leu Pro Ser Leu .365

2182

CAA TTT TTA Glu Gin

Phe ?Leu

AAT

Asn

ACA ATC

Thr lie

AAA

Lys

GAA GAC AAA GGA

Glu

Asp pys Gly

2230.

TAT ATG AGT ACA AGC. TTA Tyr Mef. Ser Thr Ser Leu 385.

TCG

Ser

AGT.GAA CGT CTT Ser

Glu

Arg Leu

GCA

Ala

GCT

Ala

TTT GGA

Phe Gly

TCT

Ser

2278

AGA AAA

Arg Lys 400

ATT ATA TTA CGA TTA He lie Leu Arg Leu

־. . .405

CAA

Gin

GTT

Vai

CCG AAA

Pro. Lys

GGA

Gly

AGT

Ser

ACG

Thr

GGT

Giy

GCG

Ala

2326

TAT

Tyr

TTA

AGT GCC ATT.GGT GGA

Leu. Ser Ala

He 420

Gly Gly

TTT GCA

Phe Ala

AGT Ser 425 .

GAA AAA Glu

GAG

Lys Glu

ATC

He

CTA CTT

Leu Leu

430 .

2374

GAT

Asp

AAA GAT AGT Lys Asp

Ser

AAA

Lys

TAT CAT

Tyr

His

AAA GTA

ATT GAT lie Asp Lys Vai Thr

440 '

ACA

GAG

Glu Vai

GTA

ATT ATT'.

He He

2422

AAA GGT GTT , Lys Gly Vai : . 450

AAG

Lys

CGA

Arg

TAT

Tyr

GTA

Vai

GTG

Vai

GAT GCA ACA TTA

Asp Ala Thr Leu

TTA ACA

Leu.Thr

AAT

Asn . 2467

TAAGGAGATG

AAAAATATGA

AGAAAAAGTT

AGCAAGTGTT GTAACGTGTA CGTTATTAGC 2527

TCCTATGTTT

TTGAATGGAA

ATGTGAATGC

TGITI'ACGCA

GACAGCAAAA CAAATCAAAT

2587

TTCTACAACA

CAGAAAAATC

AACAGAAAGA

GATGGACCGA

AAAGGATTAC TTGGGTATTA

2647

TTTCAAAGGA

AAAGATTTTA

GTAATCTTAC tatgtttgca

CCGACACGTG ATAGTACTCT

2707

TATTTATGAT

CAACAAACAG

CAAATAAACT

ATTAGATAAA

AAACAACAAG AATATCAGTC

2767

TATTCGTTGG

ATTGGTTTGA TTCAGAGTAA

AGAAACGGGA

GATTTCACAT TTAACTTATC

2827

TGAGGATGAA CAGGCAATTA TAGAAATCAA TGGGAAAATT ATTTCTAATA AAGGGAAAGA 2887 AAAGCAAGTT GTCCATTTAG AAAAAGGAAA ATTAGTTCCA ATCAAAATAG AGTATCAATC , '2947 AGATACAAAA TTTAATATTG ACAGTAAAAC ATITAAAGAA CTTAAATTAT TTAAAATAGA 3007 TAGTCAAAAC CAACCCCAGC AAGTCCAGCA AGATGAACTG AGAAATCCTG AATTTAACAA 3067 GAAAGAATCA CAGGAATTC־T TAGCGAAACC ATCGAAAATA AATCTTTTCA CTCAAAAAAT . 3127 GAAAAGGGAA ATTGATGAAG ACACGGATAC GGATGGGGAC TCTAHCCTG ACCTrrGGGA 3187 AGAAAATGGG TATACGATTC ACAATAGAAT CGCTGTAAAG TGGGACGATT CTCTAGCAAG 3247 TAAAGGGTAT ACGAAATTTG TTTCAAATCC ACTAGAAAGT CACACAGTTG GTGATCCTTA 3307 .35

TACAGATTAT GAAAAGGCAG CAAGAGATCT AGATTTGTCA AATGCAAAGG AAACGTTTAA 3367 CCCATTGGTA GCTGCTTTTC CAAGTGTGAA TGTTAGTATG GAAAAGGTGA TATTATCACC 3427 AAATGAAAAT TTATCCAATA GTGTAGAGTC TCATTCATCC ACGAATTGGT CTTATACAAA 3487 TACAGAAGGT GCTTCTGTTG AAGCGGGGAT TGGACCAAAA GGTATTTCGT .TCGGAGTTAG 35.47 CGTAAACTAT CAACACTCTG AAACAGTTGC ACAAGAATGG GGAACATCTA CAGGAAATAC 3607 TTCGCAATTC AATACGGCTT CAGCGGGATA TTTAAATGCA AATGTTCGAT ATAACAATGT 3667

AGGAACTGGT GCCATCTACG ATGTAAAACC TACAACAAGT TTTGTATTAA ATAACGATAC 3727 TATCGCAACT ATTACGGCGA AATCTAATTC TACAGCCTTA AATATATCTC CTGGAGAAAG 3,787 TTACCCGAAA ׳ AAAGGACAAA ATGGAATCGC AATAACATCA ATGGATGATT TTAATTCCCA 3847 TCCGATTACA ΤΤΑΑΑΤΑΆΑΑ AACAAGTAGA TAATCTGCTA AATAATAAAC CTATGATGTT 3907 GGA7ACA.AAC. CAMCAGATG GTGTTTATAA GATAAAAGAT ACACATGGAA ATATAGTAAC/ 3967 TGGCGGAGAA TGGAATGGTG TCATACAACA AATCAAGGCT AAAACAGCGT CTATTATTGT 4027 GGATGATGGG GAACGTGTAG. CAGAAAAACG TGTAGCGGCA AAAGATTATG AAAATCCAGA- . 4087 AGAT/kAAACA CCGTCTTTAA CTTTAAAAGA TGCCCTGAAG CTTTCATATC CAGAl’GAAAT 4147 AAAAGAAATA GAGGGATTAT TATATTATAA AAACAAACCG ATATACGAAT CGAGCGTTAT 4207 GACTTACTTA GATGAAAATA CAGCAAAAGA AGTGACCAAA CAATTAAATG ATACCACTGG 4267 GAAATTTAAA GATGTAACTC ATTTATATGA. TGTAAAACTG ACTCCAAAAA. tgaatgttac . 4327 AATCAAATTG TCTATACTTT ATGATAATGC TGAGTCTAAT GATAACTCAA TTGGTAAATG 4387 GACAAACACA AATATTGTTT CAGGTGGAAA TAACGGAAAA AAACAATATT CTTCTAATAA . 4447

TCCGGATGCT AATTTGACAT TAAATACAGA TGCTCAAGAA AAATTAAATA AAAATCGTGA

CTATTATATA AGTTTATATA TGAAGTCAGA AAAAAACACA CAATGTGAGA TTACTATAGA

TGGGGAGATT TATCCGATCA CTACAAAAAC AGTGAATGTG AATAAAGACA ATTACAAAAG

4507

4567

4627

ATTAGATATT ATAGCTCATA ATATAAAAAG TAATCCAATT TCTTCACITC ATATTAAAAC . . 4687

GAATGATGAA ATAACTTTAT. TTTGGGATGA .TATTTCTATA ACAGATGTAG CATCAATAAA 4747

ACCGGAAAAT TTAACAGATT CAGAAATTAA ACAGATTTAT AGTAGGTATG GTATTAAGTT 4807 AGAAGATGGA ATCCTTATTG ATAAAAAAGG TGGGATTCAT TATGGTGAAT TTATTAATGA V 4867 AGCTAGTTTT AATATTGAAC CATTGCAAAA TTATGTGACC AAATATGAAG TTACTTATAG 4927

2.1,355

IAGTGAGTTA GGACCAAACG tgagtgacac acttgaaagt GATAAAATTT ACAAGGATGG 4987 GACAATTAAA TTTGATTTTA CCAAATATAG TAAAAATGAA CAAGGATTAT TTTATGACAG 5047 TGGATTAAAT 'TGGGACTTTA AAATTAATGC TATTACTTAT GATGGTAAAG AGATGAATGT 5107 TTTTCATAGA ΤΑΤΑΑΤΑΆΑΤ AGTTATTATA TCTATGAAGC TGGTGCTAAA GATAGTGTAA 5167 AAGTTAATAT ACTGTAGGAT TGTAATAAAA GTAATGGAAT TGATATCGTA CTTTGGAGTG 5227 GGGGATACTT TGTAAATAGT TCTATCAGAA ACATTAGACT AAGAAAAGTT ACTACCCCCA 5287

CTTGAAZiATG AAGATTCAAC TGATTACAAA CAACCTGTTA AATATTATAA GGTTTTAACA 5347 ΑΑΑΙΑΤΐΑΑΑ CTCTTTATGT TAATACTGTA ATATAAAGAG TTTAATTGTA TTCAAATGAA 5407 GCTTTCCCAC AAAATTAGAC TGATTATCTA ATGAAATAAT CAG7?CT7\ATT TTGTAGAACA 5467 GGTCTGGTAT TATTGTACGT GGTCACTAAA AGATATCTAA TATTATTGGG CAAGGCGTTC 5527 CATGATTGAA TCCTCGAATG TCTTGCCCTT TTCATTTATT TAAGAAGGAT TGTGGAGAAA 5587 TTATGGTTTA .GATAATGAAG AAAGACTTCA CTTCTAATTT TTGATGTTAA ATAAATCAAA 5647 ATTTGGCGAT TCACATTGTT TAATCCACTG ATAAAACATA CTGGAGTGTT CTTAAAAAAT 5707 CAGCTI'TTTT CTTTATAAAA TTTTGCTTAG CGTACGAAAT TCGTGTTTTG TTGGTGGGAC 5767 CCCATGCCCA TCAACTTAAG AGTAAATTAG TAATGAACTT TCGTTCATCT GGATTAAAAT 5827 AACCTCAAAT TAGGACATGT TTTTAAAAAT AAGCAGACCA AATAAGCCTA GAATAGGTAT 5887 ΟΆΊΤΓΤΤΑΑΑ AATTATGCTG CTlTCTTTTG TTTTCCAAAT CCATTATACT CATAAGCAAC 5947 ACCCATAATG TCAAAGACTG TTTTTGTCTC ATATCGATAA GCTTGATATC GAATTCCTGC 6007 ־

AGCCCGGGGG ATCCACTAGT TCTAGAGCGG CCGCCACCGC GG

6049 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: ' (A) LENGTH: 462 amino acids (B) ,TYPE: amino acid (D) TOPOLOGY: linear' (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Arg Met Glu Gly Lys.Leu Phe Met Vai Ser Lys Lys Leu Gin

.. 15 ־... . 10 ' ' ⁵ :־. יי ¹

Vai Val Thr Lys Thr Vai Leu Leu Ser Thr Vai Phe Ser lie Ser Leu

. 30 . . 25 ' י ²⁰ .

Leu Asn Asn Glu Val lie Lys Ala Glu Gin Leu Asn He Asn Ser Gin 35 . 40' . 45.

Ser Lys Tyr Thr Asn Leu Gin Asn Leu Lys He Thr Asp Lys Val Glu . ⁵⁰ 55 'GO ) Asp Phe Lys Glu Asp Lys Glu Lys Ala Lys Glu Trp Gly Lys Glu Lys

.30 י. : 75 . ..־.־. ⁷⁰⁶⁵

Glu Lys Glu Trp . Lys Leu Thr Ala Thr Glu Lys Gly Lys Met Asn Asn 85 . . 90 . . 95 .

Phe Leu Asp Asn Lys Asn Asp He Lys Thr Asn Tyr Lys Glu lie Thr ¹.θθ . 105. . . no

Phe Ser Met Ala Gly Ser Phe Glu Asp Glu lie Lys Asp Leu Lys Glu ^{115 120}. . 125

He Asp Lys Met Phe Asp Lys Thr Asn Leu Ser Asn Ser lie He Thr ¹³⁰ . . 135 . . 140 _; ..

Tyr Lys Asn Val Glu Pro Thr Thr lie Gly Phe Asn Lys Ser Leu Thr . ¹⁴⁵. . . ' - 150 . . . 155.!60

Glu Gly Asn Thr He Asn Ser Asp Ala Met Ala Gin Phe Lys GluGin ¹⁶⁵ ' . ' ¹⁷⁰ .175

Phe . Leu Asp Arg Asp lie Lys Phe Asp Ser Tyr Leu Asp Thr HisLeu

180 1851.90

Thr Ala Gin Gin Val Ser Ser Lys Glu Arg Val He Leu Lys ValThr ¹⁹⁵ . 200 . .205

Val Pro Ser Gly Lys Gly Ser Thr Thr Pro Thr Lys Ala Gly Val He . 21° 215 . '220

Leu Asn Asn Ser Glu Tyr Lys Met Leu lie Asp Asn Gly Tyr Met Val ²²⁵ 230 . 235240

v.

/3

His Val Asp Lys Val Ser

Lys Val Val Lys Lys

Gly

Val Glu

Cys Leu

Gin lie. Glu Gly Thr Leu

Lys Lys

Ser Leu. Asp

Phe

Lys. Asn

Asp He

Asn Ala

Glu Ala His.Ser 275 .

Trp Gly ' 280

Met

Lys Asn

Tyr

Glu Glu Trp Ala

Lys Asp

Leu Thr Asp

Ser Gin . .295

Arg

Glu

Ala

Leu Asp Gly Tyr Ala Arg

Gin Asp Tyr Lys Glu lie Asn

Asn

Tyr

Leu

Arg.Asn Gin Gly Gly Ser

320 .'.־ ³¹⁵

Gly Asn Glu Lys

Leu

Asp Ala

Gin lie Lys

Asn lie Ser Asp Ala Leu

Gly Lys Lys Pro lie Pro

Glu Asn He Thr 345 '

Val

Tyr Arg Trp Cys Gly 350

Met Pro Glu?Phe .'.. 355

Gly Tyr

Gin

He

Ser Asp Pro

Leu.Pro Ser Leu Lys

Asp Phe

Glu Glu

Gin

Phe

Leu

Asn

Thr

He Lys

Glu

Asp Lys Gly Tyr.

Met Ser

Thr Ser'Leu

Ser Ser

Glu

Arg

Leu Ala,

Ala

Phe

Gly

Ser Arg

Lys. He lie Leu

Arg

Leu Gin Val Pro Lys.Gly SerThr

Gly

Ala

Leu Ser

Ala He

Gly Gly Phe

Ala Ser Glu

Lys Glu lie Leu ' 43.0

Leu

Asp .

Lys Asp

Ser Lys

Tyr His lie

Asp Lys Val 440

Thr Glu Val lie 1!_e445'

Lys

Gly Val

Lys Arg

Tyr.Val

Val

Asp Ala Thr

Leu Leu Thr Asn.

(2) INFORMATION

FOR. SEQ

ID NO:3

SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOIXOGY: linear (ii)

MOLECULE TYPE: peptide (ix)

FEATURE:

(A) NAME/KEY: Peptide (B) LOCATION: 1.20 (D) OTHER INFORMATION- /πλΙλ- .&lt;ו1^4ה ,targetting ukmahon. ./note-Signal peptide for vacuolar

1T5382/3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro He Arg Val Thr Aj

Asp Arg ’

Ala Ala Ser Thr (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2655 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single • . (0) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO . (vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus . (B) STRAIN: AB78 (C) INDIVIDUAL ISOLATE (ix) FEATURE:

. (A) NAME/KEY: CDS (B) LOCATION: 1.2652 (D) OTHER INFORMATION:

cereus

NRRL B-21058 .

/note־ Thk°i ^W(JRMATION: /product100 ־ kDa protein VIPlA(a) /note- This sequence is identical to the portion of SEO ID ncTi between and including nucleotide 2475 to 5126 ״ ° (xi) SEQUENCE DESCRIPTION; SEQ ID NO:4:

• ATG AAA AAT ATG AAG AAA

Met Lys Asn Met Lys Lys

L. 465

AAG

Lys

TTA GCA AGT GTT Leu Ala Ser 470

Val

GTA

Val

ACG

Thr

TGT ACG TTA

Cys Thr Leu :

TTA GCT.CCT Leu

Ala

480

Pro

ATG TTT TTG

Met Phe Leu

AAT

Asn

GGA AAT GTG Gly Asn

Val

AAT

Asn

GCT

Ala

GTT

Val

TAC GCA GAC Tyr Ala. Asp

AGC

Ser

AAA

Lys

ACA

Thr

AAT.CAA ATT

Asn Gin He

500'

TCT ACA

Ser Thr

ACA

Thr

CAG

Gin

AAA

Lys

AAT

Asn

CAA

Gin

CAG AAA GAG

Gin Lys Glu

ATG GAC CGA ΆΆΑ GGA TTA CTT Met Asp Arg ׳ Lys Gly Leu Leu 515

AGT AAT CTT ACT ATG TTT GCA Ser Asn

GAT CAA

Asp Gin

GGG TAT TAT TTC Gly Tyr

Tyr Phe

AAA GGA AAA GAT Lys

Gly Lys

Asp

TTT

Phe

CCG

Leu Thr Met Phe Ala Pro 530

CAA ACA GCA AAT AAA CTA Gin Thr Ala Asn Lys

. ־ 545

CAG TCT ATT CGT. TGG ATT GGT —-¹ Ser Ηθ Arg Trp lie Gly ⁵⁵⁰ 565

Gin

TTC

Phe

GGG

Gly

GAA

Glu

AAA

Lys

ATA lie

ACA TTT AAC TTA TCT Thr Phe Asn Leu

AAA ATT ATT Lys lie He

TCT

Ser

595

AAA

Lys

ITT

Leu

ACA

Thr

CGT GAT

Arg Asp

TTA GAT AAA

Leu Asp Lys

TTG ATT CAG

Leu

GAG

Ser Glu Asp

AAT AAA

Asn Lys

GAT

He Gin

GAA CAG

Glu Gin

GGG AAA GAA

Gly Lys Glu

AGT

Ser

AAA

Lys

ACT

Thr

CTT.

Leu

ATT lie Tyr

TAT240

AGT

Ser

CAA CAA GAA TAT Gin 555

Gin Glu Tyr

AAA GAA

Lys Glu

ACG GGA GAT

Thr Gly Asp

336

GCA. ATT ATA GAA ATC AAT Ala 585 lie lie

Glu lie Asn

AAG

Lys

CAA

Gin

GTT

Vai

GTC

Vai

CAT TTA .

His Leu

GGA AAA

Gly Lys

TTA GTT CCA

Leu. Vai Pro

AAT ATT GAC

Phe Asn lie Asp 625

AGT

Ser

AAA

Lys

ATC

He

ACA

Thr

AAA ATA

Lys lie

TTT AAA

Phe, Lys

GAG TAT

Glu Tyr

CAA TCA

Gin

Ser

GAT ACA Asp Thr .

480

GAA

Glu

CTT AAA

Leu Lys

TTA

Leu

TTT

Phe

AAA

Lys

GAT Asp. .640

AGT CAA

Ser Gin

AAC CAA

Asn Gin

CCC

Pro

CAG

Gin Gin Vai

CAA GTC

CAG

Gin

CAA GAT

Gin

Asp

GAA

Glu

CTG

Leu

AGA.

Arg '

AAT CCT

Asn Pro 655

GAA TTT

Glu Phe

AAC AAG. AAA Asn.Lys Lys 660

GAA

Glu

TCA.CAG

Ser Gin

TTG

GAA

Glu Phe

665 .

TTA

Leu

GCG

Ala

AAA CCA

Lys Pro

670

TCG AAA;ATA AAT

Ser Lys lie Asn

CTT

Leu

TTC

Phe

ACT

Thr Gin

CAA

AAA ATG

Lys Met

680 ־

AAA AGG Lys

Arg Glu

GAA ATT GAT GAA

He Asp Glu 685

GAC

Asp

ACG GAT ACG Thr

Asp Thr

GAT. GGG Asp Gly

GAC

Asp

TCT ATI.

Ser He

695 ,

CCT

Pro

GAC

Asp Leu

CTT

TGG

Trp

GAA GAA AAT Glu Glu Asn 700

GGG

Gly Tyr

TAT

ACG

Thr

ATT

He

CAA

Gin

AAT AGA

Arg lie Ala 710

ATC GCT

Asn

GTA AAG TGG

Vai Lys Trp Asp Asp . 715

GAC

GAT

TCT CTA

SerLeu

GCA

Ala

AGT

Ser

AAA

Lys. Gly Tyr

GGG

TAT

ACG

Thr

AAA

Lys

TTT GTT

Phe Vai

TCA AAT CCA CTA

Ser Asn Pro Leu

GAA

Glu

AGT CAC

Ser His

<img file="IL115382A_D0002.tif" />

, 118 ־

CCT TAT. ACA GAT TAT GAA

Pro Tyr Thr Asp Tyr Glu

ACA GTT

Thr Vai

GGT GAT

Gly Asp

AAG

Lys

GCA GCA AGA GAT CTA Ala Ala

Arg Asp Leu

GAT TTG

Asp Leu

TCA AAT

Ser Asn

GCA AAG GAA. ACG TTT Ala Lys Glu Thr Phe 755

AAC

Asn 760

CCA

Pro Leu Vai

TTG GTA

GCT GCT TTT

Ala Ala Phe • 765 . .

CCA AGT

Pro Ser

GTG AAT

Vai Asn

GIT AGT ATG' GAA AAG Vai Ser Met

Glu Lys

GTG

Vai

ATA TTA TCA CCA AAT GAA lie Leu Ser Pro Asn Glu 780

AAT TTA

Asn Leu . '785

TCC AAT

Ser.Asn

AGT

Ser

GTA

Vai

GAG

Glu

TCT CAT TCA Ser His Ser 790 .

TCC

Ser

ACG AAT

Thr Asn

TGG TCT TAT

Trp Ser Tyr

1008

ACA

Thr

AAT ACA.GAA Asn Thr Glu 800 .

GGT.

Gly Ala

GCT

TCT.

Ser Vai

GTT

GAA

Glu

GCG

Ala Gly

GGG

ATT GGA

CCA AAA GGT lie Gly Pro Lys Gly. 810

1056

TCG TTC

ATT lie Ser Phe 815

GGA'GTT AGC Gly Vai

Ser

GTA AAC

Vai Asn Tyr

TAT

CAA CAC

Gin His

TCT GAA

Ser Glu

ACA GTT GCA

Thr Vai Ala 830 ^;

1104

TGG

Trp Gly Thr

835,

GGA ACA

TCT ACA

Ser

Thr

GGA AAT ACT TCG CAA Gly Asn Thr Ser Gin 840

TTC

Phe

AAT. ACG

Asn Thr

GCT

Ala

1152

TCA GCG

Ser Ala

GGA TAT TTA AAT Gly Tyr Leu Asn Ala 850 .

GCA

AAT GTT CGA TAT AAC Asn Vai Arg Tyr Asn 855.

AAT GTA GGA ACT .

Asn Vai Gly Thr . . 860

1200

GGT GCC ATC

Gly Ala He

TAC GAT

Tyr Asp

GTA

Vai

AAA CCT ACA ACA AGT TTT Lys Pro Thr Thr Ser Phe 870

GTA TTA AAT AAC Vai Leu Asn Asn

^ז 875

1248

GAT ACT' ATC

Asp Thr He

880

GCA ACT

Ala

Thr

ATT lie

885

ACG GCG AAA Thr Ala Lys

TCT

Ser

AAT TCT

Asn Ser

ACA GCC TTA AAT Thr Ala. Leu Asn

1296

ATA TCT: CCT GGA GAA AGT TAC lie

Ser Pro

Gly Glu Ser Tyr 900 .

CCG

Pro

AAA

Lys Lys

AAA

GGA CAA

Gly Gin

AAT

Asn

GGA ATC

Gly He

GCA

Ala

910.

1344

ATA lie

ACA-TCA

Thr Ser

ATG

Met

GAT GAT TTT Asp Asp 915 .

Phe

AAT

Asn

TCC

Ser

CAT CCG ATT

His Pro lie

ACA

Thr

TTA AAT

Leu Asn

AAA

Lys

1392

AAA CAA GTA

Lys Gin Vai

GAT

Asp 930

AAT

Asn

CTG

Leu

CTA AAT

Leu

Asn

AAT

Asn Lys

AAA

CCT ATG

Pro Met

ATG TTG

Met Leu

GAA

Glu

ACA

Thr

1440

AAC CAA ACA

Asn Gin Thr

GAT GGT

Asp Gly

GTT

Vai

TAT

Tyr

AAG

Lys

ATA AAA

He Lys

GAT ACA

Asp Thr.His Gly

CAT GGA

AAT

Asn

ATA

He

1488

־

950 .

GTA ACT GGC'GGA GAA TGG AAT Vai Thr Gly Gly Glu Trp Asn ?60. _%5

ACA GCG

Thr Ala 975

TCT ATT ATT GTG GAT Ser

GTAGCG

Ala. Ala

GCA

Vai

ACT

Thr

TTA

Leu

He

AAA

Lys

GGT GTC ATA CAA CAA Gly Vai lie Gin Gin 970

GAT GGG. GAA CGT GTA He Vai Asp Asp Gly Glu Arg Vai ⁹⁸⁰ . .985

GAT TAT

Asp Tyr

GAA

Glu

ATC AAG lie Lys

GCA GAA

Ala Glu

AAT CCA GAA GAT AAA ACA CCG Asn Pro Glu Asp Lys Thr Pro 1000

GCT AAA

Ala Lys

AAA.CGT Lys

Arg 990

TCT

Ser 1005

TTA .

Leu

1536

1584

1632

AAA

Lys Asp Ala 1010

GAT

GCC

CTG

Leu Lys

AAG

CTT

Leu

TCA TAT CCA GAT GAA ATA Ser Tyr Pro Asp’ Glu lie 1015

AAA

Lys 1020

GAA

Glu

1680 gga tta tta tat

ATA GAG ( J______ lie Glu 'Gly Leu Leu Tyr 1025

GTT ATG ACT TAC TTA GAT Vai Met Thr Tyr Leu Asp 1040

TTA AAT GAT ACC ACT Leu.Asn 1055

Asp Thr Thr

TAT

Tyr

AAA AAC AAA CCG ATA TAC GAA .TCG ־;״ ' > Lys

Lys Asn 1030

GAA .

Glu . 1045

AAT

Ash Thr

ACA

GCA

Ala

GTA AAA ;Vai Lys

TAT GAT

Tyr Asp

ACA

Thr

AAT

Ash

Pro lie Tyr Glu Ser .1035

AGC

Ser

1728

GGGAAA

Gly Lys

1060

TTT AAA GAT. Phe

Lys Asp

GTA .

Vai . 1065

AAA

Lys

<td> GAA GTG</td><td> ACC AAA CAA</td>

<td> Glu Vai</td><td> Thr Lys Gin</td>

<td> 1050</td><td> . </td>

<td> AGT CAT</td><td> TTA TAT GAT</td>

<td> Ser His</td><td> Leu Tyr Asp</td>

1070

1776

1824

CTG ACT CCA

Leu Thr Pro׳Lys Met 1075

AAA ATG

AAT

Asn

GTT ACA .

Vai' Thr

1080

ATC lie

AAA

Lys

TTG TCT Leu

Ser.

ATA CTT He Leu 1085

1872

AAT GCT GAG TCT AAT GAT AAC TCA'ATT Asn Asp Asn Ser He . 1095

Asn Ala Glu

1090

AAT, ATT GTT Asn He Vai . 1105

AAT CCG GAT Asn Pro 1120 .

TTA

Leu 1135

AAA

Lys

ACT

Asp

Ser

GGT AAA

Gly Lys

TGG ACA AAC .

TrpThr Asn

..־ .1100

1920

TCA

Ser

GCT

Ala

AAT.AAA Asn

AAC

Asn

Lys

AAT

Asn

ACA

Thr

CAA

Gin

GGT

Gly Gly Asn Asn Gly Lys '.. . 1110 .

GGA AAT AAC GGA AAA

AAT TTG ACA TTA AAT

Asn Leu Thr . 1125.

Leu

Asn

AAA CAA TAT TCT Lys Gin Tyr Ser 1115. .

TCT.

Ser

1968

CGT

Arg Asp Tyr Tyr lie 1140

GAC TAT TAT

TGT GAG ATT Cys Glu lie

1155

ACT

Thr

ATA

ATA He

ACA AAA. ACA GTG AAT GTG

AAT

ACA

Thr Asp Ala Gin Glu Lys 1130

GAT GCT CAA. GAA

AGT

Ser .Leu Tyr Met Lys Ser

1145 .־

TTA TAT ATG

GAT GGG GAG

Asp Gly Glu 1160

AAA GAC AAT TAC

AAA .

2016

AAG TCA

GAA

Glu

1150'

2064

ATT TAT CCG lie Tyr Pro 1165 .

ATC He

2112

AAA AGA TTA GAT׳

2160

2208

Thr Thr

Lys Thr Vai

1170

Asn

Vai Asn Lys Asp Asn

1175

Tyr Lys Arg Leu Asp

י 1180 . ATT ATA

- He lie

GCT CAT Ala His 1185.

AAT

Asn

ATA

He.

AAA

AGT AAT CCA ATT‘ Lys Ser Asn Pro He '1190

TCT TCA ___ ___

Ser Ser Leu His . 1195

CTT CAT

ATT

He

AAA

Lys

ACG AAT

Thr Ash

1200

GAT Asp ’ 1215

CAG

Gin

GAT

GAA ATA Asp Glu

GTA GCA Vai Ala Ser

TCA ATA

He

ATT TAT. AGT lie Tyr

Ser

He

ACT TTA TIT Thr Leu,Phe 1205

TGG GAT

Trp Asp Asp lie Ser He 1210

AAA

Lys Pro Glu Asn Leu 1220

CCG GAA AAT

AGG

Arg Tyr Gly 1235

TAT GGT

TTA

GAT ATT TCT ATA

ACA

Thr

ACA GAT TCA GAA ATT AAA. Thr Asp Ser Glu.lie Lys 1225

ATT AAG TTA

Lys Leu : .;. 1240

He

1230 . .2256 .2304 .’

GAT

Asp Lys Lys Gly Gly 1250 '

GAA GAT GGA ATC CTT ATT Glu Asp Gly He Leu lie ' 1245

2352

AAA AAA

TTT AAT

Phe Asn

TAT AGT

Tyr

GGT

GGG ATT CAT, TAT C״ . Us His Tyr Gly Glu '1255.

GGT. GAA TTT. ATT AAT i Phe lie Ash

GAA Glu 1260

GCT AGT

Ala Ser:

2400

ATT GAA CCA lie Glu Pro 1265

TTG CAA

Leu Gin Asn Tyr Vai

׳ . 1270

AAT

TAT GTG

ACC AAA TAT Thr Lys.Tyr 1275

GAA GTT.ACT Glu Vai Thr

2448

Ser

1280

AAA Lys 1295

AGT GAG TTA Ser Glu Leu

ATT TAC AAG GAT He Tyr Lys Asp

GGA

Gly

CCA AAC GTG AGT Pro Asn Vai Ser 1285

GGG

Gly 1300

ACA ATT AAA

Thr He

Lys

GAC

Asp

ACA CTT GAA AGT GAT Thr Leu Glu ,Ser Asp 1290 . .

TTT GAT

Phe Asp.Phe Thr .1305.

TTT. ACC

AAA TAT AGT Lys Tyr

2496

2544

AAA AAT GAA CAA GGA TTA TTT TAT -- .—1 Glh Gly Leu Phe Tyr 1315

Lys Asn Glu

GAC

Asp

AGT GGA TTA AAT

Ser

1310

TGG GAC TH Ser Gly Leu Asn Tip Asp Phe ¹³²⁰ ' . 1325

TTT

2592

AAA AIT AAT C~~ ך.' Lys lie Asn Ala He Thr '1330.

GCT ATT. ACT

TAT

GAT

GGT. AAA GAG ATG AAT GTT TTT CAT Tyr Asp Gly^Lys Glu Met Asn Vai Phe His

1340

2640

AGA TAT AAT AAA. TAG Arg Tyr Asn Lys 1345 .

(2) INFORMATION FOR SEQ ID NO:5‘: .

(i) SEQUENCE CHARACTERISTICS־ ' &lt;A) LENGTH: 884 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear

2655

<img file="IL115382A_D0003.tif" />

(ii) MOLECULE TYPE: protein (xi) SEQUENCE

Met

-. 1

Lys Asn Met

Lys

DESCRIPTION: SEQ. ID NO:5:

Lys Lys Leu Ala Ser.Vai v_a! '10

Leu

Ala Pro Met

Thr Cys Thr Leu

Phe

Leu

Asn

Gly Asn Vai Asn Ala

Vai

Ser Lys Thr Asn . . 35

Tyr Ala Asp

Gin He

Ser

Ihr.Thr Gin Lys

Asn

Gin

Gin Lys Glu

Met Asp Arg Lys

Gly Leu Leu

Gly Tyr Tyr Phe

Lys

Gly Lys

Asp Phe

Ser

Asn Leu Thr

Met Phe Ala Pro

Thr Arg Asp

Ser

Thr Leu

Asp

Gin Gin Thr

He Tyr

Gin Ser . lie

Phe Thr Phe . H5

Gly Lys

He

Glu Lys

Ala Asn Lys

Arg Trp He 100

Asn Leu Ser

He Ser Asn

Gly Lys Leu

Vai

Leu

Leu Asp Lys.Lys . 90

Gin Gin Glu

..95

Gly

Glu

Leu

Asp

He Gin Ser Lys 105 .

Glu Gin Ala He

Lys Gly Lys Glu Lys Gin

-— . . 140

Glu

He

Vai

Thr Gly Asp 110 .

Glu He, Asn

Vai. His Leu

Lys Phe Asn lie. Asp . •165

Ser

Pro . He Lys He Glu : . . ' i55

Lys Thr Phe Lys Glu

170 .י.

Tyr Gin

Leu Lys

Ser Asp Thr ..

160 .

Leu

He Asp Ser Gin Asn

. 180 ־ .

Phe Lys

Asn

Pro Glu

Ser

Lys He 210.

Gin

Pro Gin Gin

Phe Asn Lys Lys Glu Ser Gin

Vai

Gin.Gin Asp.Glu '190

Glu Phe Leu Ala

Leu Arg.

Lys Pro

Asp 225

Asn Leu Phe Thr Gin Lys Met 215. ;.

Thr Asp Thr. Asp cly Asp Ser _{Ile Pr0}

Gly. Tyr Thr He Gin. Asn Arg He Ala

Lys Arg Glu,lie-Asp Glu

Asp 235

Vai

Ala Ser Lys Gly Tyr Thr Lys ׳Phe Vai ²⁶⁰ . 265

Leu Trp Glu Glii Asn

Lys Trp Asp Asp Ser Leu

255

Ser

Asn Pro Leu Glu Ser His 270 .

/3

Thr varay ״p p_ro Tyr _Thr AspTyr _{Glu lys} ,.

²⁸⁰285

Asp Leu Ser Asn Ma Lys Glu Thr _Phe a״־ _{Pto Valph(־} '300

Pro Ser Val val Ser. Met Glu Lys Val He i^u Ser Pro Asn Glu .' ³¹⁰ .. . 315 . . .

A״־ Leu Ser As״ Ser Val Glu Ser Bis Ser Ser _Thx ״־״ _Tro³²⁵ . ' 330 ' 33₅^y י‘ Clu Ala Gly He _Giy ״, _Lys³⁴⁵ .. 350 .

11־ Ser Phe Gly val Ser Val Asn Tyr _fli5 . , . ³⁶⁰ . .:. '365 ..

Gl״ Glu Trp _Gly Thr Ser Tip: Gly Asn Thr Ser Gl״ _Phe ,,.

³⁷⁵ . ' 380 ..י.

_{e1y Ihr} . 395

Gly Ala Tie Tyr Asp Val Lys Pro Thr Thr Ser Phe Val leu As״ Ash ⁴¹⁰415

Asp Thr He Ma Thr He Thr Ala Lys Ser A״־ Ser Thr Ma Leu As״ ' ⁴²⁵ ..130

He Ser Pro Gly Glu Ser 1y_r Pro Lys Lys Gly Gl״ Asn Gly He Ala .⁴⁴5 . . ...־

Tie Thr Ser Met Asp Asp Phe As״ Ser Bls Pro He Thr Leu A״־ Lys ^J33 .460 . ..³ .;׳ .

Lg Gin Val Asp Asn Uu Leu Asn As״ Lys Pro M־_t «־_t Leu Glu Thr . . /U Λ7׳ς ^4/5 : 480

A״־ Gin Thr Asp Gly Va! Tyr Lys ώ Lys Asp Thr Bls Gly Asn lie . . ⁴⁹⁰ . '.495 . .י .

val Thr Gly Gly Glu Trp-As״ Gly Val He Gin Gl״ He Lys Ma Lyf

Thr Ala Ser He He Val Asp Asp Gly Glu Ar, val Ala Glu Lys Mo .:. . ⁵²⁰ ' 525.

val Ala Ma Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu , s4n

Thr Leu Lys Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu He Lys Gl״ '⁵⁵° «δ ₅₆₀

He Glu Gly Leu leu Tyr Tyr'Lys Asn Lys Pro He Tyr Glu Ser Ser

Val

Met Thr

Leu

Val

Ztf.35

Tyr Leu Asp Glu Asn

Asn Asp Thr Thr Gly Lys Phe

Lys Leu Thr Pro Lys Met Asn ⁶¹⁰ . . 615

Tyr Asp Asn Ala Glu Ser Asn Asp ⁶²⁵ 630

Thr Asn

Asn Asn

Leu Asn

Lys

Thr lie

Lys

Asp

Tie

Pro

Lys

Asn Thr

Thr

Lys lie

Ala

Thr

Val

Gin lie . 770

Asn

Ala

Tyr

Thr Ala Lys

Lys

Val

Asn

Val Ser Gly Gly Asn Asn

645 :

Asp Ala Asn Leu Thr,Leu

660 : . 665

Asn Arg Asp Tyr Tyr Tie 68C

Gin

Thr

His

Asp

Ser

Asp

Thr

Ser

Val

He lie

Gly Lys

Asn

Ser

Cys

Glu lie

Thr lie

Val

Asn

Glu lie

Asn

Val lie

Lys

He Thr

Lys Pro

Glu Val Thr Lys Gin

Ser His

Lys Leu

Leu . Tyr Asp

Ser.Il_e Le_u

Gly Lys Trp Thr Asn

Lys Gin ׳tyr Ser Ser

Thr Asp Ala Gin Glu . 670

Leu Tyr Met Lys Ser 685

Asp Gly Glu He Tyr Pro

Asn Lys Asp

Ser Asn

Leu Phe

Glu Asn

760. .

Ser Arg tyr Gly lie Lys . 775 . '

Lys

Glu lie ^sp Asn Tyr Lys Arg Leu Asp

Pro

He Ser Ser Leu

Trp Asp Asp.Tie

Ser

Leu

Leu

A־P Lys Lys Gly Gly He _Bi_s Tyr Gly Glu . 790

Phe Asn lie Glu

Pro

Leu Gin Asn

Tyr Val

His lie lie

Thr

Thr Asp Ser .765.

Glu lie

Lys

Glu

Phe

Thr

Asp Gly lie

Leu

He

He Asn Glu

Lys Tyr Glu

Ala

Ser

Tyr Ser. Ser Glu

Leu

Gly

Pro

Asn

Val Ser

Asp

Lys lie

Tyr Lys

Asp. Gly

Thr lie

Lys' Phe

Val

Thr

Thr Leu Glu Ser

Asp Phe

Asp

Thr Lys Tyr Ser

Lys Asn . 850

Glu Gin

Gly Leu

Tyr Asp Ser

Gly Leu

Asn Trp Asp Phe

V

A

Lys He Asn Ala lie Thr Tyr Asp Gly Lys Glu. Met Asn Vai Phe His ⁸⁷⁵ ' 880'

Arg Tyr Asn Lys (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2004 base pairs (B) TYPE: nucleic acid .(C) STRANDEDNESS: single (D) TOPOLOGY:, linear ' , (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:.

(A) ORGANISM: Bacillus cereus (B) STRAIN: AB78 (C) INDIVIDUAL ISOLATE: NRRL B-21058 (ix) /note־ feature:

(A) ΝΑΜΕ/ΚΕΥ: CDS (B) LOCATION: 1.2001 (D) OTHER INFORMATION: /product80 ־ kDa protein VIPlA(a)' ^Thi^s. ^se^^{ence 1}s identical to that found in SEO id ncTi een and including nucleotide positions 3126 and 5126 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ATG

Met

CCT

Pro

AAA.AGG GAA ATT GAT Lys Arg Glu He Asp ; . . 890.

GAA GAC ACG GAT ACG GAT GGG GAC TCT ATT Glu Asp Thr Asp Thr Asp Gly Asp Ser lie ⁸⁹⁵ . .900

GTA

Vai

GAC CTT TGG GAA GAA AAT GGG TAT ACG ATT'CAA Asp Leu Trp Glu Glu Asn Gly Tyr Thr lie Gin ⁹⁰⁵ .910 .

AAT AGA ATC GCT Asn Arg He Ma 915

AAG TGG GAC GAT TCT CTA GCA Lys Trp Asp Asp Ser Leu Ala \ '920

AGT AAA GGG TAT ACG AAA TTT Ser Lys Gly Tyr Thr Lys Phe 925 95_o

GTT

Vai

TCA AAT

Ser Asn

CCA CTA GAA AGT CAC ACA GTT GGT GAT CCT Pro Leu Glu Ser His Thr Vai Gly Asp Pro yd5 . 940

TAT ACA GAT TAT . Tyr Thr Asp Tyr 945

GAA AAG GCA GCA AGA GAT Glu Lys'Ala Ala Arg Asp 950

CTA GAT TTG TCA AAT GCA AAG GAA ACG TTT Leu Asp Leu Ser Asn Ala Lys Glu Thr Phe ⁹⁵⁵ 960

GTA GCT

Vai Ala

GCT TTT CCA

Ala

y.i.W

AAC CCA TTG Asn Pro Leu 965 .

GTG ATA.TTA

Vai lie

TCA TCC

Ser Ser

Phe Pro

AGT

Ser

GTG AAT

Vai Asn

GTT AGT ATG

Vai Ser Met

GAA AAG

Glu Lys

980 .

. 288.

Leu

TCA CCA

Pro Asn

Ser

ACG

Thr Asn

1000 '

AAT

AAT

GAA AAT

Glu Asn

TTA

Leu

TCC AAT

Ser Asn

AGT

Ser

GTA GAG

Vai Glu Ser His 995.

TCT CAT.

TGG TCT

Trp Ser

TAT ACA

Tyr Thr

AAT ACA

Asn Thr 1005

GAA

Glu

GGT GCT TCT GTT Gly Ala Ser Vai 1010

GAA

Glu

GCG׳GGG ATT GGA CCA AAA ΑΣ3.: Gly

CAA CAC lie

1015

Gly Pro

Lys

GGT

Gly

ATT He 1020

TCG

Ser

TTC Phe. Gly

GGA

GCT AGC GTA AAC

Vai Ser Vai Asn Tyr 1025

TAT , 432 .

TCT GAA ACA Gin His. Ser Glu 1030

ACT TCG CAA Thr.Ser Gin .1045

CGA TAT

Arg Tyr

AAC

Asn

Thr

GTT GCA

Vai Ala

1035

CAA GAA

Gin Glu

TGG GGA

Trp Gly

ACA TCT ACA Thr Ser Thr 1040 _:

GGA AAT

Gly Asn

ACA AGT

TTC

Phe

AAT

Asn

ACG GCT TCA GCG Thr Ala Ser Ala 1050

AAT

Asn Vai

1065.

GTA

GGA 'ACT GGT GCC Gly Thr. Gly Ala

TTT GTA Thr Ser Phe

Vai

1080

TTA

Leu

AAT AAC

Asn Asn

GGA TAT

Gly Tyr

1055

TTA AAT GCA Leu Ash Ala

AAT

Asn

GTT

Vai 1060 ,528

ATC TAC GAT GTA AAA CCT He Tyr Asp . Vai Lys Pro 1070

ACA

Thr 1075 . 576.

GAT ACT' ATC Asp Thr He .1085 .

GCA ACT ACT ACG GCG AAA Ala Thr lie Thr Ala Lys 1090

624

TCT AAT TCT Ser Asn Ser 1095

ACA GCC

Thr

Ala

TTA

Leu

AAT

Asn

ATA TCT CCT _______ n״״^{Ser Pro Gly Glu Ser} ?yr Pro tys 1105

GGA GAA AGT

TAC CCG AAA

672.

' AAA GGA

Lys Gly Gin Asn 1110

CAA AAT

GGA-ATC

Gly He

GCA ATA ACA TCA ATG GAT GAT TTT Ala lie Thr Ser Mot Asp Asp Phe 11-0. וו on

1120

AAT

Asn

TCC

Ser . 720; '

CAT CCG ATT ACA His Pro He Thr 1125

TTA

Leu

AAT.

Asn 1130

AAA

Lys

AAA CAA GTA GAT AAT Lys Gin Vai Asp Asn . 1135'

CTG

Leu

CTA

Leu

AAA CCT. ATG ATG Lys Pro. Met

AATAAT

Asn 1140

Asn

Met

TTG Leu. 1 1145

GAA

Glu

ACA Thr. Asn

AAC

CAA

Gin

ACA GAT GGT Thr Asp Gly 1150

GTT

Vai

TAT AAG

Tyr Lys

1155

ATA lie

AAA GAT

Lys

Asp

ACA

Thr

CAT His 1160

GGA. AAT

Gly Asn

ATA GTA

He Vai

ACT Thr 1165 .

GGC GGA

Gly Gly

GAA

Glu

TGG

Trp

AAT GGT GTC Asn Gly Vai 1170

ATA

He

CAA

Gin

CAA

Gin

ATC AAG' GCT lie Lys Ala

AAA ACA GCG TCT ACT.ATT GTG GAT GAT GGG Ly־ Thr Ala Ser n_{־ Ile v־a tep}¹¹⁷⁵ . . 1180 1185

GAA CGT GTA

Glu Arg Vai

1190

GCA

Ala

GAA .’AAA CGT GTA GCG Glu,Lys Arg Vai 1195

Ala

GCA AAA GAT TAT GAA' AAT

Asn

Ala

Lys Asp Tyr

1200

Glu

CCA

Pro

GAA GAT AAA Glu Asp Lys 1205

ACA

Thr

CCG TCT TTA ACT

Pro

TTA

AAA

TAT

Tyr

CCA

Pro

GAT

AAA

Lys

CCG

Pro

ATA

He

GCA AAA GAA Ala Lys

Ser Leu Thr 1210

GAA ATA .

Glu lie :

1225

TAC

Tyr 1240 '

GAA

Glu

GTG ACC

Vai Thr

1255

Glu

GAT GTA :

Asp Vai

1270

AGT CAT

Ser His

TTA

Leu

AAA

Lys

TCG

Ser

ACA ATC AAA, TTG Thr lie Lys 1285

Leu

TCA

Ser

ATT. GGT lie Gly

AAA .GGA

Gly

GAT GCC CTG Leu Lys Asp Ala ’ 1215

GAA ATA GAG GGA TTA. TTA Glu He Glu Gly Leu Leu , 1230

AGC.GTT

Vai Met Thr 1245 .

Ser

CAA

ATG ACT

Leu

AAG

Lys

CTT

Leu

TCA Ser 1220

1008

AAA

Lys Gin

TTA AAT

Leu Asn 1260

TAT GAT GTA Tyr Asp Vai . . 1275

TCT ATA CTT lie Leu 1290

Ser

TGG .

Lys .Trp '

1305

ACA AAC

Thr Asn

AAA

Lys

GAT

Asp

TAT

Tyr Tyr Lys Asn 1235

TAT AAA AAC , 1056

TAC TTA GAT GAA AAT ACA Tyr Leu

Asp Glu Asn Thr

1250

1104

ACC

Thr

ACT

Thr

GGG AAA TTT AAA Gly Lys Phe Lys 1265 .'

CTG

Leu Thr Pro Lys Met 1280

TAT

Tyr Asp Asn

GAT ״AAT

ACT

CCA AAA ATG

GCT GAG TCT AAT

Ala Glu 1295

Ser Asn

1152

AAT

Asn

GTT

Vai

1200

ACA

Thr

AAT ATT

Asn lie Vai . 1310

GTT

TCA

Ser

AAA AAA CAA TAT TCT. TCT AAT Lys

Lys Gin Tyr

1320 '

Ser.Ser Asn

AAT ,CCG GAT

Asn Pro 1325 ;

GCT

Asp .'Ala

AAT

Asn Thr

ACA

GAT

Asp

1335:

GCT CAA

Ala Gin

GAA

Glu

AAA TTA

Lys leu Asn Lys 1340 ’

AAT AAA

AAT iCGT

AGT

Ser

TTA-TAT ATG AAG