AU673591B2

AU673591B2 - Photoprotected bacillus thuringiensis toxin

Info

Publication number: AU673591B2
Application number: AU63081/94A
Authority: AU
Inventors: Diana Kirsty Brebner; Veronica Estela Herrera; Samantha Ovens
Original assignee: AECI Ltd
Current assignee: AECI Ltd
Priority date: 1993-05-18
Filing date: 1994-05-16
Publication date: 1996-11-14
Anticipated expiration: 2014-05-16
Also published as: ZA943344B; AP9400646A0; AU6308194A; BR9401993A; AP498A; OA09940A; CA2123669A1

Description

-M

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): AECI LIMITED Invention Title: PHOTOPROTECTED BACILLUS THURINGIENSIS

TOXIN

The following statement is a full ,iscription of this invention, including the best method of performing it known to z-ie/us: BACKGROUND OF THE INVENTION THIS invention relates to the protection against photodegredation of either an endotoxin of a strain of Bacillus thuringiensis(Bt) or the Bt cells per se, or the spores obtained from cultures of the Bt cells after sporogenesis.

Bt is a Gram positive bacterium which produces crystal protein inclusions known as protoxins, endotoxins or deita-endotoxins. These crystals have insecticidal activity with a high degree of specificity. The crystal protein production is initiated during stage II of sporulation, after which time the cells complete sporulation and lyse, releasing spores and crystals into the culture medium.

The spores/crystals preparations have been used for many years as biopesticides. However, there are some problems with the longevity of the Bt crystals in the field. One of these is the sensitivity of the crystals to ultraviolet radiation, which results in inactivation of the toxin. As the South African environment in particular has a very high incidence of sunlight, the ability to protect the crystals from uv-damage is important. To this end, many commercial preparations of Bt are formulated to include chemicals that provide some degree of uv protection. Hov. ,ver, to date these preparations have not been very effective and increase the costs of formulation.

An article entitled "Protection from Ultraviolet Irradiation by Melanin of Mosquitocidal Activity of Bacillus thuringiensis var. israelensis", in the Journal of Invertebrate Pathology, 62, 131-136, 1993, by Yu-Tien Leu, Meng-Jiun Sui, Dar-Der Ji, I-Haun Wu, Chin-Chi Chou and Cheng-Chen Chen, discloses the efficacy of melanin in the protection of mosquito larvacidal activity of Bacillus thuringiensis var. israelensis against uv light. The melanin is produced by the fermentation of Streptomyces lividans 66 which contains a recombinant plasmid pIJ702 bearing a tyrosinase gene, and is then isolated and purified.

Thereafter an appropriate amount of the melanin powdei is added into a Bt spore suspension or delta-endotoxin preparation. The spore suspension and the delta-endotoxin preparation containing various amounts of melanin were treated with different doses of uv radiation at 253 nm wave lengths. The survival of bacteria before and after the uv radiation was measured by counting the colony forming units. Bioassay results showed that the melanin was effective as a photoprotection agent for Bt spores and the delta-endotoxin thereof. At the end of the article it is stated that the authors consider melanin to be an excellent photoprotector and could be used in protecting delta-endotoxin or other uv-sensitive substances. However, when the preparation of Bt var. israelensis is applied in the field, melanin may be easily washed off by water. To solve this problem, the studies of sub-cloning and expression of tyrosinase gene from streptomycete in the cells of Bt var.

israelensis will be carried out.

It is an object of this invention to provide a microorganism which contains in its genome both a DNA sequence which encodes a Bt endotoxin and a DNA sequence which encodes a compound which either acts as a photoprotectant or which produces a photoprotectant.

SUMMARY OF THE INVENTION According to a first aspect of the invention there is provided a microorganism containing in its genome a first DNA sequence encoding a Bacillus thuringiensis endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, the first and second DNA sequences being capable of expression in the microorganism.

i s I I The second DNA sequence may encode a compound which acts as a photoprotectant. An example of this is a carotenoid.

Alternatively the second DNA sequence may encode a compound which produces a photoprotectant. For example the compound may be an enzyme which acts on or converts a substrate to produce the photoprotectant.

Examples of this are the enzyme tyrosinase which converts tyrosine into the photoprotectant melanin, or the enzyme en:oded by the indole dioxygenase gene isolated from Rhodococcus, which produces the photoprotectant indigo.

The photoprotectant may act as a photoprotectant for the microorganism cells themselves and/or for the spores obtained from cultures of the cells after sporogenesis and/or for the endotoxin produced by the microorganism cells.

The microorganism may be any suitable microorganism including a bacterium, a fungi, an algae or a yeast. The microorganism is preferably a bacterium such as for example a strain of Bacillus thuringiensis, a strain of Bacillus subtilis, a strain of Escherichia coli (E.coli), or a strain of Pseudomonas.

Obviously, when the microorganism is a strain of Bt itself, the Bt will already include in its genome a DNA sequence encoding a Bt endotoxin.

When the microorganism is a strain of Bt, the strain is preferably asporogenous and/or autolysin deficient. Alternatively, the Bt cells can be encapsulated or the cell walls can be cross-linked using, eg. gluteraldehyde.

The photoprotectant preferably absorbs radiation i-i the uv and 300-380 nm region of the spectrum.

According to a second aspect of the invention there is provided a microorganism transformation vector carrying an expression cassette including a DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, and a promoter and optionally a I terminator.

The transformation vector may be f. r example a Bt transformation vector.

According to a third aspect of the invention there is provided a microorganism transformation vector carrying an expression cassette including a first DNA sequence encoding a Bt endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, at least one promoter and optionally a terminator for each DNA sequence.

The microorganism transformation vector as described above may be used for example for transformation of a B.subtilis, E.coli or Pseudomonas cell.

According to a fourth aspect of the invention there is provided a method of producing a microorganism which expresses a Bt endotoxin and a compound which either acts as a photoprotectant or which produces a photoprotectant including the step of transforming a cell of the microorganism by inserting into the genome of the cell a first DNA sequence encoding a Bt endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant under the transcriptional and translational control of transcriptional and translational initiation and termination regulatory regions which function in the microorganism.

According to a fifth aspect of the invention there is provided a method of conferring resistance to photodegredation on a strain of Bt including transforming a cell of the Bt strain by inserting into the genome of the Bt cell a DNA sequence which encodes for a compound which either acts as a photoprotectant or which produces a photoprotectant under the transcriptional and translational control of transcriptional and translatinnal initiation and termination regulatory regions which function in the Bt cell.

According to a sixth aspect of the invention there is provided an insecticidal i composition containing as its active ingredient a Bt endotoxin and a photoprotectant, the Bt endotoxin and the photoprotectant or a compound which produces the photoprotectant having been expressed by the same microorganism.

According to a seventh aspect of the invention there is provided an insecticidal composition containing as its active ingredient a strain of Bt which includes in its genome a DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, and/or the spores obtained from a culture of the Bt strain after sporogenesis and/or an endotoxin produced by the Bt strain after sporogenesis.

According to an eighth aspect of the invention there is provid .d a method for controlling insects, for example of the order Lepidoptera, Diptera or Coleoptera at a locus which includes the step of treating the locus to be protected with an amount of an insecticidal composition as described above.

According to a ninth aspect of the invention there is provided a recombinant DNA sequence comprising a first DNA sequence encoding a Bt endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant.

DESCRIPTION OF EMBODIMENTS The sunlight-mediated inactivation of the delta-endotoxin crystals produced by strains of Bt affects both the efficacy and economics of Bt as a biopesticide.

It has previously been established that some pigments provide protection against uv-damage and for this reason, they are often added to formulations of spores and delta-endotoxin crystals of Bt for application in the field.

Examples of compounds which have been added for this purpose are carbon black and melanin. However, these compounds have not been found to be entirely successful as they do not appear to coat the delta-endotoxin crysta!s adequately, and may wash off.

The present invention provides a microorganism which contains in its genome a first DNA sequence encoding a Bt endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, the first and second DNA sequences being capable of expression in the microorganism.

More particularly, the present invention provides a strain of Bt, preferably a commercial strain of Bt, having inserted into its genome a DNA sequence which encodes a compound which either acts as a photoprotectant or which produces a photoprotectant. This will enable the transformed strain of Bt to provide its own protection against photodegredation.

The photoprotectant may act as a photoprotectant for the microorganism cells, eg the Bt cells, per se, and/or for the spores obtained from the cultures of the cells after sporogenesis and/or for the endotoxin produced by the cells after sporogenesis.

Suitable compounds which may act as photoprotectants or radical scavengers include: Melanin which is produced by the enzyme tyrosinase by conversion of tyrosine, as well as melanin from other sources such as fungi, other bacteria and yeasts (Geis and Szaniszlo, Mycologia, 76(2) (1984) 268- 273; P Roy, K K Nayak and N K Pandey, J. Gen. Microbiol. (1989) 135, 3385-3391).

Indoles such as indigo which is produced by an enzyme encoded by the indole dioxygenase gene isolated from Rhodococcus (S Hart and D R Woods, J Gen. Micro (1992) 138, 205-209).

Carotenoids from algae, for example violaxanthin (H Senger, E I 8 Schrader and N I Bishop, Bot. Acta. 106(1993), 72-77), from fungi and bacteria ar e Flavonoids and related phenolic compou; Braun and M Trevini, Photochemnistry and Photobiology, 57 t8-323 (199.5)).

Thiol containing compounds, e g N-acetylcysteine, mercaptopropionylglycine and captorpril (LT Van Den Broeke, G M J Beyersbergen van Henegouwen, .I Photochern. Photobiol. B:Biol., 17 (1993 279 286).

Water soluble fraction of onion pigment (K Shinohara, S Iwatsuki, M Kobori, Nippon Shokuhin Kogyo Gakkaishi, Vol. 40(2), 144-149(1993)).

Cysteine and ATP (TRK Reddy, K Sarada, Phykos (1982), 21 108-114).

Serotonin from Candida. (Strakhovskaya et Izv. Akad. Nauk. SSSR, Ser. Biol. 1982(4) 624-030).

Tryptophan. (Larcomn et Photochem. Photobiol. (1981) 34 583- 587).

Green pigments in spores of Aspergillus (Brown, H-auser, Tommasi, Cor: tt and Salvo, Tetrahedron Letters (1993), 34(3), 419-422).

(11) Scytonemin in Chlorogloeo -psis sp, (F Garcia-Pichel, N D Sherry and R W Castenholz, Photochemistry and Photobiology, 17-23, (1992)).

(1)Black pigments in conidia of Aspergillus niger gof n 0 Garcia, Environ. Entomology 21(4) 913-917(1992).

(13) UVA absorbing Compounds such as Biopterin glucoside (T Matsunaga, J G Burgess, N Yamada, K Komatsu, S Yoshida, and Y Wachi, Appl.

Microbiol.Biotechnol. (1993)39:250-253).

(14) SOD and Catalase (antioxidants) sc, cs cbose cA'c\os ed in Mycosporine amino acid (MAA) like compounds (F Garcia-Pichel and R N Castenholz, AEM 59(1), 163-169 (1993) and 59(1) 170- 176(1993)).

(16) Any other compounds which absorb preferably in the uv and 300-380 nm region of the spectrum, as these are the regions which appear to cause the most damage (Pusztai et al, Biochem. J. 1991, 273:43-47).

Introduction of genes encoding for any one of these photoprotectants or the producers thereof into a s.rain of a microorganism such as Bt will enable the microorganism to produce its own photoprotection.

Dealing firstly with Bt, the introduction of a DNA sequence or gene into a commercial strain of Bt is a well-established procedure and has been achieved at high frequencies (Shurter et al, Mol. Gen. Genet. 1989, 218:177-181; Masson, et al, FEMS Microbiol. Letts. 1989, 60:205-210). Some of the vectors that have been designed and structured for B.subtilis can replicate in Bt and several of the antibiotic resistant markers are expressed. There are also reports of vectors constructed from endogenous Bt plasmids.

Several methods to transform Bt have been reported, including transformation by conjugation, transduction and protoplast formation. However, the most successful method i that of electroporation, where the cells are exposed to rapid, high voltage ;ses. This results in transient pores appearing in the cell membranes, through which DNA is able to enter the cell. There are several published methods for transforming Bt by electroporation. The method found to give the highest and most consistent transformation frequencies (Mettus A M and Macaluso A, (1990) Appl. Environ.Microbiol. 56 1128-1134) gave

I

transformation frequencies of 10 3 to 10 4 /ug DNA for the fermentor strain 152 (ATCC55267).

For the photoprotectant to be most effective, it is preferably contained within the Bt cell and thus the Bt cell must be prevented from lysing. This can be achieved by mutating the cell so that the sporulation pathway is blocked after sporulation has been initiated (after stage II of sporulation). Alternatively, the recipient Bt cells may be mutated to be autolysin minus or deficient.

Alternatively the Bt cells may be encapsulated or their cell walls cross-linked.

Mutation is via known methods and would include the use of mutagens such as nitrosoguanadine, ethylmethylsulphonate and uv. In addition, transposon mutagenesis may also produce the required genotypes.

Suitable promoters for use in Bt and other Bacillus strains include Spo 2 (from phage spo2) and amylase (sporulation-specific).

The end result is a Bt strain which produces a photoprotectant which protects i the cells of the Bt strain against photodegredation, particularly uv-inactivation and which are also asporogenous and/or autolysin deficient, encapsulated or cross-linked, thus maintaining the crystal and the photoprotectant in the cell rather than releasing it into the culture medium. These would then be released on ingestion by the insects.

A strain of Bt having inserted into its genome a DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, may be used in an insecticidal composition for the control of insects at a locus in a conventional manner.

The main advantage of the strain of Bt of the invention is that it is protected against photodegredation and thus should have a longer efficacy time.

An example of the cloning of a melanin producing tyrosinase gene into Bt will I- now be given.

I. Materials and Methods.: Bacterial strains, plasmids and media.: The locally isolated Bacillus thuringiensis (Bt) strain 152 (ATCC 55267) which was the subject of South African Patent Application number 93/2792, was used as recipient. The Dam Dcm-E.coli host, SCS 110 was obtained as competent cells from Stratagene.

The Bacillus subtilis expression vector pPL708 has been previously described (Duvall Williams Lovett Rudolph C, Vasantha N and Guyer M, Gene, 24, 171-177)). E.coli and Bt strains were grown in Luria broth Bacto-tryptone, 5g/l yeast extract, 10g/1 NaCI, pH 7,5) at 37°C. Where relevant, the LB was supplemented with antibiotics: neomycin was used at ampicillin was used at 100gg/ml.

DNA isolation and manipulation: The alkaline lysis method described by Birnboim and Doly (Nucleic Acids Research 7, 1513 (1979)), and the LiCI method (He et al, Nucleic Acids Research, 18(6), 1660(1990)) were used to i isolate DNA from E.coli. The plasmid pPL708 was isolated out of B.subtilis by the method of Lovett P.S and Keggins KM: in Wu R Methods in Enzymology, Vol 68. Academic Press, New York, 1979, pp 3 4 2 3 5 7 Small scale miniprep DNA was isolated out of Bt by the Lereclus modification of the Biraboim and Doly method (Lereclus et al: Mol Gen Genet 186, 391-398 (1982). Restriction enzymes (Boehringer Mannheim), calf alkaline phosphatase (Pharmacia) and T4 Ligase (Promega) were used according to the manufacturer's instructions. DNA fragments were isolated out of TAE gels and were purified using the GENECLEAN II Kit® (BIO101 Inc.). All other recombinant DNA techniques were as described in Sambrook, Maniatis and Fritsch, Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989).

DNA Transformation: DNA was transformed into competent cells of the E.coli strain SCS110 according to the manufacturer's instructions (Stratagene).

The Bt strain 152 was routinely transformed by electroporation (Mettus and 12 Macaluso, Appl. Environ. Micro, 56(4), 1128-1134 (1990)).

II. Results.: The melanin operon consists of two genes- mel I and rmel II, both of which are required for the production of tyrosinase (although only mel II encodes the structural gene for tyrosinase). An E.coli construct containing the Streptomyces antibioticus melanin operon from plasmid plJ702 was used as the source of the melanin genes. The Eco construct did not contain a promoter in front of the melanin operon; thus, the genes were not expressed.

However, when this construct was ligated to the B.subtilis vector pPL708 via the Pstl restriction site on each plasmid, and transformed into SCS110, the resulting colonies expressed melanin, and were brown. The constructs were confirmed by restriction mapping. As there was evidence that DNA isolated out of a Dam-Dcm-E.coli host could be successfully transformed directly into Bt (Macaluso A and Mettus A- M, J. Bacteriol. 173(3) 1453-1356 (1991) the recombinant of (pPL708 mel) isolated out of SCS110 was transformed directly into Bt strain 152 by electroporation. Transformants were selected on SLB containing neomycin, and the presence of the melanin ge e in the transformants was confirmed by Southern blotting and hybridization to the 1,4 kb BamHI/Bcll fragment which contained the melanin genes.

Although the invention is of particular application to Bt, the invention also encompasses the provision of a microorganism which contains in its genome a first DNA sequence encoding a Bt endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, the first and second DNA sequences being capable of expression in the microorganism.

The microorganism may be for example a bacterium, an algae, a fungi, a virus or a yeast.

Generally, the microorganism will be a bacterium such as for example Bacillus clsubtilis or E.coli or Pseudomonas.

In the case of a microorganism other than Bt, the microorganism must have inserted into it for expression both the gene for a Bt endotoxin and the gene for a compound which either acts as a photoprotectant or which produces a photoprotectant. Conventional techniques for the transformation of microorganisms can be used to achieve this.

An example of the cloning of a melanin producing tyrosinase gene and an endotoxin gene into E.coli will now be given.

I. Materials and methods.: Bacterial strains, plasmids and media: The E.coli hosts used as recipients of both the melanin and toxin genes were JM109 and JM105, obtained from the ATCC collection. The Dam-DcmE.coli host, SCS 110 was obtained as competent cells from Stratagene. The vector pTrc99A has been previously described (Amann et al, Gene 69, 301-315(1988)) and was obtained from Pharmacia. E.coli strains were grown in Luria broth (LB) (10g/l Bactotryptone, 5 g/1 yeast extract, 10 g/l NaCI, pH 7,5) supplemented with carbennicillin (200gg/ml) at 37°C. For production of melanin, the cells were S* grown on Luria Agar supplemented with 100 jig/ml ampicillin, 0,3 g/l tyrosine, 0,2 mM FeCI 3 0,2 mM CuSO 4 The melanin gene was induced by the addition of 1 mM Isopropylthio-B-galactoside (IPTG).

DNA isolation and manipulation: The alkaline lysis method described by Birnboim and Doly (Nucleic Acids Research 7, 1513(1979)), and the LiCI method (He et al, Nucleic Acid Research, 18(6), 1660(1990)) were used to isolate DNA from E.coli. Restriction enzymes (Boehringer Mannheim), calf alkaline phosphatase (Pharmacia), Klenow (Boehringer Mannheim) and T4 S Ligase (Promega) were used according to the manufacturers instructions.

DNA fragments were isolated from TAE agarose gels and were purified using the GENECLEAN II® kit (BIO 101 Inc.) All other recombinant DNA techniques were as described in Sambrook, Maniatis and Fritsch, Molecular Cloning: A Laboratory Manual 2nd ed. (1989).

DNA transformation: DNA was transformed into E.coli by the process of electroporation. Electroporation cells of E.coli strains JM105 and JM109 were made by growing the cells to an OD6oo of 0,5 to 1,0, chilling them for minutes on ice, and then harvesting them at 4000g for 15 minutes. The cells were washed once in water, and once in 10% glycerol, before being resuspended in 1:500 of the original volume. The cells were frozen in aliquots for up to 6 months. After being thawed on ice, the cells were electroporated using the BioRad Gene Pulser and Pulse Controller, according to the manufacturer's instructions. Thus, 40j1 of cells were mixed with 1-2g1 of DNA; the cells/DNA mixture was transferred to a chilled 0,2cm electroporation cuvette and placed in the chamber. Electroporation conditions were 25 AF, 2,5KV and the Pulse Controller was set at 200n. This resulted in a time constant of between 4 and 5 milliseconds. Immediately after the pulse, Iml of LB was added, and the cells were allowed to express for an hour at 37°C before being plated an Luria Agar with antibiotics.

Protein analysis: After incubation overnight, 1,5 ml of E.coli cells were harvested, and resuspended in 100/l of 3 x Laemmli buffer (Laemmli, Nature 227, 680-685(1970)). The proteins were denatured at 100 0 C for 10 minutes.

Samples were cooled and centrifuged at 12000xg for 10 minutes. Aliquots of the supernatant were analysed by SDS-PAGE electrophoresis on a 7,5% gel using a BioRad Mini PROTEAN II electrophoresis system. Once electrophoresis had been completed, the gel was reacted with an antibody solution in a Western Blot (Towbin et al, Proc. Natl, Acai, Sci, USA 76, 4350- 4354(1979)). Thus the gel was soaked in blotting solution (3 g/l Tris (hydroxymethyl)aminomethane; 14,4 g/l glycine; 10% methanol(technical grade)) for 1 hour with gentle shaking before being placed on nitrocellulose "i sheet which had been prewet in blotting buffer. The gel and membrane were sandwiched between two sheets of 3MM chromatography paper, and then between 2 pieces ol oponge. This entire sandwich was then loaded into a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad) and was blotted for 1 hour at 150V. Once blotting was completed, the membrane was washed in 2% milk powder in PBS for two hours at room temperature before being incubated with the diluted antibody solution (1:200 anti-protoxin antibody in PBS containing 2% milk powder) for two hours. The membrane was then washed three times for 10 minutes each in PBS containing 0,1% Triton X-100, before being soaked for two hours in the second antibody solution (alkaline phosphatase conjugated anti-rabbit solution, diluted 1:5000 in PBS plus 2% milk powder). The membrane was then washed once with PBS containing 0,1% Triton X-100 for 30 minutes, and then with PBS for 3x10 minutes.

Freshly made staining solution (30mg P-nitro blue tetrazolium chloride (NBT) in 1ml 70% N,N-dimethylformamide; 15mg 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP) in Iml 100% N,N-dimethylformamide; 98 ml carbonate buffer, pH 9.8) was added until a reaction was visible, after which the gel was rinsed in distilled water and dried between two sheets of Whatman paper in the dark.

Antibody production: The 135KDa endotoxin proteins were isolated from an SDS-PAGE gel by electroelution, and were used to immunise rabbits. Whole serum was obtained by bleeding the rabbits, and immunoglobulins were purified from the whole serum by the method of Hardie and Van Regenmortel (1977). (Isolation of specific antibody under conditions of low ionic strength. J. Immunol.Methods 15:305). The polyclonal antibodies were absorbed to E.coli cell extracts in order to reduce the background antibody activity.

UV exposure: In the case of Bt toxin, 20mg of freeze dried powder was mixed with varying amounts of melanin (0,0001%; 0,001%; 0,01%; and the mixture was resuspended in 4 ml of water. This was pipetted onto clean glass slides, and then left to dry overnight at 37°C. The slides were than exposed to 0,67 x 10' joules/m 2 of uvB light, before being resuspended in 10 ml Bioassay buffer. These samples were then tested in bioassays.

16 In the case of E.coli, the cells were incubated for 72 hours at 37°C in the presence of IPTG, CuSO 4 tyrosine and FeCI 3 When harvested, the cell pellet was brown in colour, due to the expression of the melanin gene.

Overnight cultures of E.coli did not produce brown pellets, and these cultures were used as controls.

Insect Colonies: Chilo partellus larvae were raised on the following diet: Agar 100g Water 5000ml Grated Chickpea 600g Glucose 100g Powdered milk Yeast extract Ascorbic Acid Sorbic Acid *Nipagen(Methyl-4-hydroxybenzoate/methylparaben) 1g *Cholesterol *Ether 100ml {ingredients marked were not added to media used for bioassays, but only to growth media} Chilo partellus larvae were grown on this media until they reached the second instar stage, which takes about 7 days. They were then used for bioassay tests.

Bioassays: The media indicated above was decanted into 30ml aliquots while the media was still warm (55 0 The appropriate amount of toxin was then added from stock solutions, and mixed with the media using an Ultraturrax.

S" The mixture was poured into petri dishes, and allowed to set. Once set, the media was cut into the required number of pieces, and each piece was then dispensed into a container. Ten C.partellus larvae were added to each container, and left at 25 0 C, 80% humidity for 7 days, after which the larval mortality was determined.

II. RESULTS.: The CrylA(c) gene was isolated as a 6,7kg Bam HI fragment. The Barn HI ends of the fragment were filled in using Klenow enzyme and dNTP's, and ligated using conditions that favour blunt ends to the blunt-ended Sall site of the vector pTrc99A. The vector ends were also treated with calf alkaline phosphatase to prevent recircularisation. The recombinants that contained the toxin gene were detected by gel electrophoresis of mini-prepared DNA. This result was confirmed by showing toxin expression in a Western Blot, reacted with antibodies raised to the toxin gene. One of the positive transformants which contained the desired construct, (C5) was chosen as the candidate for the introduction of the melanin gene.

The melanin operon consists of two genes- miell and inel II, both of which are required for the production of tyrosinase (although only me/Ill encodes the structural gene for tyrosinase). They were isolated as a 1,4kb BamHI/BCII fragment from an E.coli construct containing the Streptomyces antibioticus melanin operon from plasmid pIJ702. To overcome the problems of Bcll sensitivity to methylation, 'he construct was transformed into and isolated out of the Dam'Dcm- strain, SCSI10. The isolated fragment was ligated into the alkaline phosphatased BamHI site of the toxin-containing plasmid C5. This results in the melanin gene being inserted behind the strong pTrc promoter which is inducible by IPTG. Recombinants containing the melanin gene were selected by the brown pigment production in the presence of IPTG, CuSO 4 tyrosine and FeCI 3 The constructs were confirmed by mapping the DNA.

The expression of the toxin gene in the presence of melanin was confirmed by Western Blotting which indicated that the E.coli with both melanin and toxin genes showed levels of toxin expression similar to those found in E.coli with the toxin alone.

i 18 BIOASSAYS RESULTS In bioassays against second instar Chilo partellus larvae, the protective effect of melanin during ultraviolet light exposure was demonstrated by mixing pure melanin from Sepia officinalis (obtained from Sigma (M2649)) at various concentrations with Bt toxin, which was used at 120Ig/ml. The mixtures were then exposed to doses of 0,67x10 5 joules/m 2 from a Philips TL40W/12 lamp, which transmits ultraviolet light in the UVB range. This region includes the shortest wavelengths found in sunlight at the earth's surface. Thus, this lamp, with an emission peak of 313nm, simulates the damage caused by sunlight.

When the melanin-containing samples were bioassayed after being exposed to uv, the samples containing melanin at 0,1% gave 57,9% protection over controls, Those samples with melanin at 0,01% gave 26,3% protection.

In the bioassays of E.coli cells which contain the toxin and melanin genes, the melanin was shown to have a protective effect (Table 1).

o TABLE 1 Strain No of Larvae No Dead Killing Mean Killing 1 10 2 10 4 40 4 6 92,5

A

M

3 10 6 8 80 6 8 c3 4 10 6 7 70 77,5 9 9 10 6 1 10 26,7 1 Strain 1 Strain 2 Strain 3 r o o E.coli containing the toxin gene and then exposed to uv.

E.coli containing the toxin gene and not exposed to uv.

S E.coli containing the melanin and toxin genes and exposed to uv.

E.coli containing the melanin and toxin genes and not exposed to uv.

S E.coli containing only the melanin gene.

Strain 4 Strain 5 cc o Concentration used: 10 mg/ml The endotoxin gene used in the above example is the CrylA(c) gene whose nucleotide sequence is disclosed in "Biological control of Eldana Saccharina Walker (Lepidoptera:Pyralidae) using a cloned Bacillus Thuringiensis deltaendotoxin gene", by Gerardo Herrera, PhD Thesis (submitted), University of Cape Town, 1994.

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Any other suitable endotoxin gene may be used, for example the gene disclosed in Characterized full length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis susp. Kurstaki HD-73 and their toxicity to Manduce sexta, by M J Adana, M J Stauer, A Rocheleau, J Leighton, R F Barker and D V Thompson, Gene 36:289-309, (1985).

The melanin producing gene may be any suitable melanin or melanin-like producing gene such as for example the tyrosinase gene having the nucleotide sequence set out in "The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product", V Bernan, D Filipula, W Herber, M Bibb and E Katz, Gene 37 (1985), 101-110.

Suitable promoters for E.coli include lac, trp of E.coli and P, of Hybrid promoters include the tac promoter of E.coli (hybrid of trp and lac), trc promoter, tic promoter and Ipp promoter.

Suitable terminators, when such are required, for E.coli include the terminator of ribosomal RNA operon, rrn B, phage Id DNA, trp attenuator and Ipp terminator.

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Claims

1. A microorganism containing in its genome a first DNA sequence encoding a Bacillus thuringiensis endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, the photoprotectant being selected from the group consisting of melanin, an indole, a carotenoid, SOD and catalase, the first and second DNA sequences being capable of expression in he microorganism.

2. A microorganism according to claim 1 wherein the microorganism is a bacterium,

3. A microorganism according to claim 2 wherein the bacterium is a strain of Bt.

4. A microorganism according to claim 3 wherein the Bt is asporogenous and/or autolysin deficient. A microorganism according to claim 2 wherein the bacterium is E.coli.

6. A microorganism according to any one of claims 1 to 5 wherein the photoprotectant absorbs radiation in the uv and 300 to 380 nm region of the spectrum.

7. A microorganism according to any one of claims 1 to 5 wherein the compound which acts as a photoprotectant is a carotenoid.

8. A microorganism according to any one of claims 1 to 5 wherein the compound which produces a photoprotectant is selected from the group consisting of tyrosinase which produces melanin and indole dioxygenase which produces indigo.

9. A microorganism transformation vector carrying an expression cassette including a first DNA sequence encoding a Bt endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, the photoprotectant being selected from the group consisting of melanin, an indole, a carotenoid, SOD and catalase, and at least one promoter and optionally a terminator for each DNA sequence.

10. A vector according to claim 9 for E.coli. ,p RA4 (it 0 X ^W"9 22

11. A vector according to any one of claims 9 or wherein the photoprotectant absorbs radiation in the uv and

300-380 nm region of the spectrum. 12. A vector according to any one of claims 1 to wherein the compound which acts as a photoprotectant is a carotenoid. 13. A vector according to any one of claims 9 or wherein the compound which produces a photoprotectant in selected from the group consisting of tyrosinese which produces melanin and indole dioxygenase which produces indigo. 14. A method of produc:ng a microorganism which expresses a Bt endotoxin and a compound which either acts as a photoprotectant or which produces a photoprotectant S* 15 the photoprotectant being selected from the group consisting of melanin, an indole, a carotenoid, SOD and catalase, including the step of transforming a cell of the microorganism by inserting into the genome of the cell a first DNA sequence encoding a Bt endotoxin and a second DNA 20 sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant under the transcriptional and translational control of transcriptional and translational initiation and termination regulatory regions which function in the cell. 15. A method according to claim 14 wherein the microorganism is a bacterium. 16. A method according to claim 15 wherein the microorganism is E.coli. 17. A method according to any one of claims 14 to 16 wherein the photoprotectant absorbs radiation in the uv and 300 to 380 nm region of the spectrum. 18. A method according to any one of claims 14 to 16 wherein the photoprotectant is a carotenoid. 19. A method according to any one of claims 14 to 16 wherein the compound which produces a photoprotectant is selected from the group consisting of tyrosinase which produces melanin and indole dioxygenase which produces 23 indigo. A zethod of conferring resistance to photodegredation on a strain of Bt strain by inserting into the genome of the Bt cell a DNA sequence which encodes a compound which either acts as a photoprotectant or which produces a photoprotectant the photoprotectant being selected from the group consisting of melanin, an indole, a carotenoid, SOD and catalase under the transcriptional and translational coitrol of transcriptional and translational initiation and termination regulatory regions which function in the Bt cell. 21. A method according to claim 20 wherein the photoprotectant absorbs radiation in the uv and 300 to 380 m region of the spectrum. 22. A method according to claim 20 wherein the photoprotectant is a carotenoid. 23. A method according to claim 20 wherein the compound which produces a photoprotectant is selected from the group consisting of tyrosinase which produces melanin 20 and indole dioxygenase which produces indigo. 24. An insecticidal composition containing as its Sactive ingredient a Bt endotoxin and a photoprotectant the photoprotectant being selected from the group consisting of melanin, an indole, a carotenoid, SOD and catalase, the Bt endotoxin and the photoprotectant or a compound which produces the photoprotectant having been expressed by the same microorganism. An insecticidal composition according to claim 24 wherein the microorganism is a bacterium. 26. An insecticidal composition according to claim wherein the microorganism is a strain of Bt. 27. An insecticidal composition according to claim wherein the microorganism is E.coli. 28. An insecticidal composition according to any one of claims 24 to 27 wherein the photoprotectant absorbs radiation in the uv and 300 to 380 nm region of the spectrum. 24 29. An insecticidal composition according to any one of claims 24 to 27 wherein the photoprotectant is a carotenoid. An insecticidal composition according to any one of claims 24 to 27 wherein the compound which produces a photoprotectant is selected from the group consisting of tyrosinase which produces melanin an indole dioxygena'e which produces indigo. 31. An insecticidal composition containing as its active ingredient a strain of Bt which includes in its genome a DNA sequence encoding a compound which either acts .as a photoprotectant or which produces a photoprotectant, the photoprotectant being selected from the group consisting of melanin, an indole, a carotenoid, SOD and S 15 catalase. 32. An insecticidal composition according to claim 31 wherein the Bt strain is asporogenous and/or autolysin o: deficient, or encapsulated or cross-linked. 33. An insecticidal composition containing as its 20 active ingredient the spores obtained from a culture of a strain of Bt which includes in its genome a DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant after sporogenesis, the photoprotectant being selected from the group consisting of melanin, an indole, a carotenoid, SOD and catalase. 34. An insecticidal composition containing as its active ingredient an endotoxin produced by a culture of a strian of Bt which includes in its genome a DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, and the photoprotectant, the photoprotectant being selected from the group consisting of melanin, an indole, a carctenoid, SOD and catalase. An insecticidal composition according to any one of claims 31 to 34 wherein the photoprotectant absorbs radiation in the uv and 300 to 380 nm region of the spectrum. 25 36. An insecticidal composition according to any one of claims 31 to 34 wherein the photoprotectant is a carotenoid. 37. An insecticidal composition according to any one of claim 31 to 34 wherein the compound which produces a photoprotectant is selected from the group consisting of tyrosinase which produces melanin and indole dioxygenase which produces indigo. 38. A method for controlling insects at a locus which includes the step of treating the locus to be protected with an amount of an insecticidal composition according to any one of claims 24 to 37. 39. A method according to claim 38 wherein the insects are of the order Lepidoptera, Diptera or Coleoptera. A recombinant DNA sequence comprising a first DNA sequence encoding a Bt endotoxin and a second DNA sequence encoding a compound which eit' er acts :9 a photoprotectant or which produces a photoprotectant, the photoprotectant 20 being selected from the group consisting of melanin, an indole, a carotenoid, SOD and catalase. DATED THIS 5TH DAY OF SEPTEMBER 1996 AECI LIMITED By Its Patent Attorneys: GRIFFITH HACK CO., Fellows Institute of Patent Attorneys of Australia ABSTRACT This invention discloses a microorganism, preferably a bacterium, containing in its genome a first DNA sequence encoding a Bacillus thuringiensis endotoxin and a second DNA sequence encoding a compound which either acts as a photoprotectant or which produces a photoprotectant, the first and second DNA sequences being capable of expression in the microorganism. The photoprotectant is designed to protect either the microorganism itself or the endotoxin produced by the microorganism from photodegredation. The invention also covers insecticidal compositions containing the microorganism or the products thereof and the use of the insecticidal composition for controlling insects. 0 *0000