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1. WO1997041235 - NOUVELLE SOUCHE DU $i(BACILLUS THURINGIENSIS) PRODUCTRICE DE PIGMENT

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A NEW, PIGMENT-PRODUCING, STRAIN OF Bacillus thuringiensis

FIELD OF THE INVENTION
The present invention relates generally to insecticides and improved biological control of insect pests. More particularly, the present invention relates to a strain of Bacillus thuringiensis for use as an insecticide with an enhanced viability against uv inactivation.

BACKGROUND OF THE INVENTION
Bacillus thuringiensis is a gram-positive soil bacterium which is the most studied, most effective, and most often utilized microbial insecticide (see Frankenhuyzen, 1993). This bacterium, which produces large amounts of an insecticidal toxin protein that usually accumulates in the cytoplasm during sporulation to form crystals, comprises a number of different subspecies each of which produces a different toxin that can kill certain specific insects (Behtel and Bulla, 1976; and Lambert et al., 1996). For example β. thuringiensis subsp. tenebrionis (Keller and Langenbruch, 1993) is effective against coleoptera such as the boll weevil, β. thuringiensis subsp. kurstaki kills lepidoptera such as spruce budworm (Lopez-Meza et al., 1995), and β. thuringiensis subsp. israelensis kills diptera such as mosquito (Kawalek et al., 1995). Strains of this species have been recognized as potential candidates for use in the control of a variety of insect pests including mosquitoes and black flies which are vectors of several tropical diseases (Goldberg and Margalit, 1977; and Undeen and Nagel, 1978). When spores and crystals of B. thuringiensis are eaten by a susceptible insect larva, the mouth part and gut become paralyzed and the gut epithelium of the insect larva is destroyed (Hofmann et al., 1988).
Although a number of virulent and effective strains of B. thuringiensis are available commercially, until now, the known strains fail to persist in the environment and this is largely due to, among other things, photoinactivation of the insecticidal toxin by ultraviolet light (Ignoffo and Garcia, 1978). Consequently, in addition to searching for, or constructing β. thuringiensis strains with an enhanced spectrum of insecticidal activity, it is also important to develop B. thuringiensis strains that can persist in the environment for relatively long periods of time.
Several approaches to achieve photoprotection of the B. thuringiensis insecticidal toxin have included encapsulation (Dunkle and Shasa, 1988), granular formulation (Amed et al., 1973), and the addition of a variety of uv absorbing compounds (Morris, 1983; Margulies et al., 1985; and Cohen et al., 1991). However none of these have been completely successful or have achieved optimal results. Although synthetic uv chemical photostabilizers offer B. thuringiensis some protection, their use in the environment may introduce ecological problems related to soil and water pollution.

SUMMARY OF THE INVENTION
One approach to overcome the problem of photoinactivation has been described recently, where black melanin isolated from another soil bacterium has been found to provide photoprotection to β. thuringiensis toxin (Liu et al., 1993). The present invention significantly improves on this approach by providing a strain of β. thuringiensis, namely subspecies oloke, which is capable of natural production of a salmon/red melanin that can protect the organism against photoinactivation, while acting as an insecticide by virtue of insecticidal toxin contained in it. A deposit of this strain has been made with the U.S. Department of Agriculture's Northern Regional Research Laboratories (NRRL) effective March 11 , 1996, bearing accession number B21528.
In its broad aspect the present invention provides an isolated and purified organism containing salmon/red pigment. In another aspect the invention there is provided an organism isolated and purified containing salmon/red pigment and insecticidal toxin.
According to a further aspect of the invention these organisms have characteristics as follows: 1. capable of forming oval shaped spores which are located at polar ends of said organism; 2. a culture of vegetative cells consists of rod-shaped, mottle, gram-positive cells; 3. sporangium of are oval to cylindrical, not swollen, diamond-shaped to cubosidal, with a parasporal body formed in sporangium; 4. being able to liquify gelatin, hydrolyzed starch and ferment glucose, fructose and sucrose; 5. colonies are medium-sized, slightly shiny with mostly flat, mostly smooth borders; 6. in liquid medium they form heavy pellicles which tend to disintegrate into a flaky mass upon shaking; and 7. they are positive for Voges- 5 Proskauer reaction and produce acetylmethyl carbinol.
In another aspect ofthe invention E. coli transformants capable of producing both the β. thuringiensis subsp. oloke insecticidal toxin and salmon/red pigment are provided. A deposit of these transformants have also been made at NRRL effective March 11 , 1996, bearing accession number B21529.
l o According to yet a further aspect of the present invention there is provided a method of transforming an organism to obtain a transformant capable of producing Bacillus thuringiensis subsp. oloke insecticidal toxin by insertion of DNA message corresponding to said toxin into said organism and isolating a purifying said organism, as well as a method of transforming an organism to obtain a

15 transformant capable of producing salmon/red pigment by insertion of DNA message corresponding to said pigment into said organism and isolating and purifying said organism.
According to a further aspect of the present invention there is provided a method of transforming an organism to obtain a transformant capable of producing

20 Bacillus thuringiensis subsp. oloke insecticidal toxin and salmon/red pigment comprising the steps of: a. preparation of plasmid DNA containing messages for Bacillus thuringiensis subsp. oloke insecticidal toxin and salmon/red pigment, from β. thuringiensis subsp. oloke or other suitable source of DNA and preparing said plasmid in a suitable ligation mixture; b.co mbining said ligation mixture with E. coli;

25 and c. identifying, isolating and purifying said transformants. According to another aspect of this method the organism is E. coli.
According to one aspect of the present invention there is provided an insecticidal toxin isolated from B. thuringiensis subsp. oloke, or any chemical equivalent thereof.
30 In another aspect, the present invention provides a strain of B. thuringiensis which is capable of persisting longer in the environment because of enhanced photoprotection.
According to one aspect of the present invention there is provided an insecticide comprising an effective amount of B. thuringiensis subsp. oloke.
According to a further aspect of the present invention there is provided an insecticide with reduced susceptibility to photoinactivation comprising a Bacillus thuringiensis which has been treated with an effective amount of salmon/red pigment.
According to yet a further aspect of the present invention there is provided a method for conferring reduced susceptibility to photoinactivation to a natural insecticide comprising the step of mixing an effective amount of salmon/red pigment with the insecticide.
According to one aspect of the present invention there is provided a salmon/red pigment or any chemical equivalent thereof.
According to a further aspect of the present invention there is provided a salmon/red pigment produced by a salmon/red pigmented organism or any functional equivalent thereof.
In its broad aspect, the invention is a strain of B. thuringiensis which produces a compound capable of providing photoprotection to any organism which is provided with the compound. In another aspect, the invention provides a strain of β. thuringiensis which produces a salmon/red pigment which is capable of providing photoprotection from ultraviolet radiation.
According to one aspect of the present invention there is provided a composition capable of conferring reduced susceptibility to photoradiation to a photoradiation sensitive recipient comprising an effective amount of salmon/red pigment and a suitable carrier. In another aspect the recipient is a human being.

According to a further aspect of the present invention there is provided a method of conferring reduced susceptibility to photoradiation to a photoradiation sensitive recipient comprising combining an effective amount of salmon/red pigment with a suitable carrier and applying said combination topically to said recipient. In another aspect, the recipient is a human being.
In yet another aspect, the invention provides a salmon/red pigment with characteristics consistent with melanin which may be used for photoprotection as well as in the cosmetic industry as a compound with the combined value of providing colour for makeups and other such cosmetic products while at the same time providing photoprotection.
These and other aspects ofthe present invention will become evident upon reference to the following detailed description and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS
Further details of the invention are described below with the help of the examples illustrated in the accompanying drawings comprising six figures in which:

Figure 1 is a graphic representation of growth of B. thuringiensis subsp. oloke on PDW agar plates at three different temperatures.
Figure 2 is a graphic illustration of ultraviolet and visible light absorption of the salmon/red melanin isolated from a culture of β. thuringiensis subsp. oloke. Figure 3 illustrates a histogram representative of survival of β. thuringiensis subsp. oloke, B. thuringiensis subsp. kurstaki and β. thuringiensis subsp. kurstaki cells in the presence of 75 μg/mL of melanin following irradiation with uv light of varying intensity at 253 nm.
Figure 4 is a graphic illustration of the effect of pH on the solubility of crystals from β. thuringiensis subsp. oloke.
Figure 5 provides an illustration ofthe protein composition of B. thuringiensis subsp. oloke crystals determined by SDS-15%PAGE.
Figure 6 is an autoradiograph of an agarose gel electrophoresis of plasmid DNA extracted from B. thuringiensis.

DEFINITIONS
In the present specification and claims, reference will be made to phrases and terms of art which are expressly defined for use herein as follows:
As used herein, the short form "EDTA" means ethylenediaminetetraacetic acid.
As used herein, the short form "UDS-PMSF" means 2mM phenyl methyl sulfonyl fluoride in 6M urea - 1% sodium dodecyl sulfate ("SDS") - 0.5M dithiotheitol.
As used herein, the short form "BME" means β-mercaptoethanol.
As used herein, the short form "PDA" means potato dextrose agar.
As used herein, the short form "CWA" means coconut water agar.
As used herein, "coconut water" means water which contains coconut and is prepared as follows or by any equivalent method: An average size coconut is broken carefully and the water inside is collected in a beaker. Coconut milk is obtained by blending small pieces of coconut with water and then filtering with cheese cloth. The coconut milk is further clarified by centrifugation in a Sorval centrifuge using an SS-34 rotor at 8000 rpm (4°C) for 20 min. The clear filtrate is then mixed with the coconut water. The mixture is autoclaved, filtered, re-autoclaved and stored at 4°C until needed. About 200 mL of coconut water can be obtained from a single medium sized coconut.
As used herein, the shortform "uv" means ultraviolet radiation.
As used herein, the shortform "subsp." means subspecies.
As used herein, the shortform "B." means Bacillus.
As used herein, the shortform "PDW" means potato dextrose medium containing 10% coconut water.
As used herein, the expression "salmon/red pigment" and "salmon/red melanin" are used interchangeably and mean the substance, or any chemically and functionally equivalent substance, produced by a salmon/red pigment organism of the present invention, or by any other biological or non-biological process including any synthetic processes, with characteristics consistent with melanin including solubility in 1 M NaOH; precipitation in 1 M HCI or 5 mM FeCI3 and bleaching with 20% H2O2. The purified pigment shows a steady increasing absorption with no peak at wavelengths of UV light ranging from 300 to 210 nm (Figure 2).
As used herein, the expression "salmon/red pigmented organism" means Bacillus thuringiensis, subsp. oloke, or any other organism capable of producing salmon/red pigment, including those transformed or otherwise altered or naturally capable of producing salmon/red pigment.

As used herein, the expression "the strain of the present invention means Bacillus thuringiensis, subsp. oloke or any other strain of any other organism or any other organism capable of synthesizing the "salmon/red pigment".

DETAILED DESCRIPTION OF THE INVENTION
A. Characteristics of the Strain of the Present Invention
1. Morphology and biochemical properties
Isolated and purified β. thuringiensis subsp. oloke bacteria have the following characteristics:
(a) Capable of forming spores, usually in 24-48 h following growth on potato dextrose agar (PDA) supplemented with coconut water at

30°C, although as discussed below, growth is achieved at other temperatures. The spores are oval shaped with an average size of approximately 1 μm by 1.9 μm and the spores are found at the polar ends of the cell.
(b) A 24 h culture of vegetative cells consists of rod-shaped motile, gram-positive cells approximately 1.5-2.5 μm by 4.1-6.0 μm, with rounded ends.
(c) The sporangium are oval to cylindrical, not swollen, diamond-shaped to cubosidal, with a parasporal body formed in each sporangium.
(d) This organism is able to liquefy gelatin, hydrolyze starch, and after about 48 h ferment glucose, fructose and sucrose. No acid was produced when lactose, mannitol or maltose were tested.
(e) On agar plates colonies are medium sized (approximately 2-3 mm in diameter), slightly shiny, with mostly flat, mostly smooth borders and have a salmon/red colour.
(f) In liquid medium, the organism forms heavy pellicles which tend to disintegrate into a flaky mass upon shaking. Pigmented flaky mass forms at the bottom of the culture.
(g) The salmon/red pigmented organism is positive for the

Voges-Proskauer reaction; it produces acetylmethyl carbinol.

2. Antibiotic resistance
Experiments were designed and conducted to test the viability of the strain of the invention when challenged by antibiotics. As a control, the growth of β. thuringiensis subsp. kurstaki was tested and the results demonstrate that growth was inhibited by rifampicin (100 μg/mL), tetracycline (200 μg/mL), chloramphenicol (200 μg/mL), ampicillin (200 μg/mL) and penicillin (100 μg/mL) with diameters of zones of inhibition ranging from between 9 and 18 mm (See Table 1). However, β. thuringiensis subsp. kurstaki was resistant to kanamycin, carbenicillin, and streptomycin (all at 200 μg/mL). By contrast, the strain of the invention β. thuringiensis subsp. oloke, was completely resistant to all eight antibiotics just mentioned at the concentrations indicated in Table 1 where all results of these experiments are presented.

3. Growth conditions
β. thuringiensis subsp. oloke grows well on PDW agar plates at 30°C with higher viable counts than at the other temperatures tested (see Figure 1). This strain grows poorly and produces crystals and pigment poorly on both nutrient agar and Luria broth agar compared with PDW agar and CWA (Table 2). The organism produces salmon/red pigment when grown on PDA but with poor crystal formation (Table 2). When the organism is cultured on PDA containing 10% coconut water (PD), it produces the salmon/red pigment but also more than 95% of the cells form insecticidal crystal inclusions (Table 2). When the growth medium is mainly coconut water and agar, more than 99% of the cells form insecticidal crystal inclusions as well as salmon/red pigment.

4. Crystal solubility
A further aspect of characterization ofthe strain β. thuringiensis subsp. oloke is determined by evaluating the solubility of the insecticidal crystal of the strain.

This was tested in two types of buffers which would be understood by those skilled in the art to be appropriate for this purpose. These buffers are BME pH 9.2, 10.0 and 12.5 and UDS-PMSF at pH's 9.5, 10.5, 11.5 and 12.5. Experiments of this sort are also important because the behaviour of crystals of insecticidal toxin in solubilization buffers at different pH values can serve as an indication of the insecticidal activity of the protein in the crystal (Aronson, et al., 1991 ; and Du, et al., 1994). The high reducing activity of some buffers may not be compatible with 5 bioassay conditions, even if a large amount of protein can be solubilized from the crystals by these buffers. For the first set of buffers (BME), the highest amount of solubilized protein (i.e., 59.4 mg/mL) was obtained when moderate reducing conditions (i.e., 0.03 M Na2CO3-2% β-mercaptoethanol pH 10)(Aronson, et al., 1991) were used to dissolve the crystal. Using a buffer that was more reducing o and provided a higher pH (i.e., 0.8 M Na2CO3-5% BME pH 12.5), a lower amount of protein (i.e., 41.5 mg/mL) was solubilized (Table 2). With the second set of solubilization buffers (UDS-PMSF) maximal solubilization occurred at pH 11.5 (Figure 4). In summary the crystal from B. thuringiensis subsp. oloke was not solubilized by any of the buffers used for this purpose with other strains of B. 5 thuringiensis. Thus, 10 mM EDTA-50 mM NaOH used to solubilize the β. thuringiensis subsp. israelensis crystal protein (Ingle et al., 1993), 0.05N NaOH used to solubilize the crystal from β. thuringiensis subsp. thuringiensis (Meenakshi and Jayaraman, 1979) and the solubilization buffer referred to as 'cracking buffers' by Calabrese and Nickerson (1980) used for the solubilization of the crystals of 0 sixteen strains of B. thuringiensis were all ineffective against the crystals of β. thuringiensis subsp. oloke.

5. SDS-PAGE analysis of the insecticidal crystal inclusions
The solubilized protein obtained following solubilization of the crystal with 5 UDS-PMSF pH 11.5 buffer were examined by SDS-PAGE. Seven major bands with the following molecular weights were observed: 55, 47, 40, 36, 32.5, 30 and

25 kDa (Figure 5) which is completely different from all other reported

B.thuringiensis strains.
A deposit of B. thuringiensis subsp. oloke has been made with NRRL, o effective March 11 , 1996, bearing accession number B21528.

B. Reduced photoinactivation and insecticidal activity
1. Protection efficacy of the salmon/red piαment
β. thuringiensis subsp. kurstaki cells were mixed with 75 μg/mL of the salmon/red pigment and then exposed to 253 nm light at 50 J/m2. 9.5% of the cells remained viable (see Figure 3). On the other hand, when B. thuringiensis subsp. kurstaki in the absence of the salmon/red pigment was exposed to as little as 2 J/m2 of 253 nm light, 100% of the cells were killed. When β. thuringiensis subsp. oloke cells grown under conditions which promote the presence of the salmon/red pigment are irradiated with 253 nm light at 50 J/m2, the cell survival is similar to that observed for the β. thuringiensis subsp. kurstaki cells in the presence of added salmon/red pigment (see Figure 3 ).

2. Insecticidal activity
Although the salmon/red pigmented B. thuringiensis subsp. oloke was isolated from Agrotis ipsilon (Hyn), a lepidoptera; the protoxin exhibits mosquitocidal effect against the larvae of Aedes aegypti, a diptera; with an LC^of about 50 ng/mL.

C. Transformation of E. coli

Agarose gel electrophoresis of plasmid DNA extracted from β. thuringiensis subsp. oloke indicates the presence of a single large plasmid whose size is equal to or greater than about 50 kb (Figure 6). Since it was not known whether the gene encoding the biosynthesis of the salmon/red pigment was present within the plasmid or the chromosomal DNA, total cellular DNA was used to construct a clone bank of β. thuringiensis subsp. oloke DNA in the E. coli plasmid pZErO-1. Previous workers have reported the β. thuringiensis insecticidal toxin to be encoded within either the plasmid or chromosomal DNA, depending upon the strain that is examined. The crystal protein gene is located on the chromosome as well as on a plasmid in a strain of the subspecies kurstaki (Held et al., 1982) and in subspecies thuringiensis strain berliner 1715 (Klier et al., 1982). E. coli cells transformed with this clone bank were plated on low salt LB medium containing zeocin as the selective antibiotic and colonies that produced a red colour following five days of growth at 37°C were isolated and characterized. Of the five thousand colonies that were plated on this medium three were selected for further study. One of these three colonies was coloured salmon/red.

Colonies ofthe salmon/red-coloured transformant are similar in colour to the parent B. thuringiensis strain and also appear to produce visible inclusion bodies. However, this tranformant grows very poorly, producing only small colonies, in comparison with the parent B. thuringiensis subsp. oloke strain which produces large colonies on rich media. Cells from the parent B. thuringiensis strain are approximately 5.2 by 2.2 mm in size while the E. coli transformant cells are approximately 1.8 by 0.8 mm in size. In addition, the surface of the parent B. thuringiensis cells appears coarse and fibrous while the surface of the salmon/red E. coli transformant cells is smooth. A deposit of these transformants has been made at NRRL, effective March 11 , 1996, bearing accession number B215299.

The colony size of these E. coli transformants is inversely correlated with the size ofthe inclusions found within the transformed cells. We have hypothesized that transformed £. coli cells that produce the greatest amount of foreign protein (as indicated by the amount of inclusion body formation) are much more likely to be debilitated in their normal physiological functioning than are non-transformed cells (Glick, 1995). Based on observations with a number of different microorganisms, the sorts of physiological impairments that can result from a "metabolic load" being placed on transformed cells expressing high levels of foreign protein include alterations in cell size and growth rate (Glick, 1995; Glick et al., 1985; and Hong et al., 1995).

Salmon/red pigment obtained from the transformants had chemical characteristics consistent with melanin including solubility in 1 M NaOH, precipitation in 1 M HCI or 5 mM FeCI3 and bleaching with 20% H2O2. The LC50 of the solubilized crystals from the red transformants were 93 ng/mL (compared to 50 ng/L obtained for the parent β. thuringiensis subsp. oloke strain).

EXPERIMENTAL ASPECTS OF THE INVENTION
Bacterial strains and culture conditions
The strain of Bacillus thuringiensis disclosed herein was isolated and purified by Dr. J.K. Oloke from a dried cadaver of a larva of Agrotis ipsilon (Hyn.) found on the campus of Ladoke Akintola University of Technology, Ogbomoso, Nigeria. A suspension of the dried cadaver of Agrotis ipsilion in sterile distilled water was spread onto potato dextrose agar (PDA) plates. Following several days of incubation at approximately 30°C the salmon/red pigmented organism was subcultured on fresh PDA plates. Bacillus thuringiensis subsp. kurstaki was isolated from a commercial formulation of Thuricide® (ICI Canada, Inc.).

Morphology and biochemical characterization
The β. thuringiensis subsp. oloke strain was characterized as described by Heimpel and Angus (1960) to define the following properties of the organism: spore, sporangium, vegetative rods, gelatin liquefaction, nature of agar colonies, nature of broth culture, starch hydrolysis, sugar fermentation and ability to produce acetylmethyl carbinol.

Antibiotic resistance pattern
The antibiotic resistance pattern of β. thuringiensis subsp. oloke strain was compared to that of B. thuringiensis subsp. kurstaki using an agar diffusion technique as described by Oloke et al. (1988). For this test potato dextrose broth and agar containing 20% (v/v) coconut water was used for the newly isolated strain while Luria broth and agar were used for β. thuringiensis subsp. kurstaki. An eighteen hour broth culture (at 30°C) of each organism was spread on agar plates and allowed to drain. With the aid of a pair of forceps, sterilized filter paper discs were dipped into the different concentrations of each of the eight antibiotics used and layered appropriately on the surface of the agar plates. Diameters of zones of inhibition were obtained for each antibiotic in duplicate.

Growth conditions
The optimum growth temperature was estimated by obtaining viable cell counts on a medium consisting of potato dextrose broth containing 20% (v/v) coconut water (PDW) at 19, 30 and 37°C for 24 h. At each temperature serial dilutions of the growing culture were done at regular intervals between zero and 24 h and viable counts were obtained in duplicate on PDW agar plates. The growth medium composition for maximum crystal and pigment formation was investigated by streaking β. thuringiensis subsp. oloke on the following types of media: Nutrient agar (NA), LB; LB containing 5 mg/ml tyrosine; PDA; PDW and 100% coconut water agar (CWA). The plates were incubated at 30°C for about 72 h.

Pigment isolation and purification
Pigmented bacterial colonies are scraped from the surfaces of PDW agar cultures, suspended in sterile distilled water and sonicated (Braun-Sonic 2000) for 5 min on ice. The suspension is pelleted by centrifugation in a Sorval centrifuge using an SS-34 rotor at 8000 rpm (4°C) for 10 min and the pellet discarded. The pH of the supernant is adjusted to 3 before centrifugation for an additional 10 min. The pigmented pellet, as well as the pigment isolated from the transformants are purified and characterized as described (Fuqua et al., 1991 ; and Liu et al., 1993).

Protection efficacy of pigment
The protocol used to assess the photoprotective effect of the pigment followed the method described by Liu et al. (1993). Small amounts of B. thuringiensis subsp. kurstaki and β. thuringiensis subsp. oloke were separately ascepticly scraped from the surface of 72 h LB agar and PDW agar plates, respectively, then suspended in sterile distilled water at a concentration of approximately 106 cells/mL in glass petri dishes. For β. thuringiensis subsp. kurstaki two separate exposure experiments were done; viz., one containing only the cells and the other containing the cells and 75 mg/mL of the purified salmon/red pigment from B. thuringiensis subsp. oloke. These were then treated with different doses of 253-nm wavelength light, which was measured with a uvx digital radiometer (UVP, Inc.). The survival of bacteria before and after the uv radiation was measured by counting the colony-forming units. The entire experiment was repeated twice.

Purification and solubilization of the insecticidal toxin
β. thuringiensis subsp. oloke is grown with vigorous shaking for six days in 500 mL of potato dextrose broth (Difco Laboratories) containing 20% coconut water, 0.2 % NaCI and 4 mL of 50% glucose. Harvested cultures are pelleted by centrifugation and then washed once with 10 mL of 1 M KCI-5 mM EDTA, once with 10 mL of deionized water containing 5 mM phenylmethylsulfonyl fluoride (PMSF) and twice with 10 mL of deionized water before being suspended in about 10 mL of an aqueous solution of 0.2% Triton X-100 (Aronson et al., 1991). Six mL of the cell suspension is layered over 18 mL of a discontinuous gradient of equal volumes of 50, 63 and 73% (w/v) sucrose (Ingle et al., 1993). The tubes are then centrifuged at 12,000 rpm in a Sorval HB4 rotor for 60 min in a Sorval RC-2B refrigerated centrifuge at 4°C (Meenakshi and Jayaraman, 1979). The inclusion band which contains the insecticidal toxin is removed with a Pasteur pipette, and its purity ascertained by phase-contrast microscopy of samples of the preparation stained with 1% carbol fuchsin. If this band is contaminated with spores and/or cellular debris, it is repurified through a second discontinuous sucrose density gradient. The final band is diluted at least five-fold with water, pelleted at 8,000 rpm for 20 min in a Sorval SS-34 rotor at 4°C and washed twice with deionized water before being dried in a Savant Speed-Vac (Aronson et al., 1991).

For the solubilization assay, 75 mL of an intact crystal suspension (2 mg/mL in distilled water) is mixed with 75 mL of different buffers (Aronson et al., 1991; and

Du et al., 1994). The initial buffer, 0.3 M Na2CO3-β-mercaptoethanol (BME) (pH 9.2) was selected based on conditions reported by others for the solubilization of insecticidal protein from inclusions found in other β. thuringiensis strains. More reducing conditions at a higher pH were then used; and finally buffers consisting of 6 M urea-1 % SDS-0.5 M dithiothreitol-2 mM phenylmethylsulfonyl fluoride (UDS-PMSF) at pH 9.5, 10.5, 11.5 and 12.5 were used. Protein levels were determined by the method of Bradford (1976) using bovine serum albumin as a standard.

Purification and solubilization of the insecticidal toxin from transformants
The crystals of the transformant were obtained by sucrose gradient centrifugation (Aronson et al., 1991; and Ingle et al., 1993) and solubilized in 0.3 M Na2CO3-β-mercaptoethanol, pH 9.7 (Aronson et al., 1991). The solubilized crystals were dialyzed at 4°C against several changes of 0.03 M NaHCO3, pH 8.5.

Inclusions were suspended in the latter buffer.

Insecticidal activity
Aedes aegypti eggs were kindly provided by Dr. B. Diel-Jones of the

Department of Biology, University of Waterloo. In order to measure the insecticidal activity against Aedes aegypti, protoxins solubilized in 0.3 M Na2CO3-2%BME (pH 9.7) were dialyzed at 4°C against several changes of 0.03 M NaHCO3 (pH 8.5) (Aronson et al., 1991). Ten fourth instar larvae were incubated at 28°C (Khawalled et al., 1990) with the suspension of the inclusion in the latter buffer. The assays were performed in duplicate and mortality was scored after 48 h (17). LC50 values were obtained by probit analysis (Lithchfield and Wilcoxon, 1949).

SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970) with 12% separating and 4% stacking gels. Following electrophoresis the gels were stained with 0.4% Coomassie blue R-250. The molecular masses of the solubilized proteins were determined in comparison with known protein standards (Bio-Rad).

Preparation of Plasmid DNA
Plasmid DNA containing appropriate messages for β. thuringiensis subsp. oloke toxin and salmon/red pigment was prepared from β. thuringiensis subsp. oloke by the procedure of Kronstad et al. (1983). For preferential isolation of 5 plasmids 50 megadaltons (Mdal) in size and smaller, cultures were grown in 500 mL of L broth (Difco Laboratories, Detroit, Ml) supplemented with glucose to give a final concentration of 0.1% in a 2.8 L Fernback flask with shaking at 37°C; cells were harvested at an optical density at 600 nm of 0.8. For the preferential isolation of plasmids 30 Mdal and larger, cells were grown separately in two types of media: o in 500 mL coconut water supplemented with glucose and in 500 mL of SPY medium (Spizizen medium (Spizizen, 1958)) supplemented with 0.1% yeast extract and 0.1% glucose in a 2.8 L Fernback flask with shaking at 37°C; cells were harvested at an optical density at 600 nm of 0.7.

5 Preparation of Total cell DNA
Total cell DNA was prepared from B. thuringiensis subsp. oloke as described (Kronstad et al., 1983). Cells grown in coconut water medium were harvested by centrifugation, washed with a solution containing 100 mM NaCI , 10 mM Tris (pH 7.9), and 10 mM EDTA, and resuspended in 5 mL of a solution containing 150 mM o NaCI and 100 mM EDTA at pH 7.9 before being lysed by the addition of lysozyme.

The isolated DNA was dissolved in 10 mM Tris pH 7.9.

Recombinant DNA procedures
Restriction enzymes were used as recommended by the supplier (New 5 England Biolabs), and recombinant DNA procedures were carried out as described by Sambrook et al. (1989). A genomic DNA library of β. thuringiensis subsp. oloke was prepared in plasmid pZErO-1 (Invitrogen Corp., San Diego, CA) using the conditions suggested by the manufacturer. Two mL aliquots of the ligation mixture were used to transform £. coli TOP10F* cells (Invitrogen Corp., San Diego, CA). o The transformants were plated on Low Salt LB-Zeocin-IPTG medium (1 % Tryptone,

0.5%Yeast Extract, 0.5%NaCI, 1 mM IPTG and 500 μg/L Zeocin.).

Scanning Electron microscopy
Scanning electron microscopy was carried out as described by Calabrese et al. (1980). A drop of each culture suspension (~1 Oβbacteria/mL) was placed on a Formvar coated grid and excess liquid drawn off with Whatman No. 50 filter paper. The dried grid was then shadowed with a 63.5 mm Pt-Pd wire at 20 A for

20 s in the tungsten basket of a Varian PS10E vacumm evaporator.

Transmission Electron microscopy
Transmission electron microscopy was carried out as described by Bechtel and Bulla (1976). E. coli transformants were suspended in 4% glutaraldehyde in 0.01 M phosphate-buffered saline at pH 7.2 for 5 min at 4°C then pelleted and suspended in 2% agar at 55°C. The agar was immediately cooled to 4°C, cut into 1-mm cubes, and placed into fresh cold glutaraldehyde for 1 h. Samples were washed four times in cold 0.01 M phosphate buffer for a total of 80 min. After washing, the bacteria were postfixed in 1% OsO4 at 4°C for 1 h. Samples were rinsed in double-distilled water for 30 min and then stained overnight in 0.5% aqueous uranyl acetate. The bacteria were dehydrated by passing them through a graded acetone series and then embedded in Epon 812. Samples were cut with a diamond knife on a Porter-Blum MT-2b ultramicrotome, stained with lead citrate, and examined in a Philips EM 201 electron microscope operated at 60 kv. Serial sections, 150 nm thick, were placed on slotted grids previously coated with Formvar™ and carbon.

SUMMARY
The present invention provides a unique insecticidal agent with an enhanced viability against uv inactivation. The protection afforded by the salmon\red melanin ofthis insecticidal agent can be synthesized via an appropriate vehicle such as E. coli and thereby provide a means of extending this protection to other organisms.

In addition to a role as a biological "sun screen" protecting β. thuringiensis against uv inactivation, there is considerable commercial interest in melanins due to their ability to act as uv light absorbers, cation exchangers, amorphous semiconductors and novel biopolymers with drug-binding and other unique properties (Bell and Wheeler, 1986), i.e., block photoradiation. In this regard, β. thuringiensis subsp. oloke, in addition to its insecticidal properties, may serve as an inexpensive source of a unique salmon/red melanin which could be used on photoradiation sensitive recipients in a variety of consumer applications including suntan lotions. Indeed, the salmon/red pigment could be incorporated into any creams, lotions or other suitable carrier compounds or compositions and applied topically to photoradiation sensitive recipients, including humans, and animals, or any other entity or organism where it is desirable to block the efects of photoradiation. In connection with the human cosmetices market, at the present time, such "attractively coloured" melanins are produced by expensive and cumbersome chemical procedures (della-Cioppa et al., 1990).

While the invention has been particularly shown and described with reference to preferred embodiments it will be understood by those skilled in the art that various other changes in form and detail may be made without departing from the spirit and scope of the invention.

REFERENCES
The present specification refers to the following publications, each of which is expressly incorporated by reference herein.

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Table t Respone of β. thuringiensis subsp. kurstaki and β. thuringiensis
subsp. oloke cells to various antibiotics.

Zone of inhibition, mm

Antibiotic Concentration of B. thuringiensis B. thuringiensis
antibiotic μg/mL kurstaki oloke

Kanamycin 200 - Rifampicin 200 16
100 11
50 -

Tetrcycline 200 9
100 -

Carbenicillin 200 - Chloramphenicol 200 18
100 -

Ampicillin 200 20
100 19
50 18

Stretomycin 200 - Penicillin 200 18
100 16
50 _ Table 2. Effect of medium composition on the formation of insecticidal
crystal and salmon/red pigment by β. thuringiensis subsp. oloke,.

Medium Growth Pigmentation Crystal formation, % of cells

LB agar ++ light red 2

LB agar + 5 mg/mL ++ light red 2
tyrosine

NA + light red <1

PDA +++ salmon/red 3

PDW agar +++ salmon/red >95

CWA +++ salmon/red >99.5