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WO1997041235A2 - Nouvelle souche du bacillus thuringiensis productrice de pigment - Google Patents

Nouvelle souche du bacillus thuringiensis productrice de pigment Download PDF

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Publication number
WO1997041235A2
WO1997041235A2 PCT/CA1997/000286 CA9700286W WO9741235A2 WO 1997041235 A2 WO1997041235 A2 WO 1997041235A2 CA 9700286 W CA9700286 W CA 9700286W WO 9741235 A2 WO9741235 A2 WO 9741235A2
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organism
salmon
oloke
red pigment
thuringiensis subsp
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PCT/CA1997/000286
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English (en)
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WO1997041235A3 (fr
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Julius Kola Oloke
Bernard Robert Glick
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University Of Waterloo
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Priority to AU23768/97A priority Critical patent/AU2376897A/en
Publication of WO1997041235A2 publication Critical patent/WO1997041235A2/fr
Publication of WO1997041235A3 publication Critical patent/WO1997041235A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
    • A61Q1/02—Preparations containing skin colorants, e.g. pigments
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/42—Colour properties
    • A61K2800/43—Pigments; Dyes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/075—Bacillus thuringiensis

Definitions

  • 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.
  • 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).
  • ⁇ . thuringiensis subsp. tenebrionis is effective against coleoptera such as the boll weevil, ⁇ .
  • thuringiensis subsp. kurstaki kills lepidoptera such as spruce budworm (Lopez-Meza et al., 1995), and ⁇ . 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).
  • 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).
  • the present invention provides an isolated and purified organism containing salmon/red pigment.
  • an organism isolated and purified containing salmon/red pigment and insecticidal toxin is provided.
  • 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.
  • E. coli transformants capable of producing both the ⁇ . 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
  • 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 ⁇ . 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;
  • the organism is E. coli.
  • an insecticidal toxin isolated from B. thuringiensis subsp. oloke, or any chemical equivalent thereof.
  • the present invention provides a strain of B. thuringiensis which is capable of persisting longer in the environment because of enhanced photoprotection.
  • an insecticide comprising an effective amount of B. thuringiensis subsp. oloke.
  • an insecticide with reduced susceptibility to photoinactivation comprising a Bacillus thuringiensis which has been treated with an effective amount of salmon/red pigment.
  • 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.
  • a salmon/red pigment or any chemical equivalent thereof there is provided a salmon/red pigment or any chemical equivalent thereof.
  • a salmon/red pigment produced by a salmon/red pigmented organism or any functional equivalent thereof.
  • 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.
  • the invention provides a strain of ⁇ . thuringiensis which produces a salmon/red pigment which is capable of providing photoprotection from ultraviolet radiation.
  • 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.
  • the recipient is a human being.
  • 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.
  • the recipient is a human being.
  • 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.
  • 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 ⁇ . thuringiensis subsp. oloke.
  • Figure 3 illustrates a histogram representative of survival of ⁇ . thuringiensis subsp. oloke, B. thuringiensis subsp. kurstaki and ⁇ . thuringiensis subsp. kurstaki cells in the presence of 75 ⁇ g/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 ⁇ . 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.
  • EDTA ethylenediaminetetraacetic acid
  • UDS-PMSF 2mM phenyl methyl sulfonyl fluoride in 6M urea - 1% sodium dodecyl sulfate (“SDS”) - 0.5M dithiotheitol.
  • BME ⁇ -mercaptoethanol
  • the short form “PDA” means potato dextrose agar.
  • the short form “CWA” means coconut water agar.
  • 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. Coco 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.
  • subsp means subspecies.
  • the shortform "B" means Bacillus.
  • the shortform “PDW” means potato dextrose medium containing 10% coconut water.
  • 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 FeCI 3 and bleaching with 20% H 2 O 2 .
  • the purified pigment shows a steady increasing absorption with no peak at wavelengths of UV light ranging from 300 to 210 nm ( Figure 2).
  • 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.
  • 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”.
  • Isolated and purified ⁇ . 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
  • the spores are oval shaped with an average size of approximately 1 ⁇ m by 1.9 ⁇ m and the spores are found at the polar ends of the cell.
  • a 24 h culture of vegetative cells consists of rod-shaped motile, gram-positive cells approximately 1.5-2.5 ⁇ m by 4.1-6.0 ⁇ m, with rounded ends.
  • the sporangium are oval to cylindrical, not swollen, diamond-shaped to cubosidal, with a parasporal body formed in each sporangium.
  • israeltaki was resistant to kanamycin, carbenicillin, and streptomycin (all at 200 ⁇ g/mL).
  • the strain of the invention ⁇ . 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.
  • a further aspect of characterization ofthe strain ⁇ . thuringiensis subsp. oloke is determined by evaluating the solubility of the insecticidal crystal of the strain.
  • 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.
  • the highest amount of solubilized protein i.e., 59.4 mg/mL was obtained when moderate reducing conditions (i.e., 0.03 M Na 2 CO 3 -2% ⁇ -mercaptoethanol pH 10)(Aronson, et al., 1991) were used to dissolve the crystal.
  • moderate reducing conditions i.e., 0.03 M Na 2 CO 3 -2% ⁇ -mercaptoethanol pH 10.5
  • a lower amount of protein i.e., 41.5 mg/mL was solubilized (Table 2).
  • UDS-PMSF second set of solubilization buffers
  • 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
  • Agarose gel electrophoresis of plasmid DNA extracted from ⁇ . 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 ⁇ . thuringiensis subsp. oloke DNA in the E. coli plasmid pZErO-1. Previous workers have reported the ⁇ .
  • 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.
  • 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 FeCI 3 and bleaching with 20% H 2 O 2 .
  • the LC 50 of the solubilized crystals from the red transformants were 93 ng/mL (compared to 50 ng/L obtained for the parent ⁇ . thuringiensis subsp. oloke strain).
  • 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.).
  • the ⁇ . 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.
  • the antibiotic resistance pattern of ⁇ . 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).
  • 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 ⁇ . thuringiensis subsp. kurstaki.
  • An eighteen hour broth culture (at 30°C) of each organism was spread on agar plates and allowed to drain.
  • 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 ⁇ . 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).
  • 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).
  • the initial buffer 0.3 M Na 2 CO 3 - ⁇ -mercaptoethanol (BME) (pH 9.2) was selected based on conditions reported by others for the solubilization of insecticidal protein from inclusions found in other ⁇ . 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.
  • 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 Na 2 CO 3 - ⁇ -mercaptoethanol, pH 9.7 (Aronson et al., 1991).
  • the solubilized crystals were dialyzed at 4°C against several changes of 0.03 M NaHCO 3 , pH 8.5.
  • SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis 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).
  • Plasmid DNA containing appropriate messages for ⁇ . thuringiensis subsp. oloke toxin and salmon/red pigment was prepared from ⁇ . 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.
  • Mdal megadaltons
  • 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.
  • SPY medium Spizizen medium (Spizizen, 1958)
  • 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.
  • 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 FormvarTM and carbon.
  • 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.
  • ⁇ . 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.
  • 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 effectss of photoradiation.
  • photoradiation sensitive recipients including humans, and animals, or any other entity or organism where it is desirable to block the effectss of photoradiation.
  • such "attractively coloured" melanins are produced by expensive and cumbersome chemical procedures (della-Cioppa et al., 1990).
  • B. thuringiensis an environmental biopesticide: Theory and practice, (eds. P.F. Entwistle; J.S. Cory; M.J. Bailey and S. Higgs). John Wiley & Sons. New York, pp. 171-191.

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Abstract

Cette invention a trait à une souche isolée et purifiée du Bacillus thuringiensis, dénommée Bacillus thuringiensis, sous-espèce oloke, qui produit naturellement un pigment coloré saumon/rouge dont les propriétés physico-chimiques indiquent qu'il s'agit d'une forme de mélanine. La souche selon cette invention est capable d'assurer une photoprotection à des toxines insecticides, la sous-espèce du Bacillus thuringiensis, notamment, ainsi qu'à d'autres toxines sensibles à une inactivation par les rayons solaires. Cette invention a trait, en outre, à un pigment à même de conférer une photoprotection à d'autres organismes, ce pigment se caractérisant par le fait qu'il est un agent protecteur coloré contre la lumière.
PCT/CA1997/000286 1996-05-01 1997-04-30 Nouvelle souche du bacillus thuringiensis productrice de pigment WO1997041235A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU23768/97A AU2376897A (en) 1996-05-01 1997-04-30 A new, pigment-producing, strain of (bacillus thuringiensis)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9609095.6A GB9609095D0 (en) 1996-05-01 1996-05-01 A new, pigment-producing, strain of bacillus thuringiensis
GB9609095.6 1996-05-01

Publications (2)

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WO1997041235A2 true WO1997041235A2 (fr) 1997-11-06
WO1997041235A3 WO1997041235A3 (fr) 1997-12-24

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AU (1) AU2376897A (fr)
GB (1) GB9609095D0 (fr)
WO (1) WO1997041235A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021236416A1 (fr) * 2020-05-21 2021-11-25 Liberty Biosecurity, Llc Compositions provenant d'un organisme bactérien et leurs utilisations

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0654075B1 (fr) * 1992-08-27 2005-10-26 Bayer BioScience N.V. Nouvelles souches de bacillus thuringiensis et leurs proteines insecticides
AP498A (en) * 1993-05-18 1996-05-29 Aeci Ltd Photoprotected bacillus thuringiensis

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021236416A1 (fr) * 2020-05-21 2021-11-25 Liberty Biosecurity, Llc Compositions provenant d'un organisme bactérien et leurs utilisations

Also Published As

Publication number Publication date
GB9609095D0 (en) 1996-07-03
AU2376897A (en) 1997-11-19
WO1997041235A3 (fr) 1997-12-24

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