JP5145506B2 - Bacillus bacteria and uses thereof - Google Patents

Description

  The present invention relates to a novel bacterium belonging to the genus Bacillus, inclusion bodies thereof, a method for controlling diptera using the same, and the like.

Bacteria Bacillus Chu (abbreviated as Bacillus thuringiensis, hereinafter BT) phosphorus thuringiensis forms inclusion bodies containing the insecticidal protein to sporulation in Bacillus Gram-positive. It is known that pests that have eaten the inclusion bodies will die after rupture of the intestinal tract. It is known that the insecticidal activity spectrum of BT covers Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Tobigera. However, its insecticidal action is different from chemically synthesized insecticides and is specific to each insect and is not toxic to animals and plants. Therefore, a pest control agent using BT is expected as a highly safe and effective pest control agent that can replace a chemically synthesized pesticide (Non-patent Document 1: Glare and O'Callaghan, Bacillus thuringiensis : Biology, Ecology and Safety. Chichester: John Wiley & Sons (2000)). Among Diptera, BT is used against sanitary pests such as mosquitoes and gnats and unpleasant pests such as chironomids and butterflies. Among mosquitoes, squid are strongly involved in the transmission of West Nile fever as a vector for West Nile virus, so this control is strongly required. A strain of BT that is generally used for controlling Diptera is B. thuringiensis serovar israelensis (hereinafter abbreviated as Isla Elensis ). Isla Elensis has Cry4A, Cry4B, Cry10A, Cry11A, Cyt1A, and Cyt2B as insecticidal proteins, and its genes are known to be retained in one plasmid (Non-patent Document 2: C. Berry et al. al. Appl. Environ. Microbiol. 68: 5082-5095 (2002)).
However, since Isla Elensis has been quite long since it was first isolated, there are concerns about the emergence of dipterous pests of strains that have acquired resistance to it. Therefore, there is a demand for the development of new bacteria having toxicity exceeding Isla Elensis against Diptera pests, especially squid.
Glare and O'Callaghan, "Bacillus thuringiensis: Biology, Ecology and Safety" (USA), Chichester, John Wiley & Sons ,the year of 2000 C. Berry et al., Applied and environmental microbiology, 68, p5082-5095, 2002

  In view of the above circumstances, an object of the present invention is to provide a novel bacterium having strong toxicity against Diptera pests, particularly squid.

As a result of diligent studies to achieve the above object, the present inventors have found that a new Bacillus bacterium B282 isolated from river water of Yakushima is an Isla Elensis against Diptera pests, especially squids such as Chikaeka. The present inventors have found that the bacterium and inclusion bodies contained therein are useful as insecticides for dipterous pests, and have completed the following invention.
That is, the present invention relates to the following.
[1] A Bacillus bacterium represented by accession number FERM P-20949.
[2] An inclusion body of a bacterium belonging to the genus Bacillus represented by accession number FERM P-20949.
[3] A composition comprising a Bacillus bacterium represented by accession number FERM P-20949 or an inclusion body thereof.
[4] The composition according to [3], which is an insecticide for dipteran pests.
[5] The composition according to [4], wherein the Diptera pest is Culex.
[6] A method for controlling Diptera pests, which comprises applying a Bacillus bacterium represented by accession number FERM P-20949 or an inclusion body thereof in a habitat of Diptera pests.
[7] The method according to [6], wherein the Diptera pest is Culex.
[8] The following polypeptide (a), (b) or (c) or a partial peptide thereof:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 2 having 95% or more sequence identity;
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4;
(C) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6, or an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6.
[9] A polynucleotide encoding the polypeptide according to [8].
[10] An expression vector comprising the polynucleotide according to [9].
[11] A transformant comprising the expression vector according to [10].
[12] An antibody against the polypeptide according to [8].
[13] A hybridoma that produces the antibody according to [12].

  The Bacillus bacterium of the present invention and inclusion bodies thereof are useful as insecticides for dipterous pests because they have strong toxicity against dipterous pests, particularly squid.

(1. Bacteria, inclusion bodies and their uses)
The present invention provides a Bacillus bacterium (hereinafter sometimes referred to as B282) represented by accession number FERM P-20949. The Bacillus genus bacterium represented by the deposit number FERM P-20949 has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba City, Ibaraki Pref. 5th).

  The bacterium of the present invention has inclusion bodies (inclusions) having a strong toxicity to Diptera insects in the bacterium, and the bacteria themselves have a strong toxicity to Diptera insects. When the bacterium of the present invention is applied in the habitat of the larvae of the dipteran insect, the larvae are killed and the reproduction of the dipteran insect is suppressed. Diptera insects include diptera pests (sanitary pests such as mosquitoes and gnats, unpleasant pests such as chironomids and butterflies). Examples of mosquitoes include squid, such as Chikaieka, and mosquitoes, such as anopheles, Aedes aegypti. The bacterium of the present invention has a strong toxicity against mosquitoes among Diptera insects, and particularly has a particularly strong toxicity against mosquitoes such as Chikaeka compared with Isla Elensis. ing.

The bacterium of the present invention has been isolated from river water of Yakushima and has the following phenotypes under normal culture conditions:
(1) Gram-positive bacilli, most of which are 5 or more streptococci when cultured in liquid.
(2) Motility is negative.
(3) Oval sporulation occurs. Spore locations are ubiquitous.
(4) Intracellular inclusions are indefinite and have the same size as spores.
(5) The settlement form is a large settlement typical of Bacillus thuringiensis (BT), and the surface is dull.
(6) Aerobic.
(7) It has carbohydrate metabolism ability as shown in Table 1 below.

The bacterium of the present invention has a 16S rRNA gene comprising the nucleotide sequence represented by SEQ ID NO: 7. From the molecular phylogenetic analysis based on this nucleotide sequence, the bacterium of the present invention is considered to be a kind of Bacillus cereus bacterium or Bacillus thuringiensis bacterium (see Example 2.3.).

  The bacterium of the present invention can be cultured by a method known per se used for culturing Bacillus bacteria in general. For example, the bacterium of the present invention is about 28 ° C. under aerobic conditions on a meat extract agar medium (pH 7.6) in which 10 g of meat extract, 10 g of polypeptone, 2 g of sodium chloride and 20 g of agar are dissolved in 1 liter of water. It can be cultured.

The present invention also provides an inclusion body (also referred to as inclusion or crystal) of the bacterium of the present invention. Inclusion bodies are crystals of insecticidal protein and have strong toxicity to Diptera. When the inclusion body of the present invention is applied to the larvae of the dipteran insect, the larvae are killed and the reproduction of the dipteran insect is suppressed. The inclusion body of the present invention has a strong toxicity against mosquitoes among Diptera insects, and has a particularly strong toxicity against mosquitoes such as Chikaeka in comparison with Isla Elensis. doing. For example, when compared with LC ₅₀ at 24 hours incubation, the insecticidal activity of the inclusion body of the present invention against Chikaeka (larvae) is usually more than twice that of the Islaerensis inclusion body, preferably Is more than three times (for example, about four times) stronger.

  The inclusion body of the present invention can be collected from the bacterium of the present invention by a method known per se. For example, after culturing the bacterium of the present invention under the above-mentioned culture conditions for about 5 days, the bacterium is recovered and the two-phase separation method using dextran sulfate-polyethylene glycol system (NS Goodman, J. Bact., 94: 485 (1967)). By attaching to, the inclusion body of the present invention can be obtained.

  The bacterium of the present invention and inclusion bodies thereof are useful as insecticides for dipterous pests (eg, squid) because they have a strong toxicity to dipteran insects, particularly squids such as chikaeka. The insecticide of the present invention can be produced by mixing an effective amount of the bacterium of the present invention or an inclusion body thereof for killing dipterous pests with water or an appropriate known compounding agent. The content of the active ingredient in the insecticide of the present invention is not particularly limited. However, when the active ingredient is the bacterium of the present invention, it is usually about 1 ng (wet weight) / ml to 50 μg (wet weight) / ml. When the component is the inclusion body of the present invention, it is usually about 0.1 ng (protein amount) / ml to 5 μg (protein amount) / ml.

The present invention also provides a method for controlling diptera pests (eg, squid), which comprises applying the bacterium of the present invention or an inclusion body thereof in the habitat of the diptera pest larvae. When the bacterium of the present invention or an inclusion body thereof is applied (sprayed or mixed) into the habitat environment of a dipterous pest, the larva comes into contact with the bacterium of the present invention or the inclusion body or ingests the component As a result, since the larvae die, it is possible to suppress the breeding of Diptera pests. The habitat environment of the dipterous pests can be appropriately selected according to the type of the dipterous pests to be controlled. The habitat environment of the dipterous pest larvae is not particularly limited, and examples thereof include aquatic environments (rivers, lakes, swamps, seas, ducks, etc.), soils, and the like. Here, the “diptera pest larva habitat” refers to an environment in which diptera pest larvae can inhabit, and does not require that the diptera pest larvae actually live. That is, the method of the present invention includes the following:
(1) By applying the bacterium of the present invention or its inclusion body in an environment where larvae of the dipterous pests actually live, the larvae are killed and the reproduction of the dipterous pests is suppressed. And (2) dipterous pest larvae do not inhabit, but the bacterium of the present invention or its inclusion body is applied in advance to an environment where the larvae may occur in the future, To prevent the breeding of animals.

  In the method of the present invention, the dose of the bacterium of the present invention or its inclusion body is not particularly limited as long as it can control Diptera pests, and is appropriately set according to the target Diptera pests, the type of habitat, etc. can do. For example, when the bacterium of the present invention is applied to an aquatic environment, the bacterium is usually applied so that the concentration in the environment is about 1 ng (wet weight) / ml to 10 μg (wet weight) / ml. For example, when the inclusion body of the present invention is applied to an aquatic environment, the inclusion body is usually applied so that the concentration in the environment is about 1 ng (protein amount) / ml to 1 μg (protein amount) / ml. Is done.

(2. Novel insecticidal polypeptide and related invention)
The present invention provides a novel insecticidal polypeptide derived from the bacterium of the present invention. The polypeptide of the present invention may be a polypeptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. The polypeptide of the present invention can be useful, for example, for killing dipterous pests and producing the antibody of the present invention.

  In one embodiment, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is defined as represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, It can be an amino acid sequence having amino acid sequence identity. The degree of amino acid sequence identity can be, for example, about 95%, preferably about 96%, more preferably about 97%, even more preferably about 98%, most preferably about 99% or about 99.5% or more. . Amino acid sequence identity can be determined by a method known per se. For example, amino acid sequence identity (%) can be determined using a program commonly used in the art (eg, BLAST, FASTA, etc.) by default. In another aspect, identity (%) is determined by any algorithm known in the art, such as Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4: 11-17). Needleman et al.'S algorithm is incorporated into the GAP program in the GCG software package (available at www.gcg.com) and the percent identity is, for example, BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, It can be determined by using either 14, 12, 10, 8, 6 or 4 and length weight: 1, 2, 3, 4, 5 or 6. The Myers and Miller algorithms are also incorporated into the ALIGN program that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, for example, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used.

  In another embodiment, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 In which one or more amino acids may be substituted, added, deleted and / or inserted. The number of amino acids to be substituted, added, deleted and / or inserted is not particularly limited as long as it is 1 or more. For example, 1 to about 100, preferably 1 to about 50, more preferably 1 to about 30. Even more preferably, it may be 1 to about 20, most preferably 1 to about 10, 1 to about 5, or 1 or 2.

  The polypeptides of the present invention may have insecticidal activity against Diptera pests (eg, squid). When the polypeptide of the present invention is a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, the degree of insecticidal activity is SEQ ID NO: 2, It can be quantitatively equivalent to a polypeptide comprising the same amino acid sequence as the amino acid sequence represented by No. 4 or SEQ ID No. 6, but within an acceptable range (for example, about 0.1 to about 5 times, preferably about 0 .5 to about 2 times).

  The partial peptide of the present invention may be a partial peptide of a polypeptide consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. The partial peptide of the present invention has at least about 8, preferably at least 10, more preferably selected from the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Can be a peptide consisting of at least 12, more preferably at least 15, most preferably 20 or more consecutive amino acids.

  The present invention also provides a polynucleotide encoding the polypeptide of the present invention or a partial nucleotide thereof. The polynucleotide of the present invention may comprise a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. The polynucleotide of the present invention or a partial nucleotide thereof can be useful, for example, for producing the polypeptide of the present invention or a partial peptide thereof.

  In one embodiment, it may be a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. The degree of nucleotide sequence identity can be, for example, about 95%, preferably about 96%, more preferably about 97%, even more preferably about 98%, and most preferably about 99% or 99.5% or more. Nucleotide sequence identity can be determined by methods known per se. For example, nucleotide sequence identity (%) can be determined in the same manner as amino acid sequence identity (%) described above.

  In another embodiment, the nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 is the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 In which one or more nucleotides may be substituted, added, deleted and / or inserted nucleotide sequences. The number of nucleotides to be substituted, added, deleted and / or inserted is not particularly limited as long as it is 1 or more. For example, 1 to about 300, preferably 1 to about 150, more preferably 1 to about 100. Even more preferably from 1 to about 50, most preferably from 1 to about 30, from 1 to about 20, from 1 to about 10 or from 1 to about 5.

  In yet another embodiment, the nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 is the nucleotide represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. It can be a polynucleotide that can hybridize under high stringency conditions to a complementary sequence of the sequence. Hybridization conditions under high stringency conditions can be set with reference to previously reported conditions (Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999). For example, hybridization conditions under high stringency conditions include hybridization at 6 × SSC (sodium chloride / sodium citrate) / 45 ° C., then 0.2 × SSC / 0.1% SDS / 50 to 65 ° C. More than once.

  The polynucleotide of the present invention can encode a polypeptide having the insecticidal activity. When the polynucleotide of the present invention is a polynucleotide comprising a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, insecticidal action of the polypeptide encoded by the polynucleotide The degree of activity can be quantitatively equivalent to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, but within an acceptable range (for example, about 0.1 to about 5 times) , Preferably about 0.5 to about 2 times).

  The partial nucleotide of the present invention can be a nucleotide encoding the partial peptide of the present invention. Specifically, the partial nucleotide of the present invention may be a partial nucleotide of a polynucleotide consisting of a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

  The polynucleotide of the present invention can be prepared by a method known per se. For example, a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 can be cloned by extracting DNA from the bacterium of the present invention and performing PCR using appropriate primers. A polynucleotide comprising a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 can be prepared by introducing mutations into the cloned polynucleotide as described above. Examples of the mutagenesis method include a synthetic oligonucleotide-directed mutagenesis method (gapped duplex) method, a method of randomly introducing point mutations (eg, treatment with nitrous acid or sulfite), a cassette mutagenesis method, and a linker scanning method. And a mismatch primer method.

  The polypeptide of the present invention can also be produced by a method known per se. For example, the polynucleotide of the present invention produced as described above is incorporated into an expression vector, the obtained recombinant vector is introduced into an appropriate host cell to obtain a transformant, the transformant is cultured, The polypeptide of the invention may be produced and then recovered. The present invention also provides such a recombinant vector and a transformant introduced with the vector.

  The expression vector is not particularly limited as long as it can express a gene encoding the polypeptide of the present invention in any host cell and produce these polypeptides. For example, a plasmid vector, a virus vector, etc. can be mentioned.

  When a bacterium, particularly E. coli, is used as a host cell, generally an expression vector can be composed of at least a promoter-operator region, a start codon, DNA encoding the polypeptide of the present invention, a stop codon, a terminator region, and a replicable unit.

  The promoter-operator region for expressing the polypeptide of the present invention in bacteria contains a promoter, an operator and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is Escherichia coli, preferred examples include those containing Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter and the like. As the terminator region and the replicable unit, those known per se can be used.

  The transformant of the present invention can be prepared by introducing the above expression vector into a host cell.

  The host cell used for the preparation of the transformant is not particularly limited as long as it is compatible with the above expression vector and can be transformed. Natural cells or artificially used usually in the technical field of the present invention can be used. Various cells such as established recombinant cells (eg, bacteria such as E. coli, Bacillus, actinomycetes, eukaryotic cells such as yeast) are exemplified.

  The polypeptide of the present invention can be produced by culturing a transformant containing the expression vector prepared as described above in a nutrient medium.

  The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the transformant. Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include soybean cake, potato extract and the like. If desired, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins) may be contained.

  The transformant is cultured by a method known per se. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the polypeptide of the present invention is produced in large quantities. For example, when the host is a bacterium, a liquid medium containing the above nutrient source is appropriate. Preferably, the medium has a pH of 5-8. When the host is Escherichia coli, preferred media include LB media and M9 media. In such a case, the culture can be performed, for example, at about 30 to 40 ° C. for about 5 to 30 hours with aeration and stirring. When the host is Bacillus, it can be performed at about 25 to 40 ° C. for about 15 to 100 hours with aeration and stirring.

  The polypeptide of the present invention can be obtained by culturing a transformant as described above and recovering, preferably isolating and purifying from the transformant.

  As an isolation and purification method, for example, a method using solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, a method using molecular weight difference such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Methods using charges such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using hydrophobic differences such as reversed-phase high-performance liquid chromatography, isoelectricity Examples thereof include a method using a difference in isoelectric point such as point electrophoresis.

In addition, a polypeptide to which a tag (for example, histidine tag, Flag tag) or the like is added is produced in the transformant, and a substance having an affinity for the tag (for example, Ni ²⁺ resin, an antibody specific for the tag) is used. Thus, the polypeptide of the present invention can be isolated and purified more easily.

  Furthermore, the polypeptide of the present invention can be synthesized in a cell-free system. In the synthesis of the polypeptide of the present invention in a cell-free system, for example, an extract from Escherichia coli, rabbit reticulocyte, wheat germ, or the like can be used. In addition, the polypeptide of the present invention can be produced by a known organic chemical method such as a solid phase synthesis method or a liquid phase synthesis method.

  Alternatively, the polypeptide of the present invention can be purified from the inclusion body of the present invention using the isolation and purification method described above.

  Examples of the antigen used for producing the antibody of the present invention include polypeptides containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or partial peptides thereof Can be used. The partial peptide is not particularly limited as long as it has immunogenicity. For example, the partial peptide is at least about selected from the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. It may be a peptide consisting of 8, preferably at least 10, more preferably at least 12, even more preferably at least 15, most preferably 20 or more consecutive amino acids.

The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. This antibody may be not only a complete antibody molecule but also any fragment as long as it has an antigen binding site (CDR) for the polypeptide of the present invention, such as Fab, F (ab ′) ₂ , ScFv, minibody, etc. Is mentioned.

For example, polyclonal antibodies, the antigen (if necessary, bovine serum albumin, KLH (may be a K eyhole L impet H emocyanin) complexes crosslinked to a carrier protein, etc.), commercial adjuvants (e.g., (Complete or incomplete Freund's adjuvant) and administered subcutaneously or intraperitoneally 2 to 4 times every 2 to 3 weeks (the antibody titer of the partially collected serum is measured by a known antigen-antibody reaction) It can be obtained by collecting the whole blood about 3 to 10 days after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  A monoclonal antibody can be prepared by a cell fusion method. For example, the above antigen is administered to mice subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and spleen or lymph nodes are collected 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. Cell fusion may be PEG or voltage pulse. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The hybridoma producing the monoclonal antibody can be cultured in vitro, or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1 Selection of New Bacillus Bacteria Bacteria B282 was selected from the river water collected at Yakushima on July 12, 1996 by the following method.
That is, the sample (river water) was pasteurized at 65 ° C. for 30 minutes. A moderately diluted sample was applied to a meat extract agar plate and cultured at 28 ° C. for 5 days. The cultured colonies were observed with an optical microscope, colonies forming inclusion bodies with spores were isolated, and those stored in slants were used as a BT library. Each bacterium in the BT library was collected after culturing on a meat extract agar medium at 28 ° C. for 5 days.
A cell suspension suspended in sterilized water so that the wet cell weight was 250 mg / ml was used as a selection sample. 5 μl of the sample for selection was administered to a 12-well plate containing 3 ml of distilled water and 5 3rd instar larvae of Chikaeka. As a result, B282 was selected as a strain showing strong insecticidal activity.

Example 2: Analysis of novel Bacillus bacteria
2.1. Analysis of phenotype The phenotype of B282 obtained in Example 1 was analyzed. Carbohydrate metabolizing ability was determined by inoculating each well with a cell suspension adjusted to turbidity according to the manual using API 50CH and API 50CHB / CHE medium kit (Nihon Biomeryu Co., Ltd.). . The results are shown below.
・ Cell morphology:
Neisseria gonorrhoeae (almost 5 streptococci or more in liquid culture)
Motility: Negative Spore formation (Oval: Spore location unevenly distributed)
Intracellular inclusions: Amorphous Size is about the same as spores Colony form: Large colony typical of Bacillus thuringiensis (BT), with a dull surface.
Gram staining: positive, physiological properties:
Aerobic carbohydrate metabolic capacity: shown in Table 1 below.

2.2. Observation of spores and inclusion bodies Isolate B282 was cultured on a normal agar medium (meat extract 10 g, polypeptone 10 g, sodium chloride 2 g, agar 20 g, water 1 liter, pH 7.6) at 27 ° C. for 5 days. Colonies that grew after the culture were observed under a phase contrast microscope. The results are shown in FIG.
Oval sporulation was observed. Spore locations were ubiquitous. Intracellular inclusions were amorphous and were about the same size as spores.

2.3. Base sequence analysis of 16S rRNA gene B282 was cultured for 3 hours, and genomic DNA was extracted and purified from B282 using QIAGEN Genomic-tip (Qiagen) according to the manufacturer's protocol.
Using a primer set that can cover almost the entire region of the 16S rRNA gene, the gene was amplified by PCR under the following conditions.
・ Primer set:
27f: 5′-AGAGTTTGATCCTGGCTCAG-3 ′ (SEQ ID NO: 8)
1492r: 5'-GGCTACCTTGTTACGACTT-3 '(SEQ ID NO: 9)
-Reaction solution composition (in 50 μl): Premix Taq (EX Taq Version) 25 μl, primer 0.5 μM, genomic DNA 10 ng
-Reaction conditions: preheating 94 ° C 3 minutes, 30 cycles (thermal denaturation 94 ° C 30 seconds, annealing 60 ° C 30 seconds, extension 72 ° C 60 seconds), cooling 4 ° C
The nucleotide sequence of the amplified product was determined using an automatic DNA sequencer (Model 373S, Perkin-Elmer) using Big Dye terminator (Applied Biosystems). Based on the nucleotide sequence obtained, databases (GenBank, EMBL, DDBJ) The homology search used was performed. Multiple alignment was performed with ClustalW, and a phylogenetic tree was created by the neighborhood join method.
The resulting nucleotide sequence is shown in SEQ ID NO: 7. The phylogenetic tree is shown in FIG.
From the analysis based on this nucleotide sequence, B282 was determined to be a new species of the genus Bacillus, which is a kind of Bacillus cereus bacterium or Bacillus thuringiensis bacterium.

2.4. Evaluation of insecticidal activity of B282 against dipteran insects B282 was cultured at 28 ° C. for 5 days on a normal agar medium (meat extract 10 g, polypeptone 10 g, sodium chloride 2 g, agar 20 g, water 1 liter, pH 7.6). The bacteria were collected. The microbial cell weight was measured, and the microbial cell suspension prepared with distilled water so that the concentration was 250 mg / ml was used as a sample to evaluate the insecticidal activity. That is, 20 Chikaeka larvae were placed in a 6-well plate containing 5 ml of deionized water. B282 diluted to various concentrations was administered to each well. 25 ° C., after 24 hours of incubation, measuring the number of survival larvae in each well, Probit method ^{(Finney, Probit Analysis, 2 nd} edn London:. Cambridge University Press (1952).) Were calculated LC ₅₀ at .
As a result, a strong insecticidal activity of B282 against Chikaeka was observed, and its LC ₅₀ was 18.4 ng (wet weight) / ml.

2.5. Purification of inclusion bodies of B282 The purification of inclusion bodies (crystalline toxin protein) from bacterial cells is performed by a two-phase separation method using dextran sulfate-polyethylene glycol system (NS Goodman, J. Bact., 94: 485 (1967)). went.
That is, B282 was cultured for 5 days at 28 ° C. on a normal agar medium (meat extract 10 g, polypeptone 10 g, sodium chloride 2 g, agar 20 g, water 1 liter, pH 7.6) and collected. The cells were washed with a 1M sodium chloride solution, and the cells were collected again by centrifugation (washing operation). The washing operation was performed 3 times in total. 2 to 4 g of the centrifuged cells were suspended in 33 ml of distilled water, and the suspension was sonicated. The suspension was added to the lower layer solution (20% (w / v) dextran sulfate 500 (Sigma) 34 ml, 20% (w / v) polyethylene glycol 6000 (Wako) 23 ml, 3M sodium chloride 10 ml), and then the mixture. 100 ml of the upper layer liquid (0.15 g of dextran sulfate 500, 35.15 g of polyethylene glycol 6000, 8.75 g of sodium chloride, 500 ml of distilled water) was added and mixed for 5 minutes, and then the mixture was allowed to stand for 40 minutes. The upper layer was removed, 100 ml of a new upper layer solution was added, and the inclusion body was purified by repeating the operation of mixing / stationary / removing the upper layer five times. The lower layer was recovered, subjected to ammonium sulfate precipitation, and the precipitate (inclusion body) was recovered by centrifugation. The inclusion bodies were washed four times with distilled water, and the recovered inclusion bodies were suspended in distilled water and stored at −20 ° C. until use.

2.6. Evaluation of insecticidal activity of B282 inclusion bodies against dipteran insects The insecticidal activity of Culex pipiens molestus , Anopheles stephensi and Aedes aegypti on second-instar larvae is derived from B282 and Islaerensis strains The inclusion body was determined.
That is, each of the 20 larvae was placed in a 6-well plate containing 5 ml of deionized water. Purified inclusion bodies diluted to various protein concentrations were administered to each well. 25 ° C., after 24 hours of incubation, measuring the number of survival larvae in each well, Probit method ^{(Finney, Probit Analysis, 2 nd} edn London:. Cambridge University Press (1952).) Were calculated LC ₅₀ at .
The results are shown in Table 2.

For all dipteran insects tested, B282 inclusion bodies showed more insecticidal activity than Isla Ellensis inclusion bodies. In particular, the insecticidal activity of B282 inclusion bodies against Chikaeka was very strong, four times that of Isla Elensis inclusion bodies (LC ₅₀ comparison).

Example 3: Analysis of a novel insecticidal protein
3.1. Examination of protein composition contained in inclusion body of B282 In order to clarify the protein composition contained in the inclusion body of B282, SDS-polyacrylamide gel electrophoresis was performed. Isla Elensis was used as a control.
That is, 2.5. The inclusion body was purified from B282 and Isla Elensis by the method described in 1. Using the 10% separation gel and the 4% concentrated gel by the Laemmli method (UK Laemmli, Nature 227: 680-685 (1970)). Electrophoresis was performed under reducing conditions (β-mercaptoethanol). Molecular weight was determined by comparison with SDS-PAGE molecular weight standard (Bio-Rad). Quick-CBB (Wako Pure Chemical Industries, Ltd.) was used for staining.

The results are shown in FIG.
B282 contained proteins in the inclusion body corresponding to Isla Elensis insecticidal proteins Cry11A (68 kDa) and Cyt1A (28 kDa), but Cry4A (125 kDa), Cry4B (135 kDa) There was no protein of a size corresponding to. In addition, the inclusion body of B282 had a protein of about 75 kDa that the inclusion body of Isla Elensis did not have. From the above, it was shown that the inclusion body of B282 has a protein having a composition different from that of the Isla Ellensis inclusion body.

3.2. Examination of plasmid composition of B282
Using the QIAGEN Plasmid Kit (QIAGEN), all plasmids were extracted and purified from B282 and Isla Elensis, and subjected to 0.7% agarose electrophoresis.
The results are shown in FIG.
It was shown that the composition of the constitutive plasmid carried by B282 is very different from that of Isla Elensis.

3.3. Study on insecticidal protein gene of B282 (by PCR method)
In order to examine whether or not B282 retains the insecticidal protein gene retained by Isla Elensis, PCR was performed using primers that specifically amplify each gene. The conditions for PCR and electrophoresis are as follows.
Primers: Primers that specifically amplify the insecticidal protein genes ( cry4A , cry4B , cry10A , cry11A , cyt1A , cyt2B ) retained by Isla Elensis (JE Ibarra et al. Appl. Environ. Microbiol. 69: 5269 -5274 (2003)).
・ Reaction solution composition (in 50 μl): Premix Taq (EX Taq Version) (Takara Bio) 25 μl, primer 0.5 μM, template plasmid (derived from the aforementioned B282 or Isla Elensis) 10 ng
-Reaction conditions: Preheating 94 ° C 3 minutes, 30 cycles (thermal denaturation 94 ° C 30 seconds, annealing 60 ° C 30 seconds, extension 72 ° C 60 seconds), cooling 4 ° C
3% agarose electrophoresis The results are shown in FIG.
If the B282-derived plasmid as a template, cry11A, cyt1A, although amplification product was obtained with primers that specifically amplify the cyt2B, cry4A, cry4B, amplification product was not obtained by the primers for Cry10A.

3.4. Study on the insecticidal protein gene of B282 (by Southern blotting method)
In order to investigate whether B282 holds the insecticidal protein gene held by Isla Ellensis, and to analyze the structure of the plasmid, Southern blotting was performed using probes that specifically hybridize to each gene. .
That is, all plasmids derived from B282 and Isla Elensis were treated with restriction enzymes APaL I or Nco I and separated by 0.7% agarose electrophoresis. The isolated plasmid DNA was transferred to Hybond-N + nylon membrane using 0.4M NaOH transfer buffer. 3.3. PCR products were used as probes. Probe labeling, hybridization and detection were performed using reagents and methods of ECL Labeling and Detection System (GE Healthcare Bioscience).
The results are shown in FIG.
When the PCR products of cry4A , cry4B , and cry10A were used as probes, no band was detected from B282. On the other hand, when cry11A , cyt1A , and cyt2B PCR products were used as probes, bands were detected from B282. However, the detected band size was significantly different from that of Isla Elensis.

From the above, Isla Elensis holds six insecticidal protein genes ( cry4A , cry4B , cry10A , cry11A , cyt1A , cyt2B ) in one plasmid pBtoxis, whereas B282 is Isla Elensis cry11A. It was suggested that cyt1A and cyt2B- like insecticidal protein genes are retained, and that the structure of the B282 plasmid encoding the genes is significantly different from that of Isla Elensis.

3.5. Analysis of nucleotide sequence of B282-derived cry11A, cyt1A or cyt2B-like insecticidal protein gene
The entire B282 plasmid was extracted and purified using the QIAGEN Plasmid Kit (QIAGEN). Using the plasmid as a template, each gene was amplified by PCR, and the nucleotide sequence of the amplified product was determined. The used primers are as follows.
Primer set for cry11A-like gene:
5'-ATGGAAGATAGTTCTTTAGAT-3 '(SEQ ID NO: 10)
5'-CTACTTTAGTAACGGATT-3 '(SEQ ID NO: 11)
Primer set for cyt1A-like gene:
5'-CCTCAATCAACAGCAAGGGTTATT-3 '(SEQ ID NO: 12)
5'-TGCAAACAGGACATTGTATGTGTAATT-3 '(SEQ ID NO: 13)
Primer set for cyt2B-like gene:
5'-ATTACAAATTGCAAATGGTATTCC-3 '(SEQ ID NO: 14)
5'-TTTCAACATCCACAGTAATTTCAAATGC-3 '(SEQ ID NO: 15)
The PCR reaction solution composition, reaction conditions, and nucleotide sequence determination method were based on the nucleotide sequence analysis of 16S rRNA gene.

The nucleotide sequences of PCR amplification products of the cry11A- like gene, cyt1A- like gene, and cyt2B- like gene are shown in SEQ ID NOs: 1, 3, and 5, and the deduced amino acid sequences derived from the nucleotide sequences are shown in SEQ ID NOs: 2, 4, and 6, respectively. In addition, Tables 3 to 5 show a comparison between each nucleotide sequence and a sequence derived from Isla Elensis.
The amplified fragment of B282 cry11A- like gene had 99.3% homology with Isla Elensis cry11A gene at the nucleotide sequence level and 99.0% at the amino acid sequence level.
The amplified fragment of the B282 cyt1A- like gene had 98.6% homology with Isla Ellensis cyt1A gene at the nucleotide sequence level and 95.7% at the amino acid sequence level.
The amplified fragment of the B282 cyt2B- like gene had a homology of 99.7% at the nucleotide sequence level and 99.0% at the amino acid sequence level with the Isla Elensis cyt2B gene.

In Table 3, (1) shows the nucleotide sequence of the PCR fragment amplified with the cry11A specific primer using the B282 plasmid as a template, and (2) shows the corresponding nucleotide sequence of Isla Elensis cry11A .
In Table 4, (1) shows the nucleotide sequence of the PCR fragment amplified with the cyt1A- specific primer using the B282 plasmid as a template, and (2) shows the corresponding nucleotide sequence of Isla Elensis cyt1A .
In Table 5, (1) shows the nucleotide sequence of the PCR fragment amplified with the cyt2B specific primer using the B282 plasmid as a template, and (2) shows the corresponding nucleotide sequence of Isla Elensis cyt2B . The nucleotide sequence derived from B282 in Table 5 corresponds to the sequence of the complementary strand of the nucleotide sequence represented by SEQ ID NO: 5.

  The Bacillus bacterium of the present invention and inclusion bodies thereof are useful as insecticides for dipteran insects because they have strong toxicity to dipterous insects, particularly squid.

It is a phase-contrast micrograph of B282 after spore formation. The phylogenetic tree of B282 based on the 16S rRNA gene sequence is shown. The result of having analyzed the protein structure of the inclusion body by SDS-PAGE is shown. (M) Molecular weight standard; (1) Isla Elensis; (2) B282. The examination result of a plasmid composition is shown. (M) Molecular weight marker; (1) Isla Elensis; (2) B282. The results of agarose gel electrophoresis of PCR products with primers specific for each insecticidal protein gene are shown. (M) Molecular weight marker; (1) Isla Elensis; (2) B282. The analysis result of the position of each insecticidal protein gene on a plasmid is shown. (A) Construction of a plasmid fragment cleaved with a restriction enzyme on a 0.7% agarose gel. (B), (C), (D), (E), (F) and (G) show the results of Southern hybridization with cry4A , cry4B , cry10A , cry11A , cyt1A and cyt2B specific probes, respectively. Lane M: Molecular weight marker Lane 1: ApaL I treated with Isla Ellen cis-derived plasmid fragments Lane 2: APaL I B282 derived plasmid fragments were treated with Lane 3: Nco Isla treated with I Ellen cis-derived plasmid fragments Lane 4 : B282-derived plasmid fragments treated with Nco I

SEQ ID NO: 8: Primer for 16S rRNA gene SEQ ID NO: 9: Primer for 16S rRNA gene SEQ ID NO: 10: Primer for cry11A SEQ ID NO: 11: Primer for cry11A SEQ ID NO: 12: Primer sequence for cyt1A Number 13: Primer for cyt1A SEQ ID NO: 14: Primer for cyt2B SEQ ID NO: 15: Primer for cyt2B

Claims (11)

  Bacillus bacteria represented by accession number FERM P-20949.   The inclusion body of the genus Bacillus represented by accession number FERM P-20949.   A composition comprising a Bacillus bacterium represented by accession number FERM P-20949 or an inclusion body thereof.   The composition according to claim 3, which is an insecticide for dipteran pests.   The composition according to claim 4, wherein the Diptera pest is Culex.   A method for controlling Diptera pests, which comprises applying a Bacillus genus bacterium represented by accession number FERM P-20949 or an inclusion body thereof in a larval habitat of Diptera pests.   7. The method of claim 6, wherein the Diptera pest is Culex. The following (a), (b) or (c) polypeptide:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4;
(C) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6.   A polynucleotide encoding the polypeptide of claim 8.   An expression vector comprising the polynucleotide according to claim 9.   A transformant comprising the expression vector according to claim 10.