??? ?????? ?? ??(Bombyx mori)??? Bacillus? ?? (Bacillus thuringiensis : Bt)

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2003?? ???????????? ?? ??? ????
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2004. 01. 13
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Introduction
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Bacillus thuringiensis ? d-endotoxin? ?? ?? ??
??? ??

• ??? ?????? ?? ??(Bombyx mori)??? Bacillus? ??
  (Bacillus thuringiensis Bt)
• Gram-positive bacteria
• ????? insecticidal crystal protein ??
• d-endotoxin ICPs(Insecticidal crystal proteins)
• Cry1, Cry2, Cry3, Cry4 - Toxic
• cytolysin(cyt) Non-Toxic

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Bacillus thuringiensis ? d-endotoxin? ?? ?? ??
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• ?? ?? ???? RNA polymerase? ??? ??? ??, ????? ????
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• ????? ??? ???? GTP ??? ?? ??
• d-endotoxin? ??? ???? ??
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1
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delta-toxin? ?? ? ??
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Crystal protein ????
SDS-PAGE
cry gene profile ??
Multiplex PCR
Endospore ? crystal protein ???? ??
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Isolation of Bacillus thuringiensis Strain
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Insect Bioassay for B. thuringiensis Selection
M. Domestica (larvae) were obtained from a pig
farm near Chungju city (Korea)
Subcultured Bacillus thuringiensis k-1
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Table 1. Bio-toxicity of Bacillus thuringiensis
k-1 against fly larvae (M. domestica)
B. thuringiensis strains No. of larvae tested No. of the dead Mortality ()
Control 30 0 0
Bt k-1 (original amount) 30 20 66.7
Bt k-1 (supernatant ) 30 25 83.3
Larvae mortality was determined after incubation at room temperature (252?) for 72h using 105 to 106 spores/ml.  Larvae mortality was determined after incubation at room temperature (252?) for 72h using 105 to 106 spores/ml.  Larvae mortality was determined after incubation at room temperature (252?) for 72h using 105 to 106 spores/ml.  Larvae mortality was determined after incubation at room temperature (252?) for 72h using 105 to 106 spores/ml. 
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Growth curve of Bacillus thuringiensis k-1
Use of Media Nutrient Broth (NB, 0.3 beef
extract, 0.5 peptone) Culture Condition 48hr,
37?
Stationary phase
Fig. 1. Growth curve of Bacillus
thuringiensis strain k-1
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Biochemical Characteristics of B. thuringiensis
k-1

• Use of Identification Kit API CHB 50 kit and
  Medium
• Culture Condition 24hr, 37?

Table 2. Morphological and biochemical properties
of Bacillus thuringienis k-1
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Table 3. Biochemical characteristics (carbohydrates) of Bacillus thuringiensis k-1 page 1 Table 3. Biochemical characteristics (carbohydrates) of Bacillus thuringiensis k-1 page 1 Table 3. Biochemical characteristics (carbohydrates) of Bacillus thuringiensis k-1 page 1 Table 3. Biochemical characteristics (carbohydrates) of Bacillus thuringiensis k-1 page 1
Characteristics B. thuringiensis k-1 Cahracteristics B. thuringiensis k-1
Glycerol Salicine
Erythritol - Cellobiose
D-Arabinose - Maltose
L-Arabinose - Lactose -
Ribiose Melibiose -
D-Xylose - Saccharose
L-Xylose - Trehalose
Adonitol - Inuline -
Beta-Methyl-xyloside - Melezitose -
Galactose - D-Raffinose -
D-Glucose Amidon
D-Fructose Glycogen
D-Mannose Xylitol -
L-Sorbose - Beta-Gentiobiose -
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page 2 page 2 page 2 page 2
Characteristics B. thuringiensis k-1 Cahracteristics B. thuringiensis k-1
Rhamnose - D-Turanose -
Dulcitol - D-Lyxose -
Inositol - D-Tagatose -
Mannitol - D-Fucose -
Sorbitol - L-Fucose -
Alpha-Methyl-D-mannoside - D-Arabitol -
Alpha-Methyl-D-Glucoside - L-Arabitol -
N Acetyl glucosamine Gluconate -
Amygdaline 2 aceto-gluconate -
Arbutine 5 aceto-gluconate -
Esculine
, Positive -, Negative , Positive -, Negative , Positive -, Negative , Positive -, Negative
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Electron Microscopy (TEM, Transmission Electron
Micrographs)
Strain k-1 was grown for 3days in GYS medium at
30 ?
Sample was blocked in 4 agar
Sample was fixed with Karnowskys fixative and
then postfixed in 1 OsO4
Sample was dehydrated through an
ethanol-propylene oxide series and embedded in
Epon 812
Fig. 2. Transmission electron micrographs of
sporulating of Bacillus thuringiensis k-1
amplified 25,000 (A). Parasporal crystal (c) and
spore (e).
Thin sections were cut on ultramicrotome, stained
with 1 uranyl acetate and lead citrated
Photographed
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Antibiotics susceptibility of B. thuringiensis k-1
The test was performed with the use of serial
2-fold dilutions of each antibiotic as described
by Cleeland et al. The resistance of Bacillus
thuringiensis k-1 against antibiotics was
examined after cultivation for 18hr at 37 ?.
Inoculation of Bacillus thuringiensis
antibiotics
1.95ug
0ug
1.95ug
0ug
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Table 4. Determination of minimum inhibitory
concentration of various antibiotics against B.
thuringiensis k-1, B. thuringiensis subsp.
kurstaki (HD-1) and B. thueingiensis subsp.
israelensis (HD-522).
Strain k-1 Kurstaki (HD-1) Israelensis (HD-522)
Ampicillin 62.5 100 100
Kanamycin lt1.95 12.5 3.9
Oxacillin 62.5 250 7.8
Colistin 31.3 125 62.5
Tetracycline 7.9 6.25 3.9
Penicillin G 125 gt1000 gt1000
Erythromycin 250 lt1.95 lt1.95
Neomycin 7.9 3.9 3.9
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Purification of insecticidal proteins from strain
k-1
B. thuringiensis was grown for 3 days in the GYS
medium at 30? and 180rpm until complete autolysis
was achieved. Purification crystal proteins was
carried out by discontinuous sodium bromide
(NaBr) gradients of 30 to 70.
130kD
80kD
60kD
Fig. 3. SDS-PAGE of Bacillus thuringiensis k-1
crystal proteins purified. M, molecular marker
arrows indicate Cry proteins of 130, 80,
60kDa.
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Multiplex PCR for rapid determination of cry genes
The extended multiplex PCR screening is a rapid
method for detection and differentiation of
Bacillus thuringiensis field strains and for
prediction their insecticidal activities in order
to direct them for subsequent toxicity assays
against Lepidoptera, Coleoptera, and Diptera. The
10 primers contained eight forward primers and
two reverse primers for cry1 gene determination
used in this study. To identify cry4, one forward
and one reverse primer used in reaction.
Table 5. The optimal conditions of polymerase
chain reaction (PCR).
Composition Vol. unit Temp. Time cycle
Template 15ul 95 2min 1cycle
Primer (F) each 1ul 8ul 200uM
Primer (R) each 1ul 2ul 200uM 95 1min
10X buffer 8ul 52 2min 30cycle
Ex Taq. 0.5ul 0.5U 72 3min
2.5mM dNTPs 4ul 200uM
dH2O 12.5ul 72 7min 1cycle
Total volume 50ul Total volume 50ul
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Fig 4. PCR survey of B. thuringiensis k-1 and B.
thuringiensis subsp. kurstaki for cry1 contents.
Total DNA samples from B. thuringiensis k-1 and
B. thuringiensis subsp. kurstaki were analyzed by
PCR with a mixture of cry1 specific primers. Lane
M Bio basic 100bp-1kb ladder lane 1, B.
thuringiensis k-1 lane 3, B. thuringiensis
subsp. kurstaki   M Bio basic 100bp-1kb ladder
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Culture conditions for viable cell growth and
endospore formation (crystal protein)
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Table 6. Viable cell and endospore forming cell
in various culture conditions


( CFU/ml )
Time Media Viable cell Endospore
NB 2.0x108 5.8x104
MED2 1.2x108
8 BHI 4.0x108
GYS 4.6x104
PGY 2.4x108
NB 1.4x108 4.0x105
MED2 1.8x108
12 BHI 2.6x108 1.8x105
GYS 8.2x106 5.3x105
PGY 1.3x108
NB 3.6x106 1.7x106
MED2 6.4x105
16 BHI 1.1x107
GYS 1.7x107 2.0x106
PGY 1.5x106
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Enzyme activities of Bacillus thuringiensis k-1
Table. 7 Screening media and dyes used for the
detection of various enzyme activities
Enzyme Substrate (basal medium NA1) Dye Identification
Protease 2(w/v) skim milk - Clear zone
Cellulase(CMCase) 1(w/v) CMC2 0.2(w/v) congo red sol. Clear zone
Amylase 1(w/v) soluble starch 0.2(w/v) I22(w/v) KI sol. Clear zone
Xylanase 1(w/v) oat spelt xylan - Clear zone
1Nutrient agar (0.3 beef extract, 0.5 peptone,
1.5 agar)
2Carboxymethyl cellulose (medium viscosity)
Table. 8 Enzyme activities of Bacillus
thuringiensis k-1
Items Xylasnase Protease Cellulase Amylase
Enzyme activity
Size of clear zone(mm) (Width of colony / Width of clear zone) 6.5/12.0 5.0/9.0 5.0/5.5 5.0/10.0
represents excellent clear zone formation
good fair
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Isolation of the strain having multi-enzyme
activities
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Enzyme activities of Bacillus sp. strains
Table. 9 Screening media and dyes used for the
detection of various enzyme activities
Enzyme Substrate (basal medium NA1) Dye Identification
Protease 2(w/v) skim milk - Clear zone
Cellulase(CMCase) 1(w/v) CMC2 0.2(w/v) congo red sol. Clear zone
Amylase 1(w/v) soluble starch 0.2(w/v) I22(w/v) KI sol. Clear zone
1Nutrient agar (0.3 beef extract, 0.5 peptone,
1.5 agar)
2Carboxymethyl cellulose (medium viscosity)
Fig 5. Protease, cellulase, and amylase
activities of 1st isolated strains
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Table 10. Enzyme activities of 1st-selected
microorganisms
Enzyme activity3 Enzyme activity3 Enzyme activity3
Strain Growth1,2 at 37? CMCase Protease Amylase
2-4 -
2-5 -
3-1 -
3-3 -
5-1
6-7
6-9
7-3
7-10 -
7-12 -
8-5
8-7 -
9-1 - -
9-3 - - -
9-6 - -
10-4 - - -
1The rate of growth was evaluated after 12hr on
nutrient agar
2 represents excellent growth
3 represents excellent clear zone formation on
screening medium
good, fair, - no clear zone formation
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Fig 5. Protease, amylase, and cellulase
activities of the finally isolated 5-1
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Identification of the selected strain

• Use of Identification Kit API CHB 50 kit and
  Medium
• Result Bacillus subtilis

Table 11. Morphological and biochemical
properties of 5-1
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Table 12. Biochemical characteristics (carbohydrates) of 5-1 page 1 Table 12. Biochemical characteristics (carbohydrates) of 5-1 page 1 Table 12. Biochemical characteristics (carbohydrates) of 5-1 page 1 Table 12. Biochemical characteristics (carbohydrates) of 5-1 page 1
Characteristics 5-1 Cahracteristics 5-1
Glycerol Salicine
Erythritol - Cellobiose
D-Arabinose - Maltose
L-Arabinose Lactose -
Ribiose Melibiose
D-Xylose Saccharose
L-Xylose - Trehalose
Adonitol - Inuline
Beta-Methyl-xyloside - Melezitose -
Galactose - D-Raffinose
D-Glucose Amidon
D-Fructose Glycogen
D-Mannose Xylitol -
L-Sorbose - Beta-Gentiobiose -
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page 2 page 2 page 2 page 2
Characteristics 5-1 Cahracteristics 5-1
Rhamnose - D-Turanose
Dulcitol - D-Lyxose -
Inositol D-Tagatose -
Mannitol D-Fucose -
Sorbitol L-Fucose -
Alpha-Methyl-D-mannoside - D-Arabitol -
Alpha-Methyl-D-Glucoside L-Arabitol -
N Acetyl glucosamine - Gluconate -
Amygdaline 2 aceto-gluconate -
Arbutine 5 aceto-gluconate -
Esculine
, Positive -, Negative , Positive -, Negative , Positive -, Negative , Positive -, Negative
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???? ??? ?? ?? ?? ??? ??
? ?PART I

• ????? ??? ???? d-endotoxin ?? B. thuringiensis ??
  ?? ? ??(????, ???? ????) ??
• d-endotoxin ??? ??, ???? ? ?? ??? ?? ??
• ???? ????? ?? chitinase ?? ??? ?? ??

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???? ??? ???? ?? ??? ??
? ?PART II

• ? ??? ???? ?? ??? ?? ????? ?? ????? ?? ???? ? ???
  ??? ???? ??? ?? ? ?? ???? ?? ??? ???? ?? ??? ??
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• ????? ?? ???? urease??? ?? (?????? ?? ??) 5-1 ??
  ???? ? ????(Bacillus subtilis)
• ????? ???? ???? Denitrobacter sp. ? ????? ??
  ????? ? ????? ??????? ????? ????
  ?????(photosynthetic bacteria) ??

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