Bacillus thuringiensis

Presented By :
Jasmine Kaur
J-12-biotech-46
Bacillus thuringiensis (or Bt) is a facultative
anaerobic, gram-positive, soil-dwelling
bacterium, commonly used as a biological
alternative to a pesticide; alternatively, the Cry
toxin may be extracted and used as a
pesticide.
 Characteristics of Bt :

• Bt subspecies can synthesize more than one

parasporal inclusion. The parasporal inclusions are
formed by different insecticidal crystal proteins
(ICP).
• The crystals have various shapes (bipyramidal,
cuboidal, flat rhomboid, spherical or composite
with two crystal types), depending on their ICP
composition.
• During sporulation many Bt strains produce crystal
proteins (proteinaceous inclusions), called δ-
endotoxins (Cry proteins), which are encoded by cry
genes, and have insecticidal action. This has led to
their use as insecticides, and more recently to
genetically modified crops using Bt genes.
• In most strains of B. thuringiensis the cry genes are
located on the plasmid.
• Cry toxins have specific activities against insect
species of the orders Lepidoptera (moths and
butterflies), Diptera (flies and mosquitoes),
Coleoptera (beetles), hymenoptera (wasps,
bees, ants and sawflies) and nematodes.
• Different domains of the ICP are responsible for
host susceptibility (receptor recognition) and
toxicity (pore formation).
Scientific Classification:
Kingdom: Eubacteria
Phylum: Firmicutes
Class : Bacilli
Order : Bacillales
Family : Bacillaceae
Genus : Bacillus
Species: thuringiensis
(Berliner 1915)
 Genome structure
B. thuringiensis has a circular chromosome
and a GC-content of approximately
32%~35%. It has a genome size of between
5.2–5.8 Megabases. It is a facultative
anaerobic organism. It has many plasmids
and Bt's strains harbors a diverse range of
plasmids that vary in number and in size (2–
200kb).
 Cell structure and
metabolism
• B. thuringiensis is gram-positive.
• it has a thick cell wall that is comprised of
peptidoglycan (amino acid polypeptide and a sugar).
Between the cell wall and the plasma membrane is a
small section called the periplasmic space which is
essential for biosynthesis and protection.
 History :
• B. thuringiensis was first discovered in 1901 by
Japanese biologist Shigetane Ishiwata, most
abundantly found in grain dust from silos and
other grain storage facilities.
• In 1911, B. thuringiensis was rediscovered in
Germany by Ernst Berliner, who isolated it as the
cause of a disease called Schlaffsucht (excessive
sleeping) in flour moth caterpillars, collected in
the German province of Thuringia.
• Bt first became available as a commercial
insecticide in France in 1938, and in the
1950s it entered commercial use in the USA.

• Whalon and McGaughey in 1998 showed

that each strain of Bt produces a unique
crystal protein which is encoded by a single
gene located in the plasmid.
Gene Crystal shape Protein Insect
size(kDa) activity
cry I ………….. Bipyramidal 130-138 lepidoptera
[ subgroups: larvae
A(a), A(b), A(c), B, C, D,
E, F, G]
cry II …………. Cuboidal 69-71 lepidoptera
[subgroups A, B, C] and diptera

cry III….……… flat/irregular 73-74 coleoptera

[subgroups A, B, C]

cry IV………… Bipyramidal 73-134 diptera

[subgroups A, B, C, D]

cry V-IX Various 35-129 various

 Habitats :
• Many different Bt subspecies have been isolated
from dead or dying insects mostly from the orders
Coleoptera, Diptera and Lepidoptera, but many
subspecies have also been isolated from soil, leaf
surfaces and other habitats.
• The carcasses of dead insects often contain large
quantities of spores and ICPs that may enter the
environment.
 Isolation and Culture :
For Soil Sample :
• One gram of each sample is suspended in 10 ml sterile
distilled water and pasteurized at 800C for 30 min.
• For the selection of B. thuringiensis, 1 ml of each
suspension is added to 10 ml of Luria-Bertani broth
buffered with 0.25 M sodium acetate pH 6.8.
• The suspensions are incubated at 30oC for 4 h and then
heated at 80oC for 3 min.
• Suspensions were diluted and plated on T3 medium
(per liter: 3 g tryptone, 2 g tryptose, 1.5 g yeast
extract, 0.05 M sodium phosphate pH 6.8, and 0.005
g of MnCl2).
• After incubation at 30oC for 24 h, the colonies
showing similar morphology were selected and
examined under phase-contrast microscope to
determine the presence of parasporal inclusions
and spores.
For Leaf Sample :
• The leaves from different horticultural crops are
washed gently in sterile distil water to remove
dust and superficially adhering microflora.
• Then, leaves are put inside a 250mL conical flask
containing 100mL sterile distil water and rotated
at 250rpm, at 300C for 5hrs.
• The suspension was poured into sterile
centrifugation tubes and centrifuged at
10,000rpm, at 40C for 15mins.
• The pellets are washed with 5mL of LB broth buffered
with 0.25M Na-acetate and the entire contents
poured into a 100mL conical flask containing 5mL of LB
broth buffered with 0.25M Na-acetate and rotated in a
rotary shaker at 250rpm, at 300C for 4hrs.
• NOTE : The procedure given for soil sample can be
followed for leaf, seed dust and other samples also.
 Mode of Action of Bt:
• The sporulated Bt with ICP or spore-ICP complexes
must be ingested by a susceptible insect larva followed
by solubilisation, and processing from a protoxin to an
activated toxin core in the insect digestive fluid.
• The toxin core travels across the peritrophic matrix and
the C-terminal region binds to specific receptors called
cadherins on the brush border membrane of the gut
cells, resulting in pore formation by the N-terminal
domain.
• Accumulation of toxin oligomers results in toxin
insertion in the membrane, pore formation, osmotic cell
shock, septicaemia and ultimately insect death.
Limitations of Bt Sprays

•Poor Coverage
•Low efficiency
•UV degradable
THANKYOU