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 ICPcomposition.

• During sporulation many Bt strains produce crystal proteins (proteinaceous inclusions), called δ-endotoxins (Cry proteins), which are encoded by crygenes, 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 plasmidsand 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

size(kDa)

Insect

activity

cry I …………..

[ subgroups:

A(a), A(b), A(c), B, C, D,

E, F, G]

Bipyramidal 130-138 lepidoptera

larvae

cry II ………….

[subgroups A, B, C]

Cuboidal 69-71 lepidoptera

and diptera

cry III….………

[subgroups A, B, C]

flat/irregular 73-74 coleoptera

cry IV…………

[subgroups A, B, C, D]

Bipyramidal 73-134 diptera

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

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