BACILLUS THURINGIENSIS 2013.ppt

BACILLUS THURINGIENSIS 2013

1. BACILLUS THURINGIENSIS -
BIOLOGY, CLASSIFICATION,
MODE OF ACTION
Presented By
Dr. RajibKumar De
Principal Scientist
Crop Protection Division
I.C.A.R.-C.R.I.J.A.F. ,
Barrackpore.

2. Bacillus thuringiensis: Potential Microbial
Biopesticide
INTRODUCTION
Bacillus thuringiensisis Gram + ve bacteria.
Belongs to Genus 2, Bacillus in Group 18 in
Bergy’s Manual of Determinative Bacteriology
(9
th
edition.)
Special feature of Btis it produces
proteinaceous crystal in cell during
sporulation, which in turn produces a toxin
known as delta endotoxin.
According to variation in flagellum antigen
and other characteristics 70 serotypes and 83
sub spp are identified (Lacedet et al 1991.)

3. History of Bacillus thuringiensis.
Japanese biologist, Shigetane Ishiwatari while investigating the cause of the sotto disease
(sudden-collapse disease) in silkworms was first to isolate the bacterium Bacillus
thuringiensis(Bt) as the cause of the disease in 1901.
Ernst Berliner isolated a bacteria that had killed a Mediterranean flour moth in 1911, and
rediscovered Bt. He named it Bacillus thuringiensis, after the German town Thuringia where
the moth was found.
Ishiwatari had named the bacterium Bacillus sottoin 1901 but the name was later ruled
invalid.
In 1915, Berliner reported the existance of a crystal within Bt, but the activity of this crystal
was not discovered until much later.
Farmers started to use Btas a pesticide in 1920. France soon started to make
commericialized spore based formulations called Sporine in 1938. Sporine, at the time was
used primarly to kill flour moths.
In 1956, researchers, Hannay, Fitz-James and Angus found that the main insecticidal activity
against lepidoteran (moth) insects was due to the parasporal crystal.
In the US, Btwas used commercially starting in 1958.
By 1961, Btwas registerd as a pesticide to the EPA.

4. Biology of Bacillus thuringiensis
Upon sporulationB. thuringiensis, forms crystals of
proteinaceousinsecticidal δ-endotoxins(called crystal
proteins or Cry proteins), which are encoded by cry
genes.
In most strains of B. thuringiensis, the crygenes are
located on a plasmid(in other words, cryis not a
chromosomal gene in most strains).
These plasmids are usually 1-12 in number and are of
molecular weight 2-200kb.
The crystal genes also produce VIP’s, Thuringiensin
(beta-exotoxin).

5. CLASSIFICATION OF BACILLUS THURINGIENSIS
Cry toxin Insect Order
Cry I Lepidoptera
Cry II Lepidoptera & Diptera
Cry III Coleoptera
Cry IV Diptera
In 1989, Hofte and Whiteley based on diversity of amino
acid sequence and toxicity divided 42 crystal proteins into 5
groups that were further divided into 14 sub groups (13 Cry
sub groups and 1Cyt.)

6. Number of genes
32 Cry genes 113 Cry genes
2 Cytgenes
Till September 28, 2011, 280 ICP genes belonging to 34 classes have
been reported which includeGene
Crystal
shape
Protein
size(kDa)
Insect activity
Cry I [several 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

7. NEW CLASSIFICATION OF BACILLUS
THURINGIENSIS
Cry I of Hofteand Whiteleywas renamed from Cry1A to Cry1K
Cry II of Hofteand Whiteleywas renamed from Cry2A(sub groups
a, b, c)
Cry III of Hofteand Whiteleywas renamed from Cry3A(except for
CryIIIC renamed as Cry7A)
Cry IV of Hofteand Whiteleywas renamed from Cry4A(except for
Cry IV C renamed as Cry10A and Cry IV D renamed as Cry11A)
For further information regarding Bt classification please visit
http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/

8. STRUCTURAL MORPHOLOGY OF CRY PROTEINS
Cry proteins are composed of 1,100-1,200 amino-acids of 130-
140kDa mol.wt.
Full length of the cry protein is not required for exerting toxicity.
The toxin has two terminals: N-terminal and C-terminal.N-terminal C-terminal
Hydrophobic
Forms alpha –helix
Contributes toxicity
High diversity in amino acid sequence and
sequence homology is about 40-90%
Hydrophilic
Forms beta –sheets
Mainly supports formation of parasporal
crystals
Highly conserved amino acid sequence and
sequence homology is 90%

9. Relative length of Cry protoxins and position of protease digestion.
White boxes represent the protoxin and striped boxes represent the activated toxin.
Solid arrows show the amino-and carboxy-terminal cleavage sites of the activated toxins.
Doted arrows show the intramolecular cleavages.
Cleavage of Cry1A at residue 51 resulted in loss of helix α-1 and pre-pore formation.
Cleavage of Cry4B resulted in two fragments of 18 and 46 kDa,
while Cry11A resulted in two fragments of 34 and 32 kDa.

10. TOXIN ACTIVATION
1-28
th
/29
th
amino acid residues from N-terminal
side and 600
th
-1150
th
amino acid residues from
C –terminal is cleaved by trypsin enzymes of
the insect mid gut to form a toxin core of
60kDa.

11. Recent studies on the delta-endotoxin structure show that it has three
domains.
Domain Iis a bundle of 7 alpha-helices, some or all of which can
insert into the gut cell membrane, creating a pore through which ions
can pass freely.
Domain IIconsists of three antiparallel beta-sheets, similar to the
antigen-binding regions of immunoglobulins, suggesting that this
domain binds to receptors in the gut.
Domain IIIis a tightly packed beta-sandwich which is thought to
protect the exposed end (C-terminus) of the active toxin, preventing
further cleavage by gut proteases.

12. Three dimensional structures of insecticidal toxins produced by Bacillus
thuringiensisCry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa, Cry4Bb and Cyt2A.

13. MODE OF ACTION OF Bt.
1.pH requirement of alkaline condition.
2.Release of toxin
1.Solubilisation of toxins by breaking the disulphide
linkages
2.Activation of protoxinto active toxin
3.Binding of active toxin to BBMV’s receptors mainly
cadherins, APN, ALP, GPI(glycosylated phosphotidyl
inositol) of midgutepithelial cells.
4.Formation of pores in cell membrane (two models are
proposed)
1.Penknife model.
2.Umbrella model.
5.Cell lysis(two theories are proposed)
1.Proton Perilltheory
2.Osmotic cell lysistheory.

14. Receptor molecules of Cry1A proteins. CADR, cadherin receptor; APN,
aminopeptidase-N, ALP, alkaline phosphatase, GCR, 270 kDa glyco-conjugate
receptor.

15. Schematic representation of Btmode
of action

16. Mode of action of Bt

17. Model of the mode of action of Cry and Cyttoxins. Panel A, sequential interaction of Cry toxins with
different receptor molecules in lepidopteranlarvae.
(1) Solubilizationand activation of the toxin; (2). Binding of monomeric Cry toxin to the first receptor
(CADR or GCR), conformational change is induced in the toxin and α-helix 1 is cleaved;
(3) Oligomer formation; (4) Binding of oligomerictoxin to second receptor (GPI-APN or GPI-ALP), a
conformational change occurs and a molten globule state of the toxin is induced;
(5) insertion of the oligomerictoxin into lipid rafts and pore formation.
Panel B, role of Cytand Cry toxins in the intoxication of dipteran larvae. (1) Cry and Cyttoxins are
solubilized and activated; (2) Cyttoxin inserts into the membrane and Cry toxin binds to receptors located in
the membrane (ALP or Cyttoxin); (3) oligomerizationof the Cry toxin is induced; (4) oligomer is inserted
into the membrane resulting in pore formation.

18. Theories for mode of action of Bt

19. Bt: Mode of action in transgenic crops

20. Mode of action of Bt
The mode of action of the Cry1A toxin and a comparison with the Cry1AMod toxin. The main difference is
the formation of the oligomeric structure that in one case requires the interaction with the cadherin receptor
and in the other is independent of this receptor.

21. THANK YOU