B. thuringiensis is actually a very close relative of B. cereus, a common food poisoning bacterium. The main difference is that most B. thuringiensis strains carry plasmids or self-replicating extrachromosomal DNA molecules, coding for the production of the insecticidal substances. These plasmids are self-transmissible from one B. thuringiensis to another (Wilcks and others 1998) and would show even in the gut conditions clearly the highest potential of being transferred from a bacterium to another.In a plasmid pattern, two different groups of plasmids can be recognized: those that are ≤30 MDa and those that are ≥30 MDa, called megaplasmids.These Cry proteins are coded by genes (cry genes) harbored in megaplasmidsBacillus thuringiensis is a Gram-positive
Bacillus thuringiensis forms parasporal crystals during the stationary phase of its growth cycle. These crystals are specifically toxic to certain orders and species of insects, like Lepidoptera, Diptera, and ColeopteraDuring sporulation, it synthesizes a cytoplasmic inclusion containing one or more proteins that are toxic to insect larvae. Upon completion of sporulation the parent bacterium lyses to release the spore and the inclusion. In these inclusions, the toxins exist as inactive protoxins. When the inclusions are ingested by insect larvae, the alkaline pH solubilizes the crystal. The protoxin is then converted in to an active toxin after processing by the host proteases present in the midgut.It has been indicated that the activated toxin binds to insect-specific receptors exposed on the surface of the plasma membrane of midgut epithelial cells and then inserts into the membrane to create transmembrane pores that cause cell swelling and lysis and eventually death of the insect.
The Cry toxin molecules attach themselves to specific locations on the cadherin-like proteins present on the epithelial cells of the midge and ion channels are formed which allow the flow of potassium. Regulation of potassium concentration is essential and if left unchecked causes death of cells. Due to the formation of Cry ion channels sufficient regulation of potassium ions is lost and results in the death of epithelial cells. The death of such cells creates gaps in the brush border membrane. The gaps then allow bacteria and (Bt) spores to enter the body cavity resulting in the death of the organism.Cadherins (named for "calcium-dependent adhesion") are a class of type-1 transmembrane proteins. They play important roles in cell adhesion, forming adherens junctions to bind cells within tissues together. They are dependent on calcium (Ca2+) ions to function, hence their nameAlkaline phosphatase (ALP, ALKP) (EC 184.108.40.206) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is calleddephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphataseGlycoconjugates is the general classification for carbohydrates covalently linked with other chemical species such as proteins, peptides, lipids and saccharides.Glycoconjugates are very important compounds in biology and consist of many different categories such as glycoproteins, glycopeptides, peptidoglycans, glycolipids,glycosides and lipopolysaccharides. They are involved in cell–cell interactions, including cell–cell recognition; in cell–matrix interactions; in detoxification processes.Aminopeptidase N is located in the small-intestinal and renal microvillar membrane, and also in other plasma membranes. In the small intestine aminopeptidase N plays a role in the final digestion of peptides generated from hydrolysis of proteins by gastric and pancreatic proteases
Septicemia: the presence of pathogenic bacteria in the bloodstream.Even though the toxin does not kill the insect immediately, treated plant parts will not be damaged because the insect stops feeding within hours. Bt spores do not spread to other insects or cause disease outbreaks on their own.
Domain I is composed of a 7 helix bundle. This domain is the prime candidate for formation of the transmembranelytic pore. Mutations in this domain frequently yield toxins that bind receptor but fail to insert in the membrane.Domain II is believed to have a major role in receptor binding and thus in specificity determinationDomain III at the C-terminus of the molecule, is a sandwich of two twisted antiparallel beta-sheets in a so-called "jelly-roll" topology. This domain has also been implicated in the determination of insect specificity. It was originally suggested that the two twisted antiparallel beta-sheets (jelly roll) that make up domain III of the Bt toxin structure may play some role in protecting the toxins against gut proteolysis.
Delta endotoxins (δ-endotoxins, also called Cry and Cyt toxins) are pore-forming toxins produced by Bacillus thuringiensisspecies of bacteria. They are useful for their insecticidal action.During spore formation the bacteria produce crystals of this protein. When an insect ingests these proteins, they are activated by proteolytic cleavage. The N-terminus is cleaved in all of the proteins and a C-terminal extension is cleaved in some members. Once activated, the endotoxin binds to the gut epithelium and causes cell lysis by the formation of cation-selective channels, which leads to death. The activated region of the delta toxin is composed of three distinct structural domains: an N-terminal helical bundle domain (IPR005639) involved in membrane insertion and pore formation; a beta-sheet central domain involved in receptor binding; and a C-terminal beta-sandwich domain (IPR005638) that interacts with the N-terminal domain to form a channel.
Bt is susceptible to degradation by sunlight. Most formulations persist on foliage less than a week following application. Some of the newer strains developed for leaf beetle control become ineffective in about 24 hours.The highly specific activity of Bt insecticides might limit their use on Crops where problems with several pests occur, including nonsusceptible insects (aphids, grasshoppers, etc.). As strictly a stomach poison insecticide, Bt must be eaten to be effective, and application coverage must be thorough. This further limits its usefulness against pests that are susceptible to Bt but rarely have an opportunity to eat it in field use, such as codling moth or corn earworm that tunnel into plants. Additives (sticking or wetting agents) often are useful in a Bt application to improve performance, allowing it to cover and resist washing.Since Bt does not kill rapidly, users may incorrectly assume that it is ineffective a day or two after treatment. This, however, is merely a perceptual problem, because Bt-affected insects eat little or nothing before they die.Bt-based products tend to have a shorter shelf life than other insecticides. Manufacturers generally indicate reduced effectiveness after two to three years of storage. Liquid formulations are more perishable than dry formulations. Shelf life is greatest when storage conditions are cool, dry and out of direct sunlight.
ControversiesInsect ResistancePrior to the use of Bt in GMOs, issues with Bt resistance evolving in pests were unlikely due to the brief and infrequent exposures to Bt pests experienced. Following an application of Bt, the majority of pests would die, and then the Bt would degrade within a week. A farmer might apply Bt one more time, to kill any insects that hatched after the first application of Bt. Still, insects were not exposed to Bt for a very long period of time, giving them little opportunity to evolve resistance.However, with the advent and widespread use of Bt toxin by GMOs, pests have more opportunities to evolve resistance to Bt. Bt resistance in corn pests was first observed in 2011. For more information, see the article on Bt-Resistant Insects.Safety of Bt CropsA 2010 study published in the journal Reproductive Toxicology found that "3-MPPA and Cry1Ab toxin are clearly detectable and appear to cross the placenta to the fetus." 3-MPPA, or 3-methylphosphinicopropionic acid, is a metabolite of the pesticide gluphosinate ammonium, which is used in some genetically engineered crops. The Cry1Ab is the insecticidal protein produced by Bt crops. The study found that the "Cry1Ab toxin was detected in 93% and 80% of maternal and fetal blood samples, respectively and in 69% of tested blood samples from nonpregnant women." The study authors speculated this was due to consumption of contaminated meat, i.e meat from animals fed Bt corn that had retained the Cry1Ab protein in their flesh
Other concerns involve the safety of Bt transgenic crops to non-target organisms. Safety studies have demonstrated that vertebrate stomach juices rapidly inactivate Bt proteinsDr. Mezzomo and his team of Scientists from the Department of Genetics and Morphology and the Institute of Biological Sciences, at University of Brasilia recently published a study that involved Bacillus thuringensis (Bt toxin) and its effects on mammalian blood. According to the study, the “Cry” toxins that are found in Monsanto’s GMO crops like corn and soy, are much more toxic to mammals than previously thought. The study was published in the Journal of Hematology and Thromboembolic Diseases(1).Scientists tested levels ranging from 27 mg to 270 mg over a seven day period, it was remarkably evident that the Cry toxins were hemotoxic, even at the lowest doses administered. Hemotoxins destroy red blood cells, disrupt blood clotting and cause organ degeneration and tissue damage.The number of RBC’s, (red blood cells) as well as their size, were significantly reduced, and so were the levels of hemoglobin for oxygen to attach to. Every factor regarding RBC’s indicated some level of damage for all levels of toxin administered and across all cry proteins. The tests clearly demonstrated that Cry proteins resulting from the Bt toxin were cytotoxic (quality of being toxic to cells) to bone marrow cells. Studies contiually show that these proteins kill blood cells bytargeting the cell membranes of RBC’s.
Presented By M.Z.Arifeen
Department of Biotech
University of Malakand.
Date of Presentation
•Bacillus thuringiensis (Bt) is a Gram-positive, spore forming, soil bacterium.
•When resources are limited, vegetative Bt cells undergo sporulation, synthesizing
a protein crystal, the insecticidal crystal proteins (ICPs) or Cry Proteins.
•These Cry proteins are coded by genes (cry genes) harbored in megaplasmids
• For over 50 years, Bt has been applied to crops in spray form as an
insecticide, containing a mixture of spores and the associated protein crystals
• The development and commercialization of insect-resistant transgenic Bt crops
expressing Cry toxins revolutionized the history of agriculture
•High specificity and potency,
•Reduction in chemical pesticide applications,
• Increased crop yield.
• The Cry protein is made as an inactive protoxin.
• Cry protein has to be eaten to cause mortality.
• Conversion of the protoxin (e.g., 130 kDa) into the active
toxin (e.g., 68 kDa) called delta endotoxin requires the
combination of a slightly alkaline pH (7.5-8) and the action
of a specific protease(s) found in the insect gut
• The active toxin binds to protein receptors on the insect gut
epithelial cell membrane.
• The toxin forms pores in the insect gut.
B. thuringienis parasporal crystal composed of Cry1
protoxin protein. Conversion of the 130-kDa protoxin
into an active 68-kDa toxin requires an alkaline
environment (pH 7.5 to 8) and the action of a specific
protease, both of which are found in the insect gut.
The activated toxin binds to protein receptors on the
insect gut epithelial cells.
CADR= Cadherin receptor
GCR =Glyco-conjugate receptor
APN=Amino peptidase - N
Receptors for Bt Toxin
BT crystalline Toxin 200px.
Normal gut bacteria
• There are three domains in this
• Domain I and Domain II are very
• Domain III is highly variable to
attach to different receptors.
• There are over 200 known
variants for this cry or cryt
• Domain I is the prime candidate
for formation of the
transmembrane lytic pore.
• Domain II is believed to have a
major role in receptor binding
and thus in specificity
• domain III of the Bt toxin
structure may play some role in
protecting the toxins against gut
Bt action is very specific. Different strains of Bt are specific to different
receptors in insect gut wall. Bt toxicity depends on recognizing
receptors, damage to the gut by the toxin occurs upon binding to a
receptor. Each insect species possesses different types of receptors that
will match only certain toxin proteins, like a lock to a key.
The crystal proteins bind specifically to certain receptors in the
insect's intestine. Not all insects carry the same receptors allowing for
high species specificity. Humans and other vertabrates do not have these
receptors in their bodies, so the toxin is unable to affect us.
It is because of this that farmers have to be careful to match the target
pest species with a particular Bt toxin protein which is specific for that
insect. This also helps the beneficial insects because they will usually not
be harmed by that particular strain of Bt.
Japanese biologist, Shigetane Ishiwatari was
investigating the cause of the sotto disease (sudden-
collapse disease) that was killing large populations of
silkworms when he first isolated 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.
In 1915, Berliner reported the existence of a crystal
protein within Bt.
Farmers started to use Bt as a pesticide in 1920.
France soon started to make commercialized spore based formulations
called Sporine in 1938
These Bt pesticide were used in the form of spray.
Bt products such as sprays are rapidly washed away by
rain, and degrade under the sun's UV rays.
there were many insects that are not susceptible to any
of the limited number of Bt strains known at the time.
All the Bt strains known at the time were toxic to
lepidopteran (moth) larvae only.
There were also some insects that live within the plant
or underground where the Bt sprays could not reach.
• Bt plants have genes for the Bt toxins engineered to produce ICP toxic to
the pest species of concern.
• As the insect feeds on the Bt plant, it ingests the ICP and suffers the same
fate as if it ingested leaf tissue sprayed with Bt.
• At the end of 2010, an estimated 26.3 million hectares of land were planted
with crops containing the Bt gene globally(James 2011).
The chief advantages to Bt plants:
• The pests hiding inside plant parts controlled effectively;
• Multiple sprays are not needed;
• Give opportunity to get rid of chemical pesticide like
DDT, Organophosphate etc
• A disadvantage of Bt plants is that insect-specific ICPs cannot be changed
during a growing season.
•As it turns out, nature has its own biotechnologist called Agrobacterium
tumefaciens which induces the growth of tumours on woody plants.
These tumours are engineered by A.tumefaciens to produce a special
food for the bacteria (opines) that plants normally cannot make.
•These tumours arise from a unique bacterial transformation mechanism
involving the Ti-plasmid which coordinates the random insertion of a
subset of its DNA (t-DNA) containing opine synthase genes into a plant
chromosome. By replacing portions of the t-DNA sequence with genes
of interest (such as Cry).
•Researchers have been able to use this transformational mechanism and
confer new traits to many flowering plants including grasses such as
corn and rice etc.
In March 1995, the first Bt crop deregulated in the U.S. were seven lines of Colorado
Potato Beetle Resistant Bt Potato by Monsanto. Since then, many more Bt crops have
been deregulated, engineered to produce a variety of different Bt proteins from various
subspecies of Bt. Bt crops include:
European Corn Borer Resistant Corn (first deregulated in the U.S. in May 1995)
Corn Rootworm Resistant Corn (first deregulated in the U.S. in October 2002)
Lepidopteran Resistant Cotton (first deregulated in the U.S. in June 1995)
Colorado Potato Beetle Resistant Bt Potato (first deregulated in the U.S. in March 1995)
Potato Tuber Moth Resistant Bt Potato (being developed in South Africa)
Bt Soybean (first deregulated in the U.S. in October 2011, not yet sold commercially)
Lepidopteran Resistant Tomato (first deregulated in the U.S. in March 1998, not yet
Kurstaki strain (Biobit, Dipel, MVP, Steward, Thuricide, etc.):
Cabbage worm (cabbage looper, imported cabbageworm, diamondback
Tomato and tobacco hornworm.
Field and forage crop insects
European corn borer (granular formulations have given good control of first
generation corn borers).
Alfalfa caterpillar, alfalfa webworm.
Fruit crop insects
Tree and shrub insects
Spiny elm caterpillar.
Western spruce budworm.
Israelensis strains (Vectobac, Mosquito Dunks, Gnatrol, Bactimos, etc.)
San diego/tenebrionis strains (Trident, M-One, M-Trak, Foil, Novodor, etc.)
Colorado potato beetle.
Elm leaf beetle.
Cottonwood leaf beetle
• The specific activity of Bt generally is considered highly
beneficial. Unlike most insecticides, Bt insecticides do not have
a broad spectrum of activity, so they do not kill beneficial
insects. This includes the natural enemies of insects (predators
and parasites), as well as beneficial pollinators, such as
• Perhaps the major advantage is that Bt is essentially nontoxic to
people, pets and wildlife. This high margin of safety
recommends its use on food Crops or in other sensitive sites
where pesticide use can cause adverse effects.
• Used as alternative to DDT and organophosphates since
• Together with the reduction of pesticide application and cost
reduction, Bt crops have brought tremendous benefit to both the
environment and farmers .
• Expanded use of transgenic crops for insect control will likely include
more varieties with combinations of two or more Bt toxins, novel Bt
• Modified Bt toxins that have been genetically engineered to kill insects
resistant to standard Bt toxins.