Bacillus thuringiensis
Bacillus thuringiensis is a gram-positive, soil-dwelling bacterium, and is the most commonly used biological pesticide worldwide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on leaf surfaces, aquatic environments, animal feces, insect-rich environments, flour mills and grain-storage facilities. It has also been observed to parasitize moths such as Cadra calidella—in laboratory experiments working with C. calidella, many of the moths were diseased due to this parasite.
During sporulation, many Bt strains produce crystal proteins, called delta endotoxins, that have insecticidal action. This has led to their use as insecticides, and more recently to genetically modified crops using Bt genes, such as Bt corn. Many crystal-producing Bt strains, though, do not have insecticidal properties. Bacillus thuringiensis israelensis was discovered in 1976 by Israeli researchers Yoel Margalith and B. Goldberg in the Negev Desert of southern Israel. While investigating mosquito breeding sites in the region, they isolated a bacterial strain from a stagnant pond that exhibited potent larvicidal activity against various mosquito species, including Anopheles, Culex, and Aedes. This subspecies, israelensis, is now commonly used for the biological control of mosquitoes and fungus gnats due to its effectiveness and environmental safety.
As a toxic mechanism, cry proteins bind to specific receptors on the membranes of mid-gut cells of the targeted pests, resulting in their rupture. Other organisms that lack the appropriate receptors in their gut cannot be affected by the cry protein, and therefore are not affected by Bt.
Taxonomy and discovery
In 1902, B. thuringiensis was first discovered in silkworms by Japanese sericultural engineer Ishiwatari Shigetane. He named it B. sotto, using the Japanese word, here referring to bacillary paralysis. In 1911, German microbiologist Ernst Berliner rediscovered it when he isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars in Thuringia. B. sotto would later be reassigned as B. thuringiensis var. sotto.In 1976, Robert A. Zakharyan reported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore and crystal formation. B. thuringiensis is closely related to B. cereus, a soil bacterium, and B. anthracis, the cause of anthrax; the three organisms differ mainly in their plasmids. Like other members of the genus, all three are capable of producing endospores.
Species group placement
B. thuringiensis is placed in the Bacillus cereus group which is variously defined as seven closely related species: B. cereus ''sensu stricto, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. cytotoxicus; or as six species in a Bacillus cereus sensu lato: B. weihenstephanensis, B. mycoides, B. pseudomycoides, B. cereus, B. thuringiensis, and B. anthracis. Within this grouping B.t. is more closely related to B.ce. It is more distantly related to B.w., B.m., B.p., and B.cy.''Subspecies
There are several dozen recognized subspecies of B. thuringiensis. Subspecies commonly used as insecticides include B. thuringiensis subspecies kurstaki, subspecies israelensis and . Some Bti lineages are clonal.Genetics
Some strains are known to carry the same genes that produce enterotoxins in B. cereus, and so it is possible that the entire B. cereus sensu lato group may have the potential to be enteropathogens.The proteins that B. thuringiensis is most known for are encoded by cry genes. In most strains of B. thuringiensis, these genes are located on a plasmid. If these plasmids are lost it becomes indistinguishable from B. cereus as B. thuringiensis has no other species characteristics. Plasmid exchange has been observed both naturally and experimentally both within B.t. and between B.t. and two congeners, B. cereus and B. mycoides.
plcR is an indispensable transcription regulator of most virulence factors, its absence greatly reducing virulence and toxicity. Some strains do naturally complete their life cycle with an inactivated plcR. It is half of a two-gene operon along with the heptapeptide. papR is part of quorum sensing in B. thuringiensis.
Various strains including Btk ATCC 33679 carry plasmids belonging to the wider pXO1-like family. The insect parasite Btk HD73 carries a pXO2-like plasmid lacking the 35kb pathogenicity island of pXO2 itself, and in fact having no identifiable virulence factors.
The genomes of the B. cereus group may contain two types of introns, dubbed group I and group II. B.t strains have variously 0–5 group Is and 0–13 group IIs.
There is still insufficient information to determine whether chromosome-plasmid coevolution to enable adaptation to particular environmental niches has occurred or is even possible.
Common with B. cereus but so far not found elsewhere – including in other members of the species group – are the efflux pump BC3663, the N-acyl--amino-acid amidohydrolase BC3664, and the methyl-accepting chemotaxis protein BC5034.
Mechanism of insecticidal action
Upon sporulation, B. thuringiensis forms crystals of two types of proteinaceous insecticidal delta endotoxins called crystal proteins or Cry proteins, which are encoded by cry genes, and Cyt proteins.Cry toxins have specific activities against insect species of the orders Lepidoptera, Diptera, Coleoptera and Hymenoptera, as well as against nematodes. A specific example of B. thuringiensis use against beetles is the fight against Colorado Potato Beetles in potato crops. Thus, B. thuringiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops. When insects ingest toxin crystals, their alkaline digestive tracts denature the insoluble crystals, making them soluble and thus amenable to being cut with proteases found in the insect gut, which liberate the toxin from the crystal. The Cry toxin is then inserted into the insect gut cell membrane, paralyzing the digestive tract and forming a pore. The insect stops eating and starves to death; live Bt bacteria may also colonize the insect, which can contribute to death. Death occurs within a few hours or weeks. The midgut bacteria of susceptible larvae may be required for B. thuringiensis insecticidal activity.
A B. thuringiensis small RNA called BtsR1 can silence the Cry5Ba toxin expression when outside the host by binding to the RBS site of the Cry5Ba toxin transcript to avoid nematode behavioral defenses. The silencing results in an increase of the bacteria ingestion by C. elegans. The expression of BtsR1 is then reduced after ingestion, resulting in Cry5Ba toxin production and host death.
In 1996 another class of insecticidal proteins in Bt was discovered: the vegetative insecticidal proteins. Vip proteins do not share sequence homology with Cry proteins, in general do not compete for the same receptors, and some kill different insects than do Cry proteins.
In 2000, a novel subgroup of Cry protein, designated parasporin, was discovered from non-insecticidal B. thuringiensis isolates. The proteins of parasporin group are defined as B. thuringiensis and related bacterial parasporal proteins that are not hemolytic, but capable of preferentially killing cancer cells. As of January 2013, parasporins comprise six subfamilies: PS1 to PS6.
Use of spores and proteins in pest control
Spores and crystalline insecticidal proteins produced by B. thuringiensis have been used to control insect pests since the 1920s and are often applied as liquid sprays and donut pellets. They are now used as specific insecticides under trade names such as DiPel, Thuricide, and Mosquito Dunks. Because of their specificity, these pesticides are regarded as environmentally friendly, with little or no effect on humans, wildlife, pollinators, and most other beneficial insects, and are used in organic farming; however, the manuals for these products do contain many environmental and human health warnings, and a 2012 European regulatory peer review of five approved strains found, while data exist to support some claims of low toxicity to humans and the environment, the data are insufficient to justify many of these claims.New strains of Bt are developed and introduced over time as insects develop resistance to Bt, or the desire occurs to force mutations to modify organism characteristics, or to use homologous recombinant genetic engineering to improve crystal size and increase pesticidal activity, or broaden the host range of Bt and obtain more effective formulations. Each new strain is given a unique number and registered with the U.S. EPA and allowances may be given for genetic modification depending on "its parental strains, the proposed pesticide use pattern, and the manner and extent to which the organism has been genetically modified". Formulations of Bt that are approved for organic farming in the US are listed at the website of the Organic Materials Review Institute and several university extension websites offer advice on how to use Bt spore or protein preparations in organic farming.
Use of Bt genes in genetic engineering of plants for pest control
The Belgian company Plant Genetic Systems was the first company to develop genetically modified crops with insect tolerance by expressing cry genes from B. thuringiensis; the resulting crops contain delta endotoxin. The Bt tobacco was never commercialized; tobacco plants are used to test genetic modifications since they are easy to manipulate genetically and are not part of the food supply.Image:Bt plants.png|thumb|Bt toxins present in peanut leaves protect it from extensive damage caused to unprotected peanut leaves by lesser cornstalk borer larvae.