Bacillus thurigenesis

Bacillus thuringiensis (Bt). This bacterium is also a key source of genes for transgenic expression to provide pest resistance in plants and microorganisms as pest control agents in so-called genetically modified organisms (GMOs).

1. Bacillus thuringiensis Prasanna R Kovath Assistant Professor Department of Biotechnology St. Mary's College Thrissur

2. Among prokaryotes, bacteria of the genus Bacillus (family Bacillaceae ) have been used in the microbial control of pests. In this genus, the species Bacillus thuringiensis ( Bt ) is distinguished by its biopesticide potential . Bt  is a microbe naturally found in soil. It makes proteins that are toxic to immature insects (larvae). There are many types of  Bt . Each targets different insect groups. Target insects include beetles, mosquitoes, black flies, caterpillars, and moths. With  Bt  pesticides, routine testing is required to ensure that unwanted toxins and microbes are not present.  Bt  has been registered for use in pesticides by the US Environmental Protection Agency (EPA) since 1961.

3. History Bt was discovered by Ishiwatta in 1902 in reared Bombyx mori L. ( Lepidoptera,Bombycidae ) in Japan. Later it was reisolated by Berliner, in 1911, from Ephestia kuehniella Zeller (Lepidoptera, Pyralidae ) larvae in Thuringia, which gave rise to its current name. According to Van Frankenhuyzen (1993 ), the first biological control trials of Bt were conducted against Ostrinia nubilalis Hübner (Lepidoptera, Pyralidae ) between 1920 and 1930 in Europe. Between 1930 and 1940, several trials were carried out with other lepidopteran species in Europe and in the USA. Regarding the biological control of insects today, Bt is the mostly used microorganism worldwide.

4. Bt is a ubiquitous bacterium with a large enzyme complement, which allows it to be found in a variety of sites, such as: soil, insects and their habitats, stored products, plants , forest, and aquatic environments . It can remain latent in the environment even in adverse conditions for its development. Its entomopathogenic activity is highly dependent on the parasporal inclusion body that forms during sporulation, which consists of Cry proteins that are encoded by cry genes.

5. P arasporal inclusion bodies (crystals) are composed of proteins of varying quantity and quality according to the bacterial strain. Strains containing these crystals were measured as being toxic and specific to larvae of Lepidoptera, Diptera , Coleoptera , Nematoida , Protozoa,Trematoda , Acari, Hymenoptera, Hemiptera , Orthoptera , Isoptera , and Mallophaga . Transmission electron micrograph of a longitudinal section of Bacillus thuringiensis towards the end of sporulation, showing the spore (black ovoid structure) and the protein crystal with insecticidal properties ( bipyramidal inclusion). Photo: from Institut Pasteur, Station Centrale de Microscopie Électronique

6. Cellular Morphology and Physiology Bt is an aerobic Gram-positive and rod-shaped bacterium , with a vegetative cell of 1.0–1.2 μm wide and 3.0–5.0 μm in length, usually mobile by means of peritrichous flagella. The spore of this bacterium has an ellipsoidal shape but mostly are cylindrical and is located in the central or paracentral region when inside the mother cell. The species is non-strict aerobic with a temperature range of growth between 10–5 ºC and 40–45 °C. The main characteristic that distinguishes this species from the others of the same genus is the intracellular presence of a protein crystal.

7. Most strains are catalase-positive, oxidase negative, casein, gelatin and starch are hydrolyzed. Voges-Proskauer - positive and citrate is utilized as sole carbon source. Nitrate is reduced and tyrosine is decomposed. Phenylalanine is not deamineted . Most strains utilize saccharose and other sugars

8. Bacillus thuringiensis : (a, b) selective culture medium , (c) gram staining , (d) interferential phase contrast micrograph, and (e) transmission electron micrograph , Bar = μm

9. Bt has two distinct phases during cell development : vegetative cellular division and spore formation The development of the spore and crystal involves seven distinct stages : (a) phase I – formation of axial filaments (b) phase II – formation of forespore septum ( c) phase III – first appearance of parasporal crystals and formation of a forespore ( d) phases IV to VI – formation of exospore, primordial cell wall, and spore nucleoid transformation (e ) phase VII – maturation of spores and cell lysis

10. These insecticidal proteins are synthesized after stage II of sporulation and accumulate in the mother cell as a crystal , which can account for up to 25 % of dry weight of the sporulated cells T hese crystals can have different forms : B ipyramidal , P yramidal,cuboidal , Flat rhomboid, Spherical , and Rectangular . The most common form is that of a B ipyramidal crystal . The crystals may contain one or more delta-endotoxins, or Cry proteins , which have molecular weights between 30 kDa and 140 kDa , and which are converted into toxic peptides after ingestion by target pests (insects).

11. In addition to Cry proteins, Bt isolates can synthesize proteins called Cyt , which have in vitro cytolytic activity and in vivo specificity to dipterans Cyt toxins are able to affect insect midgut cells and may increase the insecticidal activity of certain Cry toxins Proteins known as beta-exotoxins are also synthesized by some Bt isolates. One of these, thuringiensin is a nonspecific and thermostable protein toxic to vertebrates Because it is nonspecific, isolates that are capable of producing, it cannot be used in the production of biopesticides , Beta-exotoxin has been identified in different subspecies Bt, such as Bt tenebrionis Bt kenyae and Bt tolworthi Bt thuringiensis , Bt kurstaki and Bt darmstadiensis

12. Vip proteins are divided into four families according to their amino acid identity . Vip1 and Vip2 proteins act as a binary toxin and are toxic to some coleopteran and hemipteran species In addition to the aforementioned proteins, Bt can produce phospholipases, proteases, chitinases , and enterotoxins . Enterotoxin is similar to that produced by B.cereus , whose ingestion in food results in intoxication. Bt produces some classes of bacteriocins , which are important for the control of pathogenic microorganisms and food contamination

13. Biochemistry and Molecular Biology : Bacillus thuringenesis In the pioneering studies found that the crystal-producing bacteria could be subdivided into six biochemical groups. In addition, the reality of this subdivision was supported by the presence of an H antigen that was specific to each group which helps in differentiating aerobic bacteria from spore formers and is widely applicable across the many genera. The commercially available biochemical tools mostly used for the identification of Bacillus spp. and correlates are the API 20E and 50CHB systems ( BioMerieux ), the VITEK systems ( BioMerieux ), and Biolog .

14. In molecular biology studies, one of the most important aspects of the Bacillus genus is its diversity. There are species that have a key role in medicine, industry and the economy. Some examples are B. cereus , B. thuringiensis , B. anthracis , B . mycoides , and B. pseudomycoides . Many studies based on molecular markers find it difficult to ultimately separate B. cereus and Bt. The most obvious difference between these two species is the presence of plasmids which encode toxic proteins for insects in Bt The differentiation between these species using molecular markers continues to be a hard task, regardless of the methods used

15. In the specific case of Bt , since its description in 1915 , the main method applied to the identification of subspecies was based on the flagellar H antigen reaction Among the tools mostly used, high- lights include that based on 16S rRNA , RAPD, RFLP, REP-PCR, ERIC-PCR , and MLST . All of them make it possible to detect differences between the analyzed strains, but a debate remains on which of these tools are the most appropriate to be used as a standard method to molecularly characterize B. thuringiensis strains and to correlate the molecular patterns with toxicity to different insect species.

16. The 16S rRNA genes are considered one of the main molecular markers for studies in bacteria diversity. This marker is widely used for phylogenetic analysis and for studies in metagenomics These genes have both conserved and variable regions which make its application possible for studies in different taxonomic levels. In recent decades, its application significantly extended our knowledge about diversity in prokaryotes. Data can be generated by sequencing some regions of this gene or by using the RFLP technique.

17. The main restriction of the use of 16S rRNA is the high similarity between the sequences of closely related species (Christensen et al. 1998), as happens in the Bacillus genus . To overcome these limitations, recent works have been using 16S rDNA analysis together with other markers, increasing the capacity to differentiate Bt strains and other species of Bacillus.

18. The analysis of repetitive regions of the genome is one of the tools successfully applied to analyze the diversity among the species of Bacillus . Its use has made possible the differentiation between Bt and B. cereus strains. The study of these regions is named fingerprint analysis and is based on amplification by PCR of the regions repetitive enterobacterial palindromic ( REP) and enterobacterial repetitive intergenic consensus (ERIC) sequence . The genetic variability found in these sequences makes its successful application possible to study the intra- and interspecific diversity of species belonging to the genus Bacillus . Its efficiency to differentiate the species is greater than the one found in the gene for 16S rRNA .

19. The additional advantages of this technique are its easy and quick implementation, its simplicity, and the fact of that the results are reliable and reproducible . REP-PCR technique to differentiate strains from different samples,is also used as atool for identification.

20. Multilocus sequence analysis (MLSA ) study the genetic diversity in pathogenic microorganisms. It is based on the analysis of (housekeeping) genes that are expressed in a constitutive way, in other words , genes which have a central role in maintaining the cell’s metabolism. The alleles are identified through the sequencing of internal fragments of these genes. The new variants are created by mutations, synonymous or not, in the nucleotide sequence. This technique is considered an excellent tool to study the inter- and intraspecific genetic variability and to study the strains evolution.

21. Nowadays, despite of the great number of studies, the main way of differentiating B. cereus from Bt is the presence of proteins ( Bt toxins) active against insect species. The studies also cannot associate molecular patterns of the chromosomal DNA with the insecticidal activity. T he best way to identify and characterize new strains of Bt is to analyze the plasmid genes responsible for the synthesis of these toxins.

22. Besides molecular patterns based on using nucleic acids, some chemical markers are considered important for the analysis and description of the inter- and intraspecific variability in bacteria. Among the chemotaxonomic tools applied to the identification of bacteria, highlights include : fatty acid methyl ester ( FAME ) matrix assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry .

23. The FAME technique is based on the analysis of short-chain fatty acids, which have between 9 and 20 carbons. The production of the fatty acids is considered a specific property of each microorganism and can be driven by environmental factors. These compounds are important for the adaptation process of microorganisms. All these properties have made the fatty acids analysis an important tool for the taxonomy of bacteria. F atty acids analysis is also a good marker for studies of the adaptation in Bacillus species, as their composition varies according to the environment .

24. The MALDI technique is considered one of the most important tools for the study of microorganisms. It is based on the use of a mass spectrophotometer to analyze the spectral patterns of ions present in macromolecules. In this technique , all proteins of the bacteria cell provide a specific pattern ( fingerprint) which can be statistically analyzed. T he results are obtained more quickly, and the process has lower cost, is less laborious, and has the same efficiency as the traditional techniques that use 16S rRNA (RFLP, AFLP, and sequencing).

25. Mode of Action of Bt Toxins The first impact of Cry toxins on the insect is cessation of feeding due to paralysis of the gut and mouthparts In addition to gut paralysis, midgut cells swell leading to an ion imbalance and death  The molecular events leading to Cry toxin-mediated insect death are controversial , but the accepted initial steps are as follows: The Bt Cry and Cyt proteins require solubilization in the insect midgut to produce protoxins that are typically about 130 kDa , 70 kDa or 27 kDa for Cyt . These in turn are proteolytically cleaved at the  C -terminus and/or at the  N -terminus by midgut proteases, generating the activated core toxin.

26. The toxin then crosses the peritrophic matrix and binds to receptors in the apical membrane of the midgut cells, with receptor binding being an important determinant of toxin specificity. Toxin insertion into the epithelial membrane forms ion channels or pores, leading to lysis of the cells, damage to the midgut epithelial tissue, and death of the larva

27. TIPS FOR APPLYING BT   Make sure you have the right strain for the pest you want to control A pH greater than 8 is what activates the toxin in the insect’s gut You will have better control if the larvae are small. There will be less damage to the plants because the little insects won’t eat as much. If you treat larger larvae, they will eat more of the plants and cause greater damage. Also, they may morph into the reproductive phase and become insensitive to the toxin . The spray will be more effective if you add a spreader or sticker to the tank mix. Use the spray within 12 hours of mixing. Make sure that you are spraying both the top and bottom surfaces of the leaves .

28. While it can survive for years in the ground if adsorbed to soil particles, Bt is rapidly inactivated by the UV radiation in sunlight. Many people spray their plants in the evening, so the toxin can work overnight before being inactivated by the sun the next day. The bacteria are sensitive to temperature and must be stored at 50-60 F. Do not expose the bacteria to hot or cold temperatures, which can kill them . Additives, such as sticking or wetting agents, are often useful in a Bt application to improve performance, allowing it to cover foliage more thoroughly and to resist washing off.

29. Unlike most insecticides, which target a broad spectrum of species, including both pests and beneficial insects, Bt is toxic to a narrow range of insects. Research suggests that Bt does not harm the natural enemies of insects, nor does it impair honeybees and other pollinators critical to agroecological systems. Bt  integrates well with other natural controls and is used for integrated pest management by many organic farmers .

30. The use of insect-resistant Bt plants can potentially reduce use of chemical insecticide sprays, which are extremely toxic and expensive .   Bt toxin applied as an insecticide or consumed with GMO food crops is considered nontoxic to humans and other mammals because they lack the digestive enzymes needed to activate the Bt protein crystals . However, any introduction of new genetic material is potentially a source for allergens, and, for this reason, certain strains of Bt are not approved for human consumption.

31. Bt Type: Controls: Bacillus thuringiensis kurstaki  ( Btk ) Most caterpillars Bacillus thuringiensis israelensis  (Bti) Mosquitoes, flies, fungus gnats Bacillus thuringiensis San Diego Specific beetles Bacillus thuringiensis tenebrionis Specific beetles Bacillus thuringiensis aizawai  (Bta) Some caterpillars The effectiveness of  Bt  may be reduced after two or three years of storage. Dry formulations last longer than liquid formulations.  Bt  products should be stored out of sunlight and in cool, dry conditions.

32. If you use Bt on your farm, the EPA will require you to take some steps to prevent resistance. One way is to alternate its use with synthetic insecticides. Another is to rotate your crops. Since different kinds of insects feed on different crops, you would be using a different type of Bt. The large variety of  Bacillus thuringiensis  strains available enables gardeners, farmers, and mosquito control experts to control an array of insect pests. Since the strains are highly specific to the insects targeted, residual effects against other organisms are not a concern.

33. What happens to  Bacillus thuringiensis ( Bt )  when it enters the body? When eaten,  Bt  is confined to the gut. It does not reproduce, and the toxin is broken down like other proteins in the diet.  Bt  leaves the body within 2 to 3 days. If breathed in,  Bt  can move to the lungs, blood, lymph, and kidneys.  Bt  is then attacked by the immune system. Levels of  Bt  decrease quickly one day after exposure.

34. What happens to  Bacillus thuringiensis ( Bt )  in the environment? Toxins created by  Bt  are rapidly broken down by sunlight and in acidic soil. Other microbes in soil can also break it down.  Bt  does not readily leach in soil. It typically remains in the top several inches of soil.  Bt  remains dormant in most natural soil conditions. However, there has been some reproduction in nutrient rich soils. On the soil surface, dormant  Bt  cells last only a few days. However , below the soil surface, they can last for months or years. The  half-life  in unfavorable soil is about 4 months.  Bt  toxins break down much faster. In one study, 12% remained after 15 days . In water,  Bt  does not readily reproduce. A study found  Bt  toxins in the air were broken down rapidly by sunlight. Forty-one percent (41%) of the toxin remained after 24 hours. On plant surfaces, sunlight breaks down  Bt ; the half-life of  Bt  toxins is 1-4 days.

35. Reference https://www.mdpi.com/2072-6651/6/10/3005/htm https://gardenerspath.com/how-to/organic/bacillus-thuringiensis/ http://npic.orst.edu/factsheets/btgen.html#products https:// www.researchgate.net/publication/41714537 Microbial Pest Control Agent BACILLUS THURINGIENSIS ( Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation , and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals) Bacillus thuringiensis as a Specific, Safe, and Effective Tool for Insect Pest Control (ROH , JONG YUL, JAE YOUNG CHOI, MING SHUN LI, BYUNG RAE JIN1, AND YEON HO JE *) https:// www.researchgate.net/publication/318152732