Crystal Morphology of Local Bacillus thuringiensis SY56.3 Isolate

Öz

Bacillus thuringiensis (Bt) is an aerobic, Gram (+) and spore-forming entomopathogenic bacterium belonging to Bacillus cereus group together with Bacillus anthracis. Bt is the most widely and effectively used biological control agent against insect pests throughout the world. Following morphological and molecular characterization of the Bt SY56.3 isolate, Scanning Electron Microscopy (SEM) analysis was performed to determine the morphology of spore and parasporal crystal proteins (Cry) in this study. A small amount of lyophilized spore-crystalline mixture of Bt SY56.3 was put on a microscope slide containing a drop of distilled water and homogenized thoroughly. Then, it was dried at room temperature while quickly passing the slide 20 times over burner flame. The slide was then fixed onto the electron microscope holder with the aid of SC7620 Mini-Sputter Coater device. The slide was made conductive by coating with Golden Poladium (Au/Pd) for 30 seconds. The samples containing the Cry proteins in the range of 25-130 kDa were then placed in scanning electron microscope (LEO440) and visualized at 20kV. The size of crystal proteins ranged between 0.3-2 μm. It was observed that Bt SY56.3 isolate had cubic, bipyramidal, circular and irregular shaped Cry proteins. Present findings revealed that Bt SY56.3 could be used for biotechnological control applications because of different functions of these different Cry proteins.

Anahtar Kelimeler

• Bacillus thuringiensis
• Cry protein
• Bioinsecticide
• Bt

Proje Numarası

FBD-08-540.

Yerel Bacillus thuringiensis SY56.3 izolatının kristal morfolojisi

Öz

Bacillus thuringiensis (Bt), Bacillus cereus grubuna ait, aerobik, Gram (+) ve spor oluşturan bir entomopatojenik bakteridir. Bt, dünya çapında böcek zararlılarına karşı en yaygın ve etkili kullanılan biyolojik kontrol ajanıdır. Bu çalışmada, Bt SY56.3 izolatının morfolojik ve moleküler karakterizasyonunun ardından, spor ve parasporal kristal proteinlerinin (Cry) morfolojisini belirlemek için Taramalı Elektron Mikroskobu (SEM) analizi yapılmıştır. Az miktarda liyofilize edilmiş Bt SY56.3 spor-kristal karışımı, bir damla saf su içeren bir mikroskop lamına konulmuş ve iyice homojenize edilmiştir. Daha sonra, lam, oda sıcaklığında 20 kez hızlıca bek alevi üzerinden geçirilerek kurutulmuştur. Ardından, lam, SC7620 Mini-Sputter Coater cihazı yardımıyla elektron mikroskobu tutucusuna sabitlenmiştir. Lam, 30 saniye boyunca Altın Poladyum (Au/Pd) ile kaplanarak iletken hale getirilmiştir. 25-130 kDa aralığında Cry proteinleri içeren örnekler daha sonra taramalı elektron mikroskobuna (LEO440) yerleştirilmiş ve 20 kV'de görüntülenmiştir. Kristal proteinlerin boyutu 0,3-2 μm arasında gözlenmiştir. Bt SY56.3 izolatının kübik, bipiramidal, dairesel ve düzensiz şekilli Cry proteinlerine sahip olduğu gözlemlenmiştir. Mevcut bulgular, bu farklı Cry proteinlerinin farklı işlevleri nedeniyle Bt SY56.3'ün biyoteknolojik kontrol uygulamalarında kullanılabileceğini ortaya koymaktadır.

Anahtar Kelimeler

• Bacillus thuringiensi
• Bt
• Cry protein
• Biyoinsektisit

Proje Numarası

FBD-08-540.

Kaynakça

1. Melo, A.L., Soccol, V.T., Soccol, C.R. (2016). Bacillus thuringiensis: Mechanism of action, resistance, and new applications. Crit. Rev. Biotechnol., 36 (2), 317–326. https://doi.org/10.3109/07388551.2014.960793
2. de Maagd, R.A., Bravo, A., Berry, C., Crickmore, N., Schnepf, H.E. (2003). Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu. Rev. Genet., 37(1), 409– 433. https://doi.org/10.1146/annurev.genet.37.110801.143042
3. [3] Lazarte, J.N., Valacco, M.P., Moreno, S., Salerno, G.L., Berón, C.M. (2021). Molecular characterization of a Bacillus thuringiensis strain from Argentina, toxic against Lepidoptera and Coleoptera, based on its whole- genome and Cry protein analysis. J. Invertebr. Pathol., 183, 107563
4. [4] Bravo, A. (2018). Biodiversity of Cry toxins produced by Bacillus thuringiensis and evolution of resistance to these toxins in different insect pests. Toxicon 149, 98–107. https://doi.org/10.1016/j.toxicon.2018.02.010
5. [5] Gothandaraman, R., Arumugam, E., Rajasekaran, R., Mannu, J., Venkatasamy, B., Eswaran, K., Subbarayalu, M. (2026). Genomic exploration of Bacillus thuringiensis strain T381: Revealing its genetic potential against lepidopteran pests. Enzyme Microb. Technol., 199, 110894
6. [6]Zhou, Y., Wu, Z., Zhang, J., Wan, Y., Jin, W., Li, Y., Fang, X. (2020). Cry80Aa1, a novel Bacillus thuringiensis toxin with mosquitocidal activity to Culex pipiens pallens. J. Invertebr. Pathol., 173, 107386. https://doi.org/10.1016/j.jip.2020.107386
7. [7] Sanchis, V., Bourguet, D. (2008). Bacillus thuringiensis: applications in agriculture and insect resistance management. A review. Agron. Sustain. Dev., 28(1), 11–20. https://doi.org/10.1051/agro:2007054
8. [8] Sanahuja, G., Banakar, R., Twyman, R.M., Capell, T., Christou, P. (2011). Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol. J., 9(3), 283–300. https://doi.org/10.1111/j.1467-7652.2011.00595.x

9. [9] Nazarian, A., Jahangiri, R., Salehi Jouzani, G., Seifinejad, A., Soheilivand, S., Bagheri, O., Keshavarzi, M., Alamisaeid, K. (2009). Coleopteran-specific and putative novel cry genes in Iranian native Bacillus thuringiensis collection. J. Invertebr. Pathol., 102, 101–109
10. [10] Crickmore, N., Berry, C., Panneerselvam, S., Mishra, R., Connor, T.R., Bonning, B.C. (2020). Bacterial Pesticidal Protein Resource Center. https://www.bpprc.org
11. [11] Pacheco, S., Gómez, I., Peláez-Aguilar, A.E., Verduzco-Rosas, L.A., García-Suarez, R., do Nascimento, N.A., Rivera-Nájera, L.Y., Cantón, P.E., Soberón, M., Bravo, A. (2023). Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis. Front. Insect Sci., 3, 1188891. https://doi.org/10.3389/finsc.2023.1188891
12. [12] Best, H.L., Williamson, L.J., Cutts, A.B., Galchenkova, M., Yefanov, O., Bryce-Sharron, N., Heath, E.A., de Wijn, R., Schubert, R., Munke, A., Henkel, A., Klopprogge, B., Scheer, T.E.S., Kremling, V., Awel, S., Pena, G., Knoska, J., Keloth, A., Maracke, J., Letrun, R., Sobolev, E., Bielecki, J., Melo, D., Kantamneni, S., Doerner, K., Kloos, M., Schulz, J., Xavier, P.L., Lauffer, M., Villanueva, M., Caballero, P., Waller-Evans, H., Lloyd- Evans, E., Uetrecht, C., Bean, R., Chapman, H.N., Crickmore, N., Rizkallah, P.J., Berry, C., Oberthür, D. (2026). The long and short of it: Distinct natural crystal packing strategies of Cry toxins from Bacillus thuringiensis. Structure, 34, 704–717. https://doi.org/10.1016/j.str.2026.01.014
13. [13] García-Cruz, K.V., Arce-Vázquez, M.B., Cruz-Monterrosa, R.G., Aguilar-Toalá, J.E., Rosas-Espejel, M., Hernández-Rodríguez, M., Pérez-Ruiz, R.V. (2026). Cry proteins from Bacillus thuringiensis: From natural insecticide to biotechnological tool for agricultural pest control. Agro Productividad. 19(1), pp: 23-37
14. [14] Li, J., Derbyshire, D.J., Promdonkoy, B., Ellar, D.J. (2001). Structural implications for the transformation of the Bacillus thuringiensis δ-endotoxins from water-soluble to membrane-inserted forms. Biochem. Soc. Trans., 29, 571–577. https://doi.org/10.1042/bst0290571
15. [15] Wang, Y., Wang, J., Fu, X., Nageotte, J.R., Silverman, J., Bretsnyder, E.C., Chen, D., Rydel, T.J., Bean, G.J., Li, K.S., Kraft, E., Gowda, A., Nance, A., Moore, R.G., Pleau, M.J., Milligan, J.S., Anderson, H.M., Asiimwe, P., Evans, A., Moar, W.J., Martinelli, S., Head, G.P., Haas, J.A., Baum, J.A., Yang, F., Kerns, D.L., Jerga, A. (2019). Bacillus thuringiensis Cry1Da7 and Cry1B.868 protein interactions with novel receptors allow control of resistant fall armyworms, Spodoptera frugiperda (J.E. Smith). Appl. Environ. Microbiol., 85(16), e00579– e619. https://doi.org/10.1128/AEM.00579-19
16. [16] Tanaka, J., Abe, S., Hayakawa, T., Kojima, M., Yamashita, K., Hirata, K., Ueno, T. (2023). Crystal structure of the in-cell Cry1Aa purified from Bacillus thuringiensis. Biochem. Biophys. Res. Commun., 685, 149144. https://doi.org/10.1016/j.bbrc.2023.149144
17. [17] Krissinel, E., Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol., 372, 774–797. https://doi.org/10.1016/j.jmb.2007.05.022
18. [18] Du, C., Martin, P.A.W., Nickerson, K.W. (1994). Comparison of disulfide contents and solubility at alkaline pH of insecticidal and noninsecticidal Bacillus thuringiensis protein crystals. Appl. Environ. Microbiol., 60(10), 3847–3853. https://doi.org/10.1128/aem.60.10.3847-3853.1994
19. [19] Tabashnik, B.E., Zhang, M., Fabrick, J.A., Wu, Y., Gao, M., Huang, F., Wei, J., Zhang, J., Yelich, A., Unnithan, G.C., Bravo, A., Soberón, M., Carrière, Y., Li, X. (2015). Dual mode of action of Bt proteins: protoxin efficacy against resistant insects. Sci. Rep., 5, 15107. https://doi.org/10.1038/srep15107
20. [20] Chicca, J., Cazeault, N.R., Rus, F., Abraham, A., Garceau, C., Li, H., Atwa, S.M., Flanagan, K., Soto, E.R., Morrison, M.S., Gazzola, D., Hu, Y., Liu, D.R., Nielsen, M.K., Urban Jr., J.F., Ostroff, G.R., Aroian, R.V. (2022). Efficient and scalable process to produce novel and highly bioactive purified cytosolic crystals from Bacillus thuringiensis. Microbiol. Spectr., 10(4), e02356–e02422. https://doi.org/10.1128/spectrum.02356-22
21. [21] Arakere, U.C., Jagannath, S., Krishnamurthy, S., Chowdappa, S., Konappa, N. (2022). Microbial bio-pesticide as sustainable solution for management of pests: achievements and prospects. In: Biopesticides Volume 2: Advances in Bio-inoculants (p. 183-200). Woodhead Publishing. https://doi.org/10.1016/B978-0-12-823355- 9.00016-X
22. [22] Knowles, B.H., Ellar, D.J. (1987). Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis delta-endotoxins with different insect specificity. Biochim. Biophys. Acta, 924, 509– 518. https://doi.org/10.1016/0304-4165(87)90167-X
23. [23] Leslie, S.A., Mitchell, J.C. (2007). Removıng gold coatıng from sem samples. Palaeontology, 50: 1459- 1461. https://doi.org/10.1111/j.1475-4983.2007.00718.x
24. [24] Kado, C.I., Liu, S.T. (1981). Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol., 145, 1365–1373. https://doi.org/10.1128/jb.145.3.1365-1373.1981
25. [25] Battisti, L., Green, B.D., Thorne, C.B. (1985). Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. J. Bacteriol., 162, 543–550. https://doi.org/10.1128/jb.162.2.543-550.1985
26. [26] Nicolas, L.J., María, P.V., Silvia, M., Graciela, L.S., Corina, M.B. (2021). Molecular characterization of a Bacillus thuringiensis strain from Argentina, toxic against Lepidoptera and Coleoptera, based on its whole- genome and Cry protein analysis, J. Invertebr. Pathol., 183, 107563
27. [27] Crickmore, N., Berry, C., Panneerselvam, S., Mishra, R., Connor, T.R., Bonning, B.C. (2020). Bacterial Pesticidal Protein Resource Center. https://www.bpprc.org
28. [28] Yılmaz, S. (2010). Molecular characterization of Bacillus thuringiensis strains isolated from different locations and their effectiveness on some pest insects. Erciyes University, Graduate School of Natural and Applied Sciences, PhD. Thesis, Kayseri, Turkey