Isolation and Screening of cry and parasporin Genes of Native Bacillus thuringiensis from Cherry Gardens in Izmir

Müjgan Kesik Oktay; Burcu Sahin; Hatice Güneş

doi:10.17776/csj.1762728

Isolation and Screening of cry and parasporin Genes of Native Bacillus thuringiensis from Cherry Gardens in Izmir

Abstract

Bacillus thuringiensis (Bt), which produces various proteins, is a versatile microorganism. In this study, we performed Bt isolation from cherry orchards in İzmir (Türkiye) and characterization of cry and parasporin (ps) genes related to the proteins that are insecticidal and cytotoxic, respectively. Forty-one soil samples were collected, and 314 Bt-like colonies were obtained from these samples. According to phase contrast microscopy examination, 80 colonies showing crystal formation were identified as ‘Bt’. Polymerase chain reaction (PCR) was performed to determine the cry and ps gene profiles of the isolates. It was found that 66 out of the 80 samples carried cry1, cry2, or cry3 genes. The most common of these genes was the cry1 (68%). In the case of parasporin gene screening, the existence of ps1, ps2, ps3, ps4, ps5, and ps6 genes was investigated. Seventeen of the 80 isolates were found positive for ps genes. Among these, the ps6 gene showed the highest frequency with 29%, followed by the ps3 gene (24%). Partial DNA sequence analysis of seven ps-positive isolates showed that 5.4, 5.7, 5.10, 7.1, and 7.3 showed 99% identity with ps6, and the other isolates 2.1 and 40.4, matched with 98% and 97% identity with ps1 and ps2, respectively. In conclusion, this study reports a new Bt collection from Türkiye. The native Bt isolates carrying cry and/or ps genes will help us select potential ones with insecticidal and cytotoxic effects that will form the basis for the production of recombinant parasporal proteins planned in future studies.

Keywords

Project Number

Scientific Research Project (BAP) from from Mugla Sıtkı Koçman University- Project number 15/152.

References

Palma, L., Muñoz, D., Berry, C., Murillo, J., & Caballero, P. (2014). Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins, 6(12), 3296–3325. https://doi.org/10.3390/toxins6123296
Soares-da-Silva, J., Pinheiro, V. C. S., Litaiff-Abreu, E., Polanczyk, R. A., & Tadei, W. P. (2015). Isolation of Bacillus thuringiensis from the state of Amazonas in Brazil and screening against Aedes aegypti (Diptera, Culicidae). Revista Brasileira de Entomologia, 59(1), 1–6. https://doi.org/10.1016/j.rbe.2015.02.001
Limbu, J., Shrestha, B. K., Shakya, J., Khatri, S. B., & Khanal, H. (2020). Isolation of Bacillus thuringiensis and its insecticidal effect against Galleria mellonella. Himalayan Journal of Science and Technology, 3–4, 96–102. https://doi.org/10.3126/hijost.v4i0.33918
Hassan, A. A., Youssef, M. A., Elashtokhy, M. M. A., Ismail, I. M., Aldayel, M., & Afkar, E. (2021). Isolation and identification of Bacillus thuringiensis strains native of the eastern province of Saudi Arabia. Egyptian Journal of Biological Pest Control, 31, 6. https://doi.org/10.1186/s41938-020-00352-8
Aynalem, B., Muleta, D., Venegas, J., & Assefa, F. (2021). Isolation, molecular characterization and pathogenicity of native Bacillus thuringiensis from Ethiopia against the tomato leafminer, Tuta absoluta: Detection of a new high lethal phylogenetic group. Microbiological Research, 250, 126807..https://doi.org/10.1016/j.micres.2021.126802
Jurat-Fuentes, J. L., Heckel, D. G., & Ferré, J. (2021). Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis. Annual Review of Entomology, 66, 121–140. https://doi.org/10.1146/annurev-ento-052620-073348
Ragasruthi, M., Balakrishnan, N., Murugan, M., Swarnakumari, N., Harish, S., & Sharmila, D. J. S. (2024).Bacillus thuringiensis (Bt)-based biopesticide: Navigating success, challenges, and future horizons in sustainable pest control. Science of the Total Environment, 954, 176594. https://doi.org/10.1016/j.scitotenv.2024.176594
Liu, L., Wu, L., & Eickhorst, T. (2018). Accumulation of Cry1Ab/Ac proteins released from transgenic Bt-rice in the rhizosphere of a paddy soil. Rhizosphere, 6, 39–46. https://doi.org/10.1016/j.rhisph.2018.02.002

Mizuki, E., Park, Y. S., Saitoh, H., Yamashita, S., Akao, T., Higuchi, K., & Ohba, M. (2000). Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillus thuringiensis. Clinical and Diagnostic Laboratory Immunology, 7(4), 625–634. https://doi.org/10.1128/CDLI.7.4.625-634.2000
Santos, E. N., Menezes, L. P., Dolabella, S. S., Santini, A., Severino, P., Capasso, R., Zielinska, A., Souto, E. B., & Jain, S. (2022). Bacillus thuringiensis: From biopesticides to anticancer agents. Biochimie, 192, 83–90. https://doi.org/10.1016/j.biochi.2021.10.003
Suárez-Barrera, M. O., Visser, L., Rondón-Villarreal, P., Herrera-Pineda, D. F., Alarcón-Aldana, J. S., Van den Berg, A., Orozco, J., Pinzón-Reyes, E. H., Moreno, E., & Rueda-Forero, N. J. (2021). Genetic Modification Approaches for Parasporins Bacillus thuringiensis Proteins with Anticancer Activity. Molecules, 26(24),7476. https://doi.org/10.3390/molecules26247476
Kitada, S., Abe, Y., Shimada, H., Kusaka, Y., Matsuo, Y., Katayama, H., Okumura, S., Akao, T., Mizuki, E., Kuge, O., Sasaguri, Y., Ohba, M., & Ito, A. (2006). Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis. Journal of Biological Chemistry, 281(36), 26350–26360. https://doi.org/10.1074/jbc.M602589200
Ammons, D. R., Short, J. D., Bailey, J., Hinojosa, G., Tavarez, L., Salazar, M., & Rampersad, J. N. (2016). Anti-cancer Parasporin Toxins are Associated with Different Environments: Discovery of Two Novel Parasporin 5-like Genes. Current Microbiology, 72(2), 184–189. https://doi.org/10.1007/s00284-015-0934-3
Krishnan, V., Domanska, B., Elhigazi, A., Afolabi, F., West, M. J., & Crickmore, N. (2017). The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts. Biochemical Journal, 474(10), 1591–1602. https://doi.org/10.1042/BCJ20170122
Santana, M. A., Moccia-V, C. C., & Gillis, A. E. (2008). Bacillus thuringiensis improved isolation methodology from soil samples. Journal of Microbiological Methods, 75(2), 357–358. https://doi.org/10.1016/j.mimet.2008.06.008
Cinar, C., Apaydin, O., Yenidunya, A. F., Harsa, S., & Gunes, H. (2008). Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey. Journal of Applied Microbiology, 104(2), 515–525. https://doi.org/10.1111/j.1365-2672.2007.03571.x
Ben-Dov, E., Zaritsky, A., Dahan, E., Barak, Z., Sinai, R., Manasherob, R., Khamraev, A., Troitskaya, E., Dubitsky, A., Berezina, N., & Margalith, Y. (1997). Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis. Applied and Environmental Microbiology, 63(12), 4883–4890. https://doi.org/10.1128/aem.63.12.4883-4890.1997
Lenina, N. K., Naveenkumar, A., Sozhavendan, A. E., Balakrishnan, N., Balasubramani, V., & Udayasuriyan, V. (2014). Characterization of parasporin gene harboring Indian isolates of Bacillus thuringiensis. 3 Biotech, 4(5), 545–551. https://doi.org/10.1007/s13205-013-0190-9
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R., & Dean, D. H. (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, 62(3), 775–806. https://doi.org/10.1128/MMBR.62.3.775-806.1998
Quesada-Moraga, E., García-Tóvar, E., Valverde-García, P., & Santiago-Alvarez, C. (2004). Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain. Microbiological Research, 159(1), 59–71. https://doi.org/10.1016/j.micres.2004.01.011
Iriarte, J., Porcar, M., Lecadet, M., & Caballero, P. (2000). Isolation and characterization of Bacillus thuringiensis strains from aquatic environments in Spain. Current Microbiology, 40(6), 402–408. https://doi.org/10.1007/s002840010078
Şahin, B., & Güneş, H. (2025). Isolation and Characterization of Bacillus thuringiensis Isolates from Hazelnut Orchards in Türkiye. Turkish Journal of Agriculture-Food Science and Technology, 13(4), 991-997.https://doi.org/10.24925/turjaf.v13i4.991-997.7415
Ramalakshmi, A., & Udayasuriyan, V. (2010). Diversity of Bacillus thuringiensis isolated from Western Ghats of Tamil Nadu State, India. Current Microbiology, 61(1), 13–18. https://doi.org/10.1007/s00284-009-9569-6
Lone, S. A., Malik, A., & Padaria, J. C. (2017). Selection and characterization of Bacillus thuringiensis strains from northwestern Himalayas toxic against Helicoverpa armigera. MicrobiologyOpen, 6(6), e00484. https://doi.org/10.1002/mbo3.484
Polanczyk, R.A., Zanuncıo, J.C., Alves, S.B. (2009) Relationship between Chemical Properties of the Soil and the Occurrence of Bacillus thuringiensis. Ciência Rural, 39(1), 1-5. https://doi.org/10.1590/S0103-84782009000100001
Carozzi, N. B., Kramer, V. C., Warren, G. W., Evola, S., & Koziel, M. G. (1991). Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles. Applied and Environmental Microbiology, 57(11), 3057–3061. https://doi.org/10.1128/aem.57.11.3057-3061.1991
Baig, D. N., & Mehnaz, S. (2010). Determination and distribution of cry-type genes in halophilc Bacillus thuringiensis isolates of Arabian Sea sedimentary rocks. Microbiological Research, 165(5), 376–383. https://doi.org/10.1016/j.micres.2009.08.003
Shishir, A., Roy, A., Islam, N., Rahman, A., Khan, S. N., & Hoq, M. M. (2014). Abundance and Diversity of Bacillus thuringiensis in Bangladesh and their cry Genes Profile. Frontiers in Environmental Science, 2(20).https://doi.org/10.3389/fenvs.2014.00020
Uemori, A., Maeda, M., Yasutake, K., Ohgushi, A., Kagoshima, K., Mizuki, E., & Ohba, M. (2007). Ubiquity of parasporin-1 producers in Bacillus thuringiensis natural populations of Japan. Naturwissenschaften, 94, 34–38. https://doi.org/10.1007/s00114-006-0153-7
Aboul-Soud, M. A. M., Al-Amri, M. Z., Kumar, A., Al-Sheikh, Y. A., Ashour, A. E., & El-Kersh, T. A. (2019). Specific Cytotoxic Effects of Parasporal Crystal Proteins Isolated from Native Saudi Arabian Bacillus thuringiensis Strains against Cervical Cancer Cells. Molecules, 24(3), 506. https://doi.org/10.3390/molecules24030506
Chubicka, T., Girija, D., Deepa, K., Salini, S., Meera, N., Raghavamenon, A. C., Divya, M. K., & Babu, T. D. (2018). A parasporin from Bacillus thuringiensis native to Peninsular India induces apoptosis in cancer cells through intrinsic pathway. Journal of Biosciences, 43(2), 407–416.https://doi.org/10.1007/s12038-018-9759-0
Moazamian, E., Bahador, N., Azarpira, N., & Rasouli, M. (2018). Anti-cancer Parasporin Toxins of New Bacillus thuringiensis Against Human Colon (HCT-116) and Blood (CCRF-CEM) Cancer Cell Lines. Current Microbiology, 75(8), 1090–1098. https://doi.org/10.1007/s00284-018-1479-z

Details

Primary Language

English

Subjects

Industrial Biotechnology (Other)

Journal Section

Research Article

Authors

Müjgan Kesik Oktay
0000-0002-0857-4872
Türkiye

Burcu Sahin
0000-0002-5701-1371
Türkiye

Hatice Güneş ^*
0000-0001-5191-365X
Türkiye

Publication Date

April 29, 2026

Submission Date

August 12, 2025

Acceptance Date

March 27, 2026

Published in Issue

Year 2026 Volume: 47 Number: 2

DOI

https://doi.org/10.17776/csj.1762728

IZ

https://izlik.org/JA56PG55GF

Cite

RIS / Bibtex

APA

Kesik Oktay, M., Sahin, B., & Güneş, H. (2026). Isolation and Screening of cry and parasporin Genes of Native Bacillus thuringiensis from Cherry Gardens in Izmir. Cumhuriyet Science Journal, 47(2), 230-235. https://doi.org/10.17776/csj.1762728