Isolation and Molecular Characterization of Bacillus thuringiensis Harboring Putative ps Genes from Bangladesh

Nasima Aktar^a, Md. Asaduzzaman Shishir^a,b, Shakila Nargis Khan^a, and Md. Mozammel Hoq^a*

Abstract

Parasporin, another type of δ- endotoxin from Bacillus thuringiensis (Bt), renowned for its highly specific and preferential toxicity against different cancer cells, could be a potentially safe anticancer therapeutic. Considering the current cancer situation, isolation and molecular characterization of parasporin-producing Bt isolates were aimed. Hence, Bt isolates were obtained through a series of tests viz. heat treatment, acetate selection, starch hydrolysis, lecithinase activity, and microscopy for parasporal proteins. Then the biochemical properties, plasmid, parasporin gene, and δ- endotoxin protein profiles were checked. Primers were designed for ps gene detection and the molecular identification of the isolates was performed by 16S rRNA gene sequence analysis. From the tested samples (n=128), 91 Bt isolates were obtained and of them, 28 non-hemolytic Bt isolates were selected as potential parasporin producers. Plasmid profile analysis of these isolates revealed that non-hemolytic Bt isolates harbor low molecular weight plasmid (6-8 Kb) unlike the Bt with insecticidal properties. Through PCR detection, amplicons of the proteins could be obtained from indigenous Bt to desired size for ps2 and ps3 genes were yielded by two isolates namely Bt DSc5 and Bt DSf3. The crude δ- endotoxin profiles and Proteinase-K digestion generated fragments of these isolates resembled previous reports. Finally, the 16S rRNA gene sequence analysis of these two isolates followed by blastn analysis confirmed them as B. thuringiensis. It is, therefore, anticipated that effective anticancer make them useful in cancer therapy.

References

Aktar, Nasima, Muhammad Manjurul Karim Rahman, Shakila Nargis Khan, Mustafizur Rahman, Anowara Begum, and Md. Mozammel Hoq. 2019. “In Silico Studies of Parasporin Proteins: Structural and Functional Insights and Proposed Cancer Cell Killing Mechanism for Parasporin 5 and 6.” Microbial Bioactives 2 (1): 82–90. https://doi.org/10.25163/microbbioacts.21007A0621280219.

Aronson, A. I., D. J. Tyrell, P. C. Fitz-James, and L. A. Bulla. 1982. “Relationship of the Syntheses of Spore Coat Protein and Parasporal Crystal Protein in Bacillus Thuringiensis.” Journal of Bacteriology 151 (1): 399–410. https://doi.org/10.1128/jb.151.1.399-410.1982.

Beegle, Clayton C., and Takashi Yamamoto. 1992. “History of Bacillus Thuringiensis Berliner Research and Development.” The Canadian Entomologist 124 (04): 587–616. https://doi.org/10.4039/Ent124587-4.

“Bergey’s Manual of Determinative Bacteriology.” 1958. Journal of the American Pharmaceutical Association (Scientific Ed.) 47 (2): 156. https://doi.org/10.1002/jps.3030470235.

Bradford, Marion M. 1976. “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.” Analytical Biochemistry 72 (May): 248–54. https://doi.org/10.1016/0003-2697(76)90527-3.

Ferdous, Umme Tamanna, Md. Asaduzzaman Shishir, Shakila Nargis Khan, and Md. Mozammel Hoq. 2018. “Bacillus Spp.: Attractive Sources of Anti-Cancer and Anti-Proliferative Biomolecules.” Microbial Bioactives 1 (1): E033–45. https://doi.org/10.25163/microbbioacts.11005B0408130818.

González-Figueredo, Carlos, René Alejandro Flores-Estrella, and Oscar A. Rojas-Rejón. 2019. “Fermentation: Metabolism, Kinetic Models, and Bioprocessing.” In Current Topics in Biochemical Engineering. IntechOpen. https://doi.org/10.5772/intechopen.82195.

Gonzalez, Eric, Jose C Granados, John D Short, David R Ammons, and Joanne Rampersad. 2011. “Parasporins from a Caribbean Island?: Evidence for a Globally Dispersed Bacillus Thuringiensis Strain,” 1643–48. https://doi.org/10.1007/s00284-011-9905-5.

González, JoséM., Howard T. Dulmage, and Bruce C. Carlton. 1981. “Correlation between Specific Plasmids and δ-Endotoxin Production in Bacillus Thuringiensis.” Plasmid 5 (3): 351–65. https://doi.org/10.1016/0147-619X(81)90010-X.

Hastowo, S., B.W. Lay, and M. Ohba. 1992. “Naturally Occurring Bacillus Thuringiensis in Indonesia.” Journal of Applied Bacteriology 73 (2): 108–13. https://doi.org/10.1111/j.1365-2672.1992.tb01695.x.

Ishii, Takaaki, and Michio Ohba. 1994. “Haemolytic Activity Associated with Parasporal Inclusion Proteins of Mosquito-Specific Bacillus Thuringiensis Soil Isolates: A Comparative Neutralization Study.” FEMS Microbiology Letters 116 (2): 195–99. https://doi.org/10.1111/j.1574-6968.1994.tb06700.x.

Ito, Akio, Yasuyuki Sasaguri, Sakae Kitada, Yoshitomo Kusaka, Kyoko Kuwano, Kenjiro Masutomi, Eiichi Mizuki, Tetsuyuki Akao, and Michio Ohba. 2004. “A Bacillus Thuringiensis Crystal Protein with Selective Cytocidal Action to Human Cells.” Journal of Biological Chemistry 279 (20): 21282–86. https://doi.org/10.1074/jbc.M401881200.

Jensen, Rasmus Bugge, and Kenn Gerdes. 1995. “Programmed Cell Death in Bacteria: Proteic Plasmid Stabilization Systems.” Molecular Microbiology. https://doi.org/10.1111/j.1365-2958.1995.mmi_17020205.x.

Kang, Donghoon, Yong Song Gho, Myungkoo Suh, and Chulhun Kang. 2002. “Highly Sensitive and Fast Protein Detection with Coomassie Brilliant Blue in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis” 23 (11): 1511–12.

Katayama, Hideki, Haruo Yokota, Tetsuyuki Akao, Osamu Nakamura, Michio Ohba, Eisuke Mekada, and Eiichi Mizuki. 2005. “Parasporin-1, a Novel Cytotoxic Protein to Human Cells from Non-Insecticidal Parasporal Inclusions of Bacillus Thuringiensis.” Journal of Biochemistry 137 (1): 17–25. https://doi.org/10.1093/jb/mvi003.

Kronstad, James W., and H.R. Whiteley. 1986. “Three Classes of Homologous Bacillus Thuringiensis Crystal-Protein Genes.” Gene 43 (1–2): 29–40. https://doi.org/10.1016/0378-1119(86)90005-3.

Martin, P A, and Russell S Travers. 1989. “Worldwide Abundance and Distribution of Bacillus Thuringiensis Isolates.” Applied and Environmental Microbiology 55 (10): 2437–42. http://aem.asm.org/content/55/10/2437.

Meadows, M. P., D. J. Ellis, J. Butt, P. Jarrett, and H. D. Burges. 1992. “Distribution, Frequency, and Diversity of Bacillus Thuringiensis in an Animal Feed Mill.” Applied and Environmental Microbiology.

Mizuki, E, M Ohba, T Akao, S Yamashita, H Saitoh, and Y S Park. 1999. “Unique Activity Associated with Non-Insecticidal Bacillus Thuringiensis Parasporal Inclusions: In Vitro Cell-Killing Action on Human Cancer Cells.” Journal of Applied Microbiology 86 (3): 477–86. https://doi.org/10.1046/j.1365-2672.1999.00692.x.

Mizuki, E, M Ohba, T Ichimatsu, S H Hwang, K Higuchi, H Saitoh, and T Akao. 1998. “Unique Appendages Associated with Spores of Bacillus Cereus Isolates.” Journal of Basic Microbiology 38 (1): 33–39. http://www.ncbi.nlm.nih.gov/pubmed/9542106.

Mizuki, Eiichi, Yu Shin Park, Hiroyuki Saitoh, Tetsuyuki Akao, Kazuhiko Higuchi, Eiichi Mizuki, Y U Shin Park, Hiroyuki Saitoh, and Satoko Yamashita. 2000. “Parasporin , a Human Leukemic Cell-Recognizing Parasporal Protein of Bacillus Thuringiensis Parasporin , a Human Leukemic Cell-Recognizing Parasporal Protein of Bacillus Thuringiensis.” https://doi.org/10.1128/CDLI.7.4.625-634.2000.Updated.

Murad, F., K. A. Alam, R. M. Mazumdar, S. Islam, M. N. Nipa, A. Iqbal, and H. R. Bhuiyan. 2010. “Anti-Bacterial Activity of the Extract of Terminalia Arjuna Against Multi Antibiotic Resistant Vibrio Cholerae.” Journal of Scientific Research 3 (1): 129. https://doi.org/10.3329/jsr.v3i1.6094.

Nadarajah, Vishna Devi, D. Ting, K. K. Chan, S. M. Mohamed, K. Kanakeswary, and H. L. Lee. 2008. “Selective Cytotoxic Activity against Leukemic Cell Lines from Mosquitocidal Bacillus Thuringiensis Parasporal Inclusions.” The Southeast Asian Journal of Tropical Medicine and Public Health 39 (2): 235–45. http://www.ncbi.nlm.nih.gov/pubmed/18564708.

Nagamatsu, Yasunori, Shoutarou Okamura, Hiroyuki Saitou, Tetsuyuki Akao, and Eiichi Mizuki. 2010. “Three Cry Toxins in Two Types from Bacillus Thuringiensis Strain M019 Preferentially Kill Human Hepatocyte Cancer and Uterus Cervix Cancer Cells.” Bioscience, Biotechnology, and Biochemistry 74 (3): 494–98. https://doi.org/10.1271/bbb.90615.

Ohba, Michio, and Keio Aizawa. 1986. “Insect Toxicity of Bacillus Thuringiensis Isolated from Soils of Japan.” Journal of Invertebrate Pathology. https://doi.org/10.1016/0022-2011(86)90158-8.

Ohba, Michio, Eiichi Mizuki, and Akiko Uemori. 2009. “Parasporin, a New Anticancer Protein Group from Bacillus Thuringiensis.” Anticancer Research 29 (1): 427–33.

Okassov, Almas, Armen Nersesyan, Sakae Kitada, and Alexander Ilin. 2015. “Parasporins as New Natural Anticancer Agents: A Review.” Journal of Balkan Union of Oncology 20 (1): 5–16.

Okumura, S., M. Ohba, E. Mizuki, N. Crickmore, J.-C. Côté, Y. Nagamatsu, S. Kitada, H. Sakai, K. Harata, and T. Shin. 2010. “Parasporin Nomenclature.” 2010. http://parasporin.fitc.pref.fukuoka.jp/.

Poornima, K, P Selvanayagam, and R Shenbagarathai. 2010. “Identification of Native Bacillus Thuringiensis Strain from South India Having Specific Cytocidal Activity against Cancer Cells” 4: 348–54. https://doi.org/10.1111/j.1365-2672.2010.04697.x.

Poornima, Kkani, Viswanathan Saranya, Periyasamy Abirami, Chandrasekaran Binuramesh, Ponnusamy Suguna, Peter Selvanayagam, and Rajaiah Shenbagarathai. 2012. “Phenotypic and Genotypic Characterization of B.t.LDC-391 Strain That Produce Cytocidal Proteins against Human Cancer Cells.” Bioinformation 8 (10): 461–65. https://doi.org/10.6026/97320630008461.

Prado, Odimári Pricila Pires do, Lucia Maria Zeoula, Lucimar Pontara Peres de Moura, Selma Lucy Franco, Sara Barbosa de Paiva, and Pedro Braga Arcuri. 2010. “Isolation and Expeditious Morphological, Biochemical and Kinetic Characterization of Propolis-Tolerant Ruminal Bacteria.” Revista Brasileira de Zootecnia. https://doi.org/10.1590/s1516-35982010000900025.

Rampersad, Joanne, Ayub Khan, and David Ammons. 2002. “Usefulness of Staining Parasporal Bodies When Screening for Bacillus Thuringiensis.” Journal of Invertebrate Pathology 79 (3): 203–4. https://doi.org/10.1016/S0022-2011(02)00018-6.

Saitoh, Higuchi, Mizuki, Hwang, and Ohba. 1998. “Characterization of Mosquito Larvicidal Parasporal Inclusions of a Bacillus Thuringiensis Serovar Higo Strain.” Journal of Applied Microbiology 84 (5): 883–88. https://doi.org/10.1046/j.1365-2672.1998.00426.x.

Shishir, Asaduzzaman, Arpita Roy, Namista Islam, Alamgir Rahman, Shakila Nargis Khan, and Md Mozammel Hoq. 2014. “Abundance and Diversity of Bacillus Thuringiensis in Bangladesh and Their Cry Genes Profile.” Frontiers in Environmental Science 2 (June): 20. https://doi.org/10.3389/fenvs.2014.00020.

Shishir, Md. Asaduzzaman, Asma Akter, Md. Bodiuzzaman, Nasima Aktar, Mushfiqur Rahman, Shakil Ahmed Khan, Mohammad Ilias, Shakila Nargis Khan, and M. Mozammel Hoq. 2012. “Molecular Characterization of Indigenous Bacillus Thuringiensis Kurstaki Isolates with Effective Pesticidal Activity against Bactrocera Cucurbitae from Bangladesh.” In Proceddings of 1st AFSA Conferences, edited by Ryohei Kada, Jin-ichi Sasaki, Chowdhury Rafiqul Ahsan, and Md. Latiful Bari, 1:143–48. Osaka, Japan: Asian Food Safety & Security Association (AFSSA). http://www.afsahome.org/wp-content/uploads/2017/05/Food_Safety_and_Food_Security_2012-00.pdf.

Shishir, Md. Asaduzzaman, Asma Akter, Md. Bodiuzzaman, M. Aftab Hossain, Md. Musfiqul Alam, Shakil Ahmed Khan, Shakila Nargis Khan, and M. Mozammel Hoq. 2015. “Novel Toxicity of Bacillus Thuringiensis Strains against the Melon Fruit Fly, Bactrocera Cucurbitae (Diptera: Tephritidae).” Biocontrol Science 20 (2): 115–23. https://doi.org/10.4265/bio.20.115.

Travers, R S, P A Martin, and C F Reichelderfer. 1987. “Selective Process for Efficient Isolation of Soil Bacillus Spp.” Applied and Environmental Microbiology 53 (6): 1263–66. http://www.ncbi.nlm.nih.gov/pubmed/16347359%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC203852.

Trevors, J. T. 1984. “A Rapid Method for the Isolation of Plasmid DNA from Bacteria.” Biotechnology Letters 6 (7): 457–60. https://doi.org/10.1007/BF00129309.

WHO media center. 2017. “Cancer Fact Sheet.” 2017. http://www.who.int/mediacentre/factsheets/fs297/en/.