Identification of bacillus thuringiensis based on biologycal tests and novel biomarkers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATE THESIS TOPIC: IDENTIFICATION OF BACILLUS THURINGIENSIS BASED ON BIOLOGICAL TESTS AND NOVEL BIOMARKERS Student : Do Duc Manh Student code : 620513 Class : K62CNSHE Supervisor : Nong Thi Hue, Ph.D Ha Noi-2022 ACKNOWLEDGEMENTS This thesis would have been possible without the advice and support of many people, especially the teachers and staffs at the faculty of Biotechnology I would like to express my sincere gratitude to all of them I would like to thank all the people inside and outside the Faculty of Biotechnology, especially the department of Plant Biotechnology, for their great support in completing my graduation thesis I would like to express my heartfelt thanks and respect to my academic supervisor Dr Nong Thi Hue, for her enthusiastic support and useful suggestions for my graduation thesis I would like to express my thankfulness to Dr Ninh Thi Thao, Dr Dinh Truong Son, and all the lecturers and staff at the Department of Plant Biotechnology for their nice advice and greatly helps during my study I want to thank my family and friends for all their help, encouragement, and supporting me over the past years I would not be able to finish this experiment without supports from all of you Due to the limited time to implement the thesis, my thesis cannot avoid errors I look forward to receiving your comments and suggestions to improve my thesis I sincerely thank! Ha Noi, May 22, 2022 Student Do Duc Manh i COMMITMENT I hereby declare that this is my research work The figures and image results, which are presented in the thesis, are truthful and have not been published in other scientific studies I hereby declare that all research contents, conclusions, and information in my graduation thesis are entirely honest and unpublished Ha Noi, May 22, 2022 Student Do Duc Manh ii OUTLINE ACKNOWLEDGEMENTS i COMMITMENT ii OUTLINE iii LIST OF ABBREVIATIONS vi LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT ix PART I INTRODUCTION 1.1 Preface 1.2 Objectives and Requirements 1.2.1 Objectives 1.2.2 Requirements PART II: LITERATURE OVERVIEW 2.1 Overview of Bacillus Thuringiensis 2.1.1 Taxonomy 2.1.2 Distribution of Bt 2.1.3 Morphological characteristics 2.1.4 Biochemical characterization 2.1.5 Classification of Bt subspecies 2.2 Bt toxicity and crystalline protein mode of action 2.2.1 Bt toxicity 2.2.2 Action mechanism of crystalline proteins on insects 10 2.3 History of Bt and its commercial applications 11 2.4 Research for identification of Bacillus thuringiensis 14 2.4.1 Conventional methods 14 PART III MATERIALS AND METHODS 18 iii 3.1 Materials 18 3.1.1 Bacterial isolates 18 3.1.2 PCR-based molecular markers 19 3.2 Location and time 20 3.3 Research contents 20 3.3.1 Identification of Bacillus thuringiensis by morphology and biochemical properties 20 3.3.2 Determination of method for bacterial DNA extraction 20 3.3.3 Identification of Bacillus thuringiensis based on species-specific PCR analysis 20 3.3.4 Confimation of Bacillus thuringiensis by crystalline protein staining 21 3.4 Research methodology 21 3.4.1 Bacteria cultures preparation 21 3.4.2 Morphological characteristics 21 3.4.3 Biochemical assays 22 3.4.4 Bacterial DNA extraction 23 3.4.5 Species-Specific PCR assays 26 3.4.6 Electrophoresis 27 3.4.7 Crystalline Protein Staining 28 3.4.8 Medium and chemical 29 PART IV RESULTS AND DISCUSSION 31 4.1 Identification of Bacillus thuringiensis by morphology and biochemical properties 31 4.1.1 Colony morphological characterization 31 4.1.2 Cell morphological characterization 33 4.1.3 Biochemical assays 34 4.2 Determination of method for baterial DNA extraction 36 iv 4.3 Identification of Bacillus thuringiensis based on species-specific PCR analysis 41 4.4 Confirmatory identification of Bacillus thuringiensis by crystalline protein staining 47 PART V CONCLUSIONS AND PROPOSALS 50 5.1 Conclusions 50 5.2 Proposals 50 REFERENCES 51 APPENDIX 56 v LIST OF ABBREVIATIONS Bt : Bacillus Thuringiensis DNA : Deoxyribonucleic acid PCR : Polymerase chain reaction GM : Genetically Modified UV : Ultraviolet LB : Luria Bertani broth CTAB : Cetyl trimethylammonium bromide RNA : Ribonucleic acid EDTA : Ethylenediaminetetraacetic acid SDS : Sodium dodecyl sulfate βME : 2-Mercaptoethanol MYP : Mannitol egg yolk polymyxin vi LIST OF TABLES Table 2.1 Current classification of 67 Bacillus thuringiensis subspecies based on their flagellar (H) antigens Table 3.1 Bacterial strains used in the study 18 Table 3.2 PCR primers used in this study 19 Table 3.3 Components of PCR reaction 26 Table 3.4 Thermal cycler conditions 27 Table 4.1 Colony morphological characteristics of Bt isolates on T3 solid medium 31 Table 4.2 Biochemical test results of isolates of Bt bacteria 35 Table 4.3 OD260/280 index results and concentration of isolates 38 Table 4.4 Estimated time for bacterial DNA extraction 41 vii LIST OF FIGURES Figure 2.1 Spore–crystal morphology of Bacillus Thuringiensis Figure 2.2a 3D structure of Cry 2Aa protein Figure 2.2b Mechanism of action of toxins 10 Figure 3.1: Systemic representation of Bacillus thuringiensis identification 21 Figure 4.1 Morphology of B thuringiensis colonies isolates on T3 solid medium 32 Figure 4.2 Cell morphological characteristics of Bt isolates 33 Figure 4.3a Lecithinase test results 35 Figure 4.3b Catalase test results 35 Figure 4.3c Results of sucrose fermentation 36 Figure 4.4a Heat treatment method for bacterial DNA extraction 39 Figure 4.4b CTAB method for bacterial DNA extraction 40 Figure 4.4c Lysozyme and CTAB-based method for bacterial DNA extraction 40 Figure 4.5a PCR product with primers specific for the XRE gene 42 Figure 4.5b PCR product with primers specific for the gyrB gene 43 Figure 4.5c PCR product with primers specific for the GroEL gene 44 Figure 4.5d PCR product with primers specific for the Cry2 gene 45 Figure 4.6 PCR products with combination of XRE, gyrB, GroEL primers 46 Figure 4.7 Spores and crystals of some Bt isolates 48 viii ABSTRACT Bacillus thuringiensis (Bt) is commonly used as a biopesticide worldwide, due to its ability to produce insecticidal protein crystals during sporulation The aim of the study was to use specific biomarkers to identify Bacillus thuringiensis A total of 11 strains, comprising of reference Bt, isolated Bt and non-Bt groups (Bacillus subtilis, Bacillus amyloliquefaciens, Staphylococcus aureus and E.coli were successfully identified by applying biological test and valid biomarkers (XRE, groEL and gyrB) Cry2 is the most common crystal protein presented in B thuringiensis was used to confirm the presence of Bt The collective identification results revealed all isolates belong to B thuringiensis In detail: - The results of the morphological and biochemical analysis showed that all the isolated Bt were Gram-positive rods and spore former, having a positivreactionson with lecithinase, catalase and sucrose that are basic characteristics of Bacillus species - The total DNA was extracted by three methods including the CTAB method, heat treatment method, and lysozyme based-method The result indicated the boiling-based method appeared to be simple and efficient in getting total DNA for PCR reaction - Four species-specific genes, including XRE (transcriptional regulator), GroEL (chaperonin protein), GyrB (topoisomerase enzyme), and Cry2 (crystal protein), were investigated All the genes showed 100% specificity towards detection of B thuringiensis with, specific band size and no specificity towards any of the strains of non-Bt group Therefore, accompanying with morphological and biochemical analysis, PCR reaction targeting XRE, GroEL GyrB can be used as a reliable molecular tool to identify B thuringiensis ix Figure 4.5d PCR product with primers specific for the Cry2 gene Blank control: DW (distilled water); 4T1, 4T4 , 4D4: positive control; non-Bt groups: ATCC79530 (Bacillus subtilis), NT 1.8 (B amyloliquefaciens), ATCC 25923 (S aureus ), ATCC 85922 (E.coli), (standard strain): And the isolates are V, E, S, B, T1, DT: Bt isolates 100bp DNA ladder The results in this study are consistent with the findings of Wei et al (2019) and Chellian et al., (2019) showing the high specificity of these primers for identifying B thuringiensis Chellian et al., (2019) used six selective genes (lipoprotein-lipo, methyltransferase-MT, S-layer homology domain protein-BC, flagellar motor protein-motB, transcriptional regulator-XRE, crystal protein-cry2) and two housekeeping genes (chaperonin protein-GroEL and topoisomerase enzyme-gyrB) to discriminate Bacillus thuringiensis from Bacillus cereus group Assessment of 120 strains, both GroEL and gyrB showed 100% specificity towards detection of both B thuringiensis in artificially spiked vegetable samples All the eight genes revealed no specificity towards any of the non-Bacillus strains Analysis of 120 above strains, the results from Wei et al (2019) showed that 97.5% targeting XRE, and 83.3% targeting cry2 45  PCR test for identification of Bacillus thuringiensis using mixture of primers In order to simplify PCR assay, multiplex PCR for simultaneous identification of microorganisms including Bacillus thuringiensis will be targeted In this PCR trial test using, primer pair XRE, gyrB and GroEL were individually combined in one reaction The PCR reaction were conducted using DNA template of strains including 4T1 (standard strain), V (isolation), and NT 1.8 (B amyloliquefaciens) (non-Bt group) The annealing temperature (Ta) was 55oC The result showed that there were no secondary structure such as primer-dimer or cross-dimer or any nonspecific band found Specific band ~246 bp fragment (XRE gene), ~ 600 bp (GroEL) and ~221bp (gryB gene) were still amplified and detected only in standard B thuringiensis strains (4T1) and Bt isolate (V), but not non-Bt group NT1.8 (B Amyloliquefaciens) Next, multiplex PCR assay using different species-specific primers for other Bacillus group and non-Bt group will be examined GroEL gene (500bp) XRE gene (246bp) gryB gene (221bp) Figure 4.6 PCR products with combination of XRE, gyrB, GroEL primers 46 Blank control: DW (distilled water); 4T1: positive control; non-Bt groups: NT 1.8 (B amyloliquefaciens) (standard strain): And the isolates are V: Bt isolates 100bp DNA ladder 4.4 Confirmatory identification of Bacillus thuringiensis by crystalline protein staining Determination of the ability to produce toxic crystals and spores of putative Bt strains It has been well-documented that B thuringiensis selectively produces insecticidal crystal proteins, which can be utilized as an appropriate marker for proper identification In the present study, PCR bands of 700 bp corresponding to cry2 gene were achieved for Bt isolates and the standard isolates Therefore, we expected presence of crystal proteins in all the Bt isolates To test the ability to produce toxic crystals and spores, colonies on T3 medium were tested for the endospore production ability through Coomassie brilliant blue solution and observed under the microscope The results of spores and crystals of all the Bt isolates were shown in Figure 4.7 Description of crystals and spores The isolates showed crystal and spore characteristics like those of standard Bt strains, including: - Crystals are rhombic, round, eccentric, and many other shapes Full-color capture - Cylindrical or ovoid spores, one end is colored, and the other is nonstaining, whether internal or external - Cells are entirely stained and cylindrical 47 4T1 V T1 E DT S B Figure 4.7 Spores and crystals of some Bt isolates 4T1: Standard strain; V,B,T1, S,E,DT: Bt isolates The results of crystal protein staining showed the presence of these crystal proteins in Bt isolates It can be clearly seen that there were similarities in crystal shape and spore characteristics similar to those of standard Bt strains Al the 48 isolates produced rhombohedral crystals - the most common shape of crystals protein of Bt Collectively, all the results (morphological and biochemical characteristics, species-specific PCR, and crystal protein staining) affirmed isolates as B thuringiensis 49 PART V CONCLUSIONS AND PROPOSALS 5.1 Conclusions (1) Morphological and biochemical analysis indicates that Bt isolates displayed a single colony type, gram positive, showed positive activity with lethicinase, sucrose and catalase tests and formed spores that are the characteristics of Bacillus species, especially Bacillus thuringiensis (2) Heat treatment method appeared to be simple, efficient and convinient method for bacterial DNA extraction (3) Species-specific PCR analysis with XRE, GroEL, GyrB and Cry2 gene show high specificity towards B thuringiensis (4) There was the presence of crystal proteins in Bt isolates using crystal protein staining, affirming isolates as B thuringiensis 5.2 Proposals  Futher biochemical tests should be included - Esculin test - Salicin fermentation test  Develop a multiplex PCR assay for identification of Bacillus thuringiensis 50 REFERENCES Vietnamese references Đái Duy Ban (2006), Công nghệ gen, Nhà xuất khoa học kỹ thuật Ngơ Đình Bính, Nguyễn Quang Châu, Nguyễn Ánh Nguyệt (2002), Thu nhận huyết miễn dịch cho phân loại Bacillus thuringiensis, Kỷ yếu 2001 -2002, Viện Công nghệ Sinh học, trang 296 – 302 Ngơ Đình Bính (2005),Giáo trình thuốc trừ sâu sinh học Ngơ Đình Bính cộng sự, 35 năm nghiên cứu phát 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41 (2): pp117-122 55 APPENDIX Appendix Catalase test results of six strains 56 Appendix Test results on sucrose fermentation medium of six strains 57 Appendix Lecithinase test results of six strains 58 Appendix Crystals and spores of six putative Bt strains 59