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INFORMATION TO USERS The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9031070 Hyperexpression ofBacillus a thuringiensis delta-endotoxin gene in Escherichia coli and localization of its specificity domain Ge, Zhixing Albert, Ph.D. The Ohio State University, 1990 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 HYPEREXPRESSION OF A Bacillus thuringiensis DELTA-ENDOTOXIN GENE IN Escherichia coli AND LOCALIZATION OF ITS SPECIFICITY DOMAIN DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Albert Zhixing Ge, B.S., M.S. ***** The Ohio State University 1990 Dissertation Committee: Approved by Charles J. Daniels Donald H. Dean Robert M. Pfister ^ Q v ijx J L ^ GP.-dyK^ William R. Strohl Advisor Department of Microbiology To The Memory Of My Father To My Mother ACKNOWLEDGMENTS I wish to express my sincere appreciation to my advisor, Dr. Donald H. Dean, for taking me as his student, and for his continuous support, encouragement, and patience throughout the course of this study. It was under his guidance and care that I evolved to become a scientist with some business sense, and I don’t know where else I could have spent my past five years that were more enjoyable and worthwhile. I would also like to thank Drs. Charles J. Daniels, Robert M. Pfister, and William R. Strohl for their valuable advice and serving on my Dissertation Committee. Thanks also go to the past and present members of Dr. Dean’s lab: Brian Almond, Thomas Boyle, Karen Butsch, Chih-I Cheng, George Chen, April Curtiss, Ching Dai, Mary Beth Dunn, Michael Grouse, John Hopper, In-Seok Kwak, Mi Lee, Jim McLinden, Dave Rivers, Josanne Sabourin, and Comrade Dan Zeigler for their assistance, ideas, understanding, and friendship. Finally, I want to thank my brother and sister for their encouragement and support whenever I needed them, and from which I gained confidence and comfort. VITA November 7,1956 Born - Shanghai, China 1978 -1980 ......... Shanghai No.2 Medical University Shanghai, China 1981 -1982 ......... B.S., Microbiology, Eastern Michigan University, Ypsilanti, Michigan 1983-1984 ......... Graduate Research Associate, Departments of Chemical Engineering, and Molecular and Cellular Biology The University of Michigan Ann Arbor, Michigan 1982-1984 ......... M.S., Bioengineering, The University of Michigan, Ann Arbor, Michigan 1985-198 6 ......... Graduate Teaching Assistant, Department of Microbiology, The Ohio State University Columbus, Ohio 1986-198 8 ......... M.B.A., Marketing, College of Business The Ohio State University Columbus, Ohio 1986-1989 ......... Graduate Research Associate Department of Microbiology The Ohio State University Columbus, Ohio PUBLICATIONS Ge, A.Z., Shivarova, N.I., and Dean, D.H.:Location of the Bombyxmori Specificity Domain on a Bacillus thuringiensis delta-endotoxin Protein. Proc. Natl. Acad. Sci. USA 86 (1989) 4037-4041. Ge, A.Z., Pfister, P.M. and Dean, D.H.: Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli : properties of the product. Gene, (1990). Milne, R., Ge, A.Z., Rivers, D., and Dean, D.H.: Specificity of Insecticidal Crystal Proteins, Implications For Industrial Standardization. L. Hickle and W. Fitch (eds), American Chemical Society Symposium (1990). FIELD OF STUDY Microbiology v TABLE OF CONTENTS Dedication ii Acknowledgments iii Vita iv List of Tables viii List of Figures ix Introduction 1 Literature Review 4 B. thuringiensis and its insecticidal crystal protein 4 The diversity of B. thuringiensis crystal proteins 6 Classification of B. thuringiensis and its crystal protein genes 7 Extrinsic factors affecting the specificity of crystal proteins 10 Intrinsic factors affecting the specificity of crystal proteins 16 Mode of action of B. thuringiensis delta-endotoxin 21 References 24 Part One 31 Introduction 32 Material and methods 34 Strain and growth condition 34 Delta-endotoxin gene, plasmids and restriction enzyme 34 Cloning, transformation and electrophoresis 36 Crystal toxin purification 36 Protein samples preparation 37 Enzyme-linked immunosorbant assay (ELISA) 37 Site-directed mutagenesis and DNA sequencing 38 Results 39 Effects of promoters on expression of cry gene 39 Effect of E. coli host strains on expression 45 Effect of other genetic elements on expression 45 Physical and biological properties of crystals made in E. coli 52 Discussion 52 Conclusion 60 References 61 vi Part Two 64 Introduction 65 Material and Methods 72 Original of delta-endotoxin genes, host and vectors 72 Site-directed mutagenesis and other molecular genetics techniques 72 Construction of site-directed mutants, substitution mutants and overexpression mutants 73 Purification of overexpressed crystal proteins 76 Bioassays 77 Results 78 Specificity of delta-endotoxin 78 Location of specificity domain for B. mori 80 Discussion 84 Conclusion 92 References 93 vii LIST OF TABLES TABLE PAGE 1.1 Classification of crystal protein toxin genes of B. thuringiensis 9 1.2 Midgut pH and host plant for lepidopteran larvae 11 2.1 Escherichia coli strains and genotypes 35 2.2 Expression of crylA(c)73 in Escherichia coli 44 2.3 Expression of crylA(c)73 by Ptac/pKK223-3 in various host strains 46 2.4 Percentage of plasmid (pUC & pBR) retention in Escherichia coli strain JM103 53 3.1 Specificity of insecticidal crystal proteins on selected insects 79 viii LIST OF FIGURES FIGURES PAGE 1.0 Positions of five conserved amino acids blocks on Bacillus thuringiensis delta-endotoxins 17 2.1 Cloning scheme of cry1A(c)73 into pKK223-3, pUC19 and pET-3a 40 2.2 Polyacrylamide gel electrophoresis of overexpressed proteins 42 2.3 Microscopic pictures of E. coli strain overexpressing crylA(c)73 and B. thuringiensis HD-73 47 2.4 The mutated 5’DNA sequence of crylA(c)73 50 2.5 Escherichia coli cell expressing crylA(c)73 54 3.1 Comparison of amino acid sequences of cryiA(a), crylA(b), and crylA(c) 67 3.2 Amino acid sequences of the five conserved blocks on B. thuringiensis delta-endotoxins 69 3.3 Construction of substitution mutations and expression plasmid of cry genes 74 3.4 Creation of different hybrid delta-endotoxins between CrylA(a)1 and CrylA(c)73 81 3.5 Computer based plots of protein secondary structures 87 ix INTRODUCTION Bacillus thuringiensis is a microorganism of considerable economic importance. The key commercial value of B. thuringiensis centers on the delta-endotoxin produced by the cells during their life cycle. B. thuringiensis delta-endotoxin can be crystallized within the sporangium and has been shown to be toxic to many damaging insects such as Manduca sexta (tobacco horn worm), Trichoplusia ni (cabbage looper), Heliothis zea (corn ear worm), Aedes aegypti (mosquitoes), and Leptinosarsa decemlineata (Colorado potato beetle) (Hall et al., 1977; de barjac, H., 1978; Dulmage, et al., 1981; Aronson, et al., 1986; Andrews, et al., 1987; McPherson, et al., 1988). With its natural protein nature and broad insecticidal spectrum, B. thuringiensis delta-endotoxin has demonstrated a great potential to replace some synthetic, chemical insecticides that may have long term detrimental consequence to our environment. The mode of action of delta-endotoxin (crystal protein) is an intriguing problem yet to be solved. Crystal proteins from various strains of B. thuringiensis have molecular weight ranging from 25,000 to 130,000 daltons, and possess different host specificities against insects. Current studies on crystal proteins reveal that despite size differences, there are conserved and variable domains on different crystal proteins. The functional significance of the conserved and variable domains is not clear, and is a subject of research pursued by many groups 1 2 around the world. It is speculated that the variable domains on crystal proteins may associate with protein-receptor interaction and as a result, affect the specificity of crystal proteins. As a first attempt to understand the functional domains of crystal proteins, it is desirable to know what differentiates the specificities among crystal proteins. One of the goals of this research, therefore, is to define the specificity domains of crystal proteins. To accomplish this goal, two B. thuringiensis crystal proteins (CrylA(a) and CrylA(c)) were chosen as model proteins. CrylA(a) and CrylA(c) are both lepidoptera specific, and they share more than 80% overall sequence homology. Both toxins have about the same activity against M. sexta. However CrylA(a) is more than 100 times active than CrylA(c) against Bombyx mori (silk worm). CrylA(c), on the other hand, is about 55 times more effective toward Heliothis virescens (tobacco bud worm) than CrylA(a) (Ge et al., 1989).