Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1996 The yC toplasmic N,n'-Diacetylchitobiase Gene From Vibrio Parahaemolyticus: Sequence Analysis, Protein Secretion, and Secretion System Development. Michael Hongli Wu Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Wu, Michael Hongli, "The yC toplasmic N,n'-Diacetylchitobiase Gene From Vibrio Parahaemolyticus: Sequence Analysis, Protein Secretion, and Secretion System Development." (1996). LSU Historical Dissertations and Theses. 6171. https://digitalcommons.lsu.edu/gradschool_disstheses/6171 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced 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 comer 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. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 THE CYTOPLASMIC N,iV-DLACETYLCHITOBIASE GENE FROM VIBRIO PARAHAEMOLYTICUS: SEQUENCE ANALYSIS, PROTEIN SECRETION, AND SECRETION SYSTEM DEVELOPMENT A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biochemistry by Michael Hongli Wu B.S., Beijing Forestry University, P. R. China, 1982 M.S., University of California, Riverside, CA, 1989 May 1996 UMI Number: 9628325 Copyright 1996 by Wu, Michael Honglr All rights reserved. UMI Microform 9628325 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ©Copyright 1996 Michael Hongli Wu All rights reserved ACKNOWLEDGMENTS I am extremely grateful to my major professor, Dr. Roger A. Laine, for his understanding, patience, encouragement, and general support dur­ ing this study and the preparation of manuscripts and this dissertation. He never seems to run out of new ideas that sometimes, somewhere, will shine some light on someone's project in the lab. I am also indebted to other members of my advisory committee, Drs. William H. Daly (Chemistry), Donal F. Day (Audubon Sugar Institute), Ding S. Shih (Biochemistry), Patrick J. DiMario (Biochemistry), Jan L. VanSteenhouse (Veterinary Pathology, School of Veterinary medicine) for their valuable suggestions, corrections, and other efforts during re­ viewing this dissertation. In addition, I also highly appreciate Drs. Simon H. Chang (Biochemistry) and Jesse M. Jaynes (Formerly Biochemistry) for their general support and technical assistance to my research program while on my committee. My gratitude is extended to Dr. Betty C. R. Zhu for her assistance and valuable suggestions that are always available. Last, but certainly not the least, I would like to express my gratitude to my wife, Kathy G. Wu. Without her understanding, patience and support, this study would not have been completed. I'm also in debt to my two sons, Peter and Clifford, with whom I should have played more as I al­ ways promised. TABLE OF CONTENTS ACKNOWLEDGMENTS.........................................................................................iii LIST OF FIGURES.......................................................................................................v ABSTRACT.................................................................................................................vi CHAPTER 1 REVIEW OF THE LITERATURE....................................................1 1.1 INTRODUCTION.................................................................................... 1 1.2 CH ITIN...................................................................................................... 2 1.3 CHITIN UTILIZATION BY MARINE BACTERIA........................ 3 1.4 PROTEIN SECRETION.........................................................................8 1.5 OVEREXPRESSION OF CLONED GENES......................................29 1.6 PCR AND RECOMBINANT DNA....................................................36 CHAPTER 2 SEQUENCING AND SEQUENCE ANALYSIS..................... 48 2.1 INTRODUCTION..................................................................................48 2.2 MATERIALS AND METHODS............................................................48 2.3 RESULTS................................................................................................. 50 2.4 DISCUSSION........................................................................................... 65 2.5 SUMMARY............................................................................................. 69 CHAPTER 3 PROTEIN SECRETION................................................................... 71 3.1 INTRODUCTION.................................................................................. 71 3.2 MATERIALS.......................................................................................... 72 3.3 EXPERIMENTAL PROCEDURES AND RESULTS.....................73 3.4 DISCUSION............................................................................................ 91 3.5 SUMMARY............................................................................................. 95 CHAPTER 4 CONCLUSIONS.............................................................................. 98 REFERENCES......................................................................................................... 101 APPENDIXES............................................................................................................127 A.l SEQUENCING AND PCR PRIMERS...........................................127 A.2 SIGNAL SEQUENCES USED IN THIS STUDY........................128 A.3 SECRETION VECTOR SEQUENCES...........................................129 A.4 GENOTYPES OF SOME HOST STRAINS...................................131 A.5 STRUCTURES OF RELEVENT COMPOUNDS.........................132 A.6 LIST OF ABBREVIATIONS...........................................................137 VITA ...........................................................................................................................138 LIST OF FIGURES Fig. 1 Chi tin degradation pathways in vibrios .....................................................6 Fig. 2 Construction of the tac promoter ............................................................30 Fig. 3 Vectorette and its application..................................................................40 Fig. 4 Construction of a possible universal vectorette .................................. 41 Fig. 5 Restriction map and nucleotide sequencing strategy ...................... 52 Fig. 6 Nucleotide sequence of the chitobiase gene ..........................................53 Fig. 7 Amino acid alignment of three vibrio chitobiases ................................ 56 Fig. 8 Amino acid sequence pileup ....................................................................60 Fig. 9 Hydrophobicity analysis .............................................................................67 Fig. 10 In-frame connection by recombinant PCR .............................................. 75 Fig. 11 Two clones for the secretion of cytoplasmic chitobiase .................... 77 Fig. 12 Secretion and protein leakage .....................................................................79 Fig. 13 Silent mutagenesis .....................................................................................84 Fig. 14 Construction of pVerSec and pVerSec-SE ............................................85 Fig. 15 Application of secretion vectors ..................................................................87 Fig. 16 Effects of EDTA on protein protection and host growth ................... 88 Fig. 17 Chitobiase activity and EDTA concentration ...................................... 89 v ABSTRACT The nucleotide sequence of the gene encoding the cytoplasmic