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Isolation and Characterization of a Rhodobacter Capsulatus Hemc Mutant

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Isolation and Characterization of a Rhodobacter Capsulatus Hemc Mutant Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 2001 Isolation and Characterization of a Rhodobacter Capsulatus hemC Mutant. Karen Coleman Sullivan Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Sullivan, Karen Coleman, "Isolation and Characterization of a Rhodobacter Capsulatus hemC Mutant." (2001). LSU Historical Dissertations and Theses. 251. https://digitalcommons.lsu.edu/gradschool_disstheses/251 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. 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. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ISOLATION AND CHARACTERIZATION OF A RHODOBACTER CAPSULATUS HEMC MUTANT 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 Biological Sciences by Karen Coleman Sullivan B.S., Nicholls State University, 1991 May 2001 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3010384 UMI___ ® UMI Microform 3010384 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION In Loving Memory of my Uncle Mike Sullivan and To my Wonderful Family, Troy, Brandon, and Cole. ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS It is impossible to adequately express my deep appreciation for my best friend, my husband, Troy. Without your love, support, and encouragement I would never have completed this degree program. Thank you for being so patient and understanding. I would like to express my love and gratitude to my children, Brandon and Cole, who have spent countless hours at LSU waiting for Mom to finish her work. You can be proud now that Mom has finally completed “20th grade”. Special thanks to my parents for their love and encouragement. I am deeply indebted to you for taking care of Brandon and Cole when they had chicken pox so that I could go to class and for babysitting whenever I needed some time to regain my sanity. Also, thanks to my in-laws for their unending encouragement. Your love and support mean the world to me. I would like to thank my advisor, Dr. Alan Biel, for advice and encouragement. I appreciate all the time and effort that you have put into my graduate career. Thanks for putting up with my occasional tears. I will never be able to eat at McDonalds without thinking of you. Thanks also to my other committee members: Dr. LaBonte, Dr. Jarosik, Dr. Dimario, and especially Dr. Achberger for his eternal patience and kindness. There are several former members of our lab who will always have a special place in my heart: Keith Canada, Alok Khanna, and Katya Kanazireva. Thank you all for teaching me so much and for friendships I will never forget. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS DED IC ATIO N.............................................................................................................ii ACKNOWLEDGMENTS....................................................................................... iii LIST OF TABLES.................................................................................................. vi LIST OF FIGURES.................................................................................................. vii ABSTRACT.............................................................................................................. viii INTRODUCTION ...................................................................................................... 1 Tetrapyrrole Pathway of R. cap su/a tu...................................................... s 2 Tetrapyrrole Biosynthesis Regulation ...................................................... 13 Oxygen Regulation of Bacteriochlorophyll Synthesis ............................ 16 Oxygen Regulation of the Tetrapyrrole Pathway ..................................... 17 MATERIALS AND METHODS .............................................................................. 22 Strains and Plasmids ...................................................................................22 M e d ia ............................................................................................................ 22 Growth Conditions.......................................................................................22 Chromosomal DNA Isolation ..................................................................... 25 Plasmid DNA Isolation ................................................................................ 25 Restriction Enzyme Digestion ................................................................... 26 DNA Ligation ................................................................................................26 Fill-in Reaction ............................................................................................. 27 Nick Translation ........................................................................................... 27 Agarose Gel Electrophoresis ..................................................................... 28 DNA Recovery from Agarose Gels .......................................................... 28 Electroporation ............................................................................................. 28 Conjugation ..................................................................................................29 Southern Hybridization .............................................................................. 30 Protein Determination ................................................................................ 31 Protein Determination by Dye Binding ......................................... 31 Lowry Protein Determination...........................................................31 Preparation of Cell Extracts ........................................................................32 Quantitation of Bacteriochlorophyll .......................................................... 33 Aminolevulinate Synthase Assay ...............................................................33 Porphobilinogen Synthase Assay .............................................................34 Porphobilinogen Deaminase Assay .......................................................... 35 Measurement of Porphyrin Accumulation ................................................35 Measurement of Porphobilinogen Accumulation ...................................36 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS ................................................................................................................. 37 Crossfeeding Studies.................................................................................... 37 Growth of E. coli hem Mutants Using Exogenous Protoporphyrin and H em in...............................................................................................................37 Growth of an R. capsufatus hemA Mutant on Exogenous Hemin .... 42 Isolation of an R. capsufatus hemC M u ta n t .............................................. 42 Chromosomal Location of the Kanamycin Cartridge ...............................45 Bacteriochlorophyll
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