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Molecular Cloning, Sequencing, and Regulation of the Rhodobacter Capsulatushemb Gene

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Molecular Cloning, Sequencing, and Regulation of the Rhodobacter Capsulatushemb Gene Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1995 Molecular Cloning, Sequencing, and Regulation of the Rhodobacter capsulatushemB Gene. Karl Joseph Indest Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Indest, Karl Joseph, "Molecular Cloning, Sequencing, and Regulation of the Rhodobacter capsulatushemB Gene." (1995). LSU Historical Dissertations and Theses. 6021. https://digitalcommons.lsu.edu/gradschool_disstheses/6021 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 afreet 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. A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 MOLECULAR CLONING, SEQUENCING, AND REGULATION OF THE Rhodobacter capsulatus hemB GENE. 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 Microbiology by Karl Joseph Indest B. A., University of New Orleans, 1990 August 1995 UMI Number: 9609095 UMI Microform 9609095 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 Aim Arbor, MI 48103 Dedication This work is gratefully dedicated to my loving wife, Lisa, for inspiring me to pursue my dream. Acknowledgements Words cannot express the deep appreciation and gratitude I have for my friend and wife, Lisa (the real hemB). Without her encouragement, emotional support, and love, I would have not been able to successfully complete this degree. I would like to express a deep hearted thanks to my family for their encouragement and support (emotional & financial) throughout the years. They taught the importance of a good education, but now they want me to stop going to school and get a 'real' job. I would like to express a sincere thanks to Alan J. Biel, my graduate advisor. His support (financial & financial) and encouragement have made my graduate career a successful one. I deeply appreciate the time and effort he has invested in training me, in the preparation of manuscripts (including this one), and in listening to me speak in incomplete sentences while nodding in approval. I would like to thank my committee members for their advice over the years: Drs Battista, Achberger, Gayda, Shih, and Fuxa. A special thanks goes out to Dr. Battista. Without his recommendation, I would have never rentedThe Evil Dead Part 1 & 2. I would like to extend a large token of gratitude to my fellow colleagues in the lab: Georgia Ineichen, Kayta Kanazireva, Keith Canada, Alok Khanna, and David Huang. Thanks for putting up with me on a day to day basis without killing me (especially Georgia). Special thanks to my colleagues in the Battista lab: Valerie Mattimore, Kumar Udupa, and Sharon Kelly. Finally, a special thanks to Cindy Henk and Valerie Mattimore for helping me with slides and photographs. KJI iv Table of Contents Page Dedication ....................................................................................... ii Acknowledgments........................................................................... iii Abstract .......................................................................................... vii Introduction..................................................................................... 1 Formation of aminolevulinate and its conversion into protoporphyrin IX.............................................................. 2 Regulation of tetrapyrrole biosynthesis............................ 13 Materials and Methods ................................................................. 20 Strains and plasmids. ....................................................... 20 Media ............................................................................... 20 Growth conditions............................................................ 20 Nucleic acid isolation ........................................................ 25 Restriction digest, ligations, nick translations, and other recombinant DNA techniques........................................... 27 Agarose gel electrophoresis............................................. 28 Transformation ................................................................ 29 Electroporation ...................................................... 29 Matings ............................................................................ 30 Southern analysis ............................................................ 30 Dot blot analysis .............................................................. 32 DNA sequencing.............................................................. 33 Pulsed Field Gel Electrophoresis (PFGE)........................ 33 Protein determination....................................................... 35 Porphobilinogen synthase assay ...................................... 35 Chloramphenicol acetyltransferase assay ........................ 36 Porphyrin determination................................................... 36 Thin layer chromatography ............................................... 37 Results......................................................................................... 38 Cloning of theR. capsulatus hemB gene........................ 38 Restriction analysis of the hemB region........................... 39 Expression of pCAP142(hemEP) in SB1003.......... 42 Southern analysis ............................................................ 43 Chromosomal location of theR. capsulatus hemB gene.. 45 Sequencing of theR. capsulatus hemB gene.................. 48 Regulation of theR. capsulatus hemB gene................... 54 v Dot blot analysis ............................................................. 54 Construction of a hemB-cat transcriptional fusion plasmid ........................................................................... 56 Effects of oxygen on transcription of thehemB-cat fusion plasmid ................................................................ 56 Overexpression of thehemB gene in AJB456............... 58 Discussion.................................................................................... 63 Literature Cited............................................................................. 71 Vita ............................................................................................... 86 vi Abstract The common tetrapyrrole pathway in the photosynthetic bacterium, Rhodobacter capsulatus, is responsible for the synthesis of bacteriochlorophyll, heme, vitamin B-12, and siroheme. The common portion of this pathway is regulated up to 100-fold by changes in oxygen tension. This regulation is accomplished by controlling the intracellular level of porphobilinogen. One way in which porphobilinogen levels could be controlled is by regulating the synthesis of porphobilinogen. Porphobilinogen synthase, encoded by thehemB gene, is the second enzyme in the common tetrapyrrole pathway that catalyzes the dimerization of 5-aminolevulinic acid to form the monopyrrole porphobilinogen. In order to further investigate the mechanism by which oxygen regulates porphobilinogen levels, theR. capsulatus hemB gene was cloned and sequenced. The R. capsulatus hemB gene was cloned by complementation of an Escherichia coli hemB mutant. Sequence analysis of the R. capsulatus hemB gene revealed that the putative porphobilinogen synthase has a metal-binding domain that more closely resembles that found in plant porphobilinogen synthases. The locations of thehemB and hemA genes on theR. capsulatus chromosome indicate that these gene do not form an operon. Oxygen mediated transcriptional regulation of the R. capsulatus hemB gene was measured by dot blot analysis of mRNA from cells grown under 3% and
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