DEFORMATION 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. Kgher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appealing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell InfoTmation Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 MOLECULAR AND ENZYMOLOGICAL ANALYSIS OF L-HISTIDINE NON-UTILIZING MUTANTS OF STREPTOMYCES GRISEUS DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in the Graduate School of the Ohio State University By KanurV. Srinivasan, B.Sc., M.S. The Ohio State University 1997 Dissertation Committe Approved By Charles J. Daniels Kathleen E. Kendrick, Adviser Joseph A. Krzycki Adviser F. Robert Tabita Department of Microbiology UMI Number: 9721164 UMI Microform 9721164 Copyright 1997, 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 ABSTRACT Histidine ammonia-lyase (histidase) catalyzes the non-oxidative (3- eiimination of the a-amino group of L-histidine to yield urocanate and ammonia. Two Hut" mutant strains of Streptomyœs griseus, SKK896 and SKK906, that were unable to utilize L-histidine as nitrogen source do not make histidase. hutHÿ^ contained a missense mutation in the histidase structural gene (A223N histidase) and restored histidase activity to transformants of SKK896 and SKK906. In contrast, hutHgog (S426F histidase) restored histidase activity only to SKK896 transformants. In recombinant strains containing the mutant hutH alleles integrated in the chromosome, the identified mutations were responsible for the Hut' phenotypes of SKK896 and SKK906. The missense mutation in SKK896 might be responsible for the reduced amount of hutH transcript in this strain. A223N histidase was active when synthesized in E. coli but had a higher K^, and reduced thermostability when compared to the wild-type histidase. S426F histidase was inactive and highly unstable when expressed in E. coli. DEDICATION To my parents III ACKNOWLEDGMENTS I would like to express my deep sense of gratitude to my advisor, Dr. Kathleen E. Kendrick, for her encouragement and guidance during my stay in this lab. I Also thank my Committee members, Drs, Charles Daniels, Joseph Krizycki, and F. Robert Tabita for their advice and suggestions regarding this project. I am indebted to my colleagues, past and present, for their help and suggestions. I thank my friend G. Convey for his help. Finally I owe my gratitude to my family for their faith and unfailing support during this endeavor. IV VITA March 14.1964................. Bom—Madras, India 1982-1986 .......................... B.Sc in Agricultural Sciences Bangalore, India 1986-1989 .......................... M.S. In Agricultural Microbiology Bangalore, India 1990-present......................Graduate Teaching and Research Associate, Department of Microbiology, The Ohio State university, Columbus, Ohio PUBLICATION Wu P.C., K.V., Srinivasan, and K.E. Kendrick. 1995. Regulated expression of the histidase structural gene In Streptomyœs griseus. J. Bacteriol. 177:854-857. FIELDS OF STUDY Major Field: Microbiology Studies in gene regulation in streptomycetes. Dr. Kathleen E. Kendrick TABLE OF CONTENTS ABSTRACT.........................................................................................................................ii DEDICATION................................................................................................................... iii ACKNOWLEDGMENTS.............................................................................................. iv VITA ....................................................................................................................................V LIST OF TABLES............................................................................................................. x LIST OF FIGURES.........................................................................................................xii CHAPTERS; 1 INTRODUCTION.................................................................................................. 1 Metabolism of amino acids in Streptomyœs...................................................1 Catabolism of L-histidine .................................................................................... 6 The active site of histidase ................................................................................9 hwf gene organization and regulation ............................................................14 Kinetic properties of streptomycete histidase ..............................................24 Goals of this project ........................................................................................... 27 2 MATERIALS AND METHODS .......................................................................28 Bacterial strains ..................................................................................................28 Plasmid vectors ..................................................................................................28 VI Media and culture conditions ...........................................................................31 Recombinant DNA techniques .........................................................................34 isolation and manipulation of DNA .....................................................34 Ligation reactions ..................................................................................35 Nucleotide sequence determination ............................................................. 35 PCR amplification of DNA fragments ................................................ 35 Dideoxy chain-termination m ethod .....................................................36 fmo/method .......................................................................................... 38 ABI Prism Dye Terminator Cycle sequencing method ...................38 Transformation ................................................................................................... 39 Preparation of cell extracts and enzyme a ssa y s ......................................... 41 Preparation of crude extracts ............................................................. 41 Assay of histidase and urocanase activities .....................................41 Immunoblot techniques ....................................................................................42 SDS-PAGE and immunoblot assay .................................................... 42 Preparation of monospecefic antibodies ........................................... 43 Generation of recombinant strains ..................................................................43 Protoplast fusion ....................................................................................43 Allele exchange......................................................................................44 Hybridization reactions ....................................................................................45 Colony hybridization ............................................................................. 45 Slot Blot hybridization ...........................................................................46 vii Northern hybridization ...........................................................................47 Overexpression of histidase ...........................................................................50 Streptomycin bioassay ...................................................................................... 51 3 RESULTS .......................................................................................................... 52 Characterization of SKK906 ........................................................................ 52 Enzyme and immunoblot analyses .....................................................52 Characterization of the mutation in SKK906 ................................... 53 Complementation studies ....................................................................57 Characterization of/jufHgos ..................................................................62 Characterization of revenants of SKK906 ....................................... 69 Allele exchange......................................................................................75