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 free, 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., m ^ s, drawings, charts) are reproduced by sectioning the original, b%inning 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 Infoimation Company 300 North Zed> Road, Ann Aibor NO 48106-1346 USA 313/761-4700 800/521-0600 ORGANIZATION AND REGULATION OF THE RHODOBACTER CAPSULATUS CO2 FIXATION GENES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By George Carl Paoli, B.S., M.S. ***** The Ohio State University 1997 Dissertation Committee Approved by: F.R. Tabita, Adviser W.R. Strohl K.E. Kendrick viser C.J. Daniels Department of Microbiology UMI Number: 9721152 UMI Microform 9721152 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 Rhodobacter capsulatus is a nonsulfur purple photosynthetic bacterium that fixes CO2 primarily via the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. R. capsulatus is one of only a few bacterial species known to synthesize two forms of the key CBB cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). The organization of several genes encoding CBB cycle enzymes {ebb genes) in R. capsulatus was determined. The R. capsulatus form I and form II RubisCO genes, cbbLcbbS and cbbM, respectively, were functionally expressed in Escherichia colt and Rhodobacter sphaeroides. Several of the ebb genes were sequenced. Phylogenetic analysis revealed that the R. capsulatus form I RubisCO genes and the genes immediately upstream and downstream of cbbLcbbS were acquired by R. capsulatus as a result of a horizontal gene transfer. A combination of ebb gene disruption strains and ebb promoter fusion constructs was employed to examine aspects of ebb gene regulation in R. capsulatus. Chemoautotrophic growth of mutant strains of R. capsulatus, and a spontaneous mutant of Rhodobacter sphaeroides, that synthesize only form I or form II RubisCO was examined. The results of these experiments suggest that these strains might be useful in providing a system for biological selection for RubisCO with desirable biochemical properties. Ill Dedicated to Renee, Cassie, and Courtney IV ACKNOWLEDGEMENTS I wish to thank my adviser. Dr. F. Robert Tabita for his support and patience, as well as the training I received while working on these studies. Thanks also to Dr. Janet L. Gibson for her advice and input. For his contributions to this work, I thank Dr. Jessup Shively. I wish to express my gratitude to Drs. Charles E. Daniels, Kathleen E. Kendrick, and William R. Strohl for their time and input, both as advisory members of my dissertation committee and as teachers. Thanks to past and present members of Dr. Tabita's laboratory, too many to list, with whom working has been a pleasure. To my parents. Lido and Mary Ann Paoli, my thanks for continued support and encouragement, and to my wife Renee, for her sacrifice and support. VTTA December 6, 1963 ............................................ Bom - Hancock, Michigan 1986 ................................................................ B.S., Michigan Technological University, Houghton, Michigan 1988................................................................ M.S., Michigan Technological University, Houghton, Michigan 1988 - Present................................................. Graduate Teaching and Research Associate, The Ohio State University, Columbus, Ohio PUBLICATIONS Tabita, F.R., J.L. Gibson, D.L. Falcone, X. Wang, L.-A. Li, B.A. Read, K.C. Terlesky, and G.C. Paoli. 1991. Current studies on the molecular biology and biochemistry of CO? fixation in phototrophic bacteria, p.469-479. In J.C. Murrell and D.P. Kelly (ed.). Microbial Growth on C| Compounds. Intercept Ltd, Andover, U.K. Paoli, G.C., N. Strom-Morgan, J.M. Shively, and F.R. Tabita. 1995. Expression of the cbbLcbbS and cbbM genes and distinct organization of the ebb Calvin cycle structural genes of Rhodobacter capsulatus. Arch. Microbiol. 164:396-405. Gibson, J.L., Y. Qian, G.C. Paoli, J.M. Dubbs, H. Xu. H.V. Modak, K.M. Horken, T.M. Wahlund, G.M.F. Watson, and F.R. Tabita. 1996. Molecular control and biochemistry of CO? fixation in photosynthetic bacteria. In, M.E. Lidstrom and F.R. Tabita (ed.). Microbial Growth on Ci Compounds. Kluwer Academic Publishers, Dordrecht, The Netherlands. FIELDS OF STUDY Major Field: Microbiology vi TABLE OF CONTENTS ABSTRACT.............................................................................................................. ii DEDICATION.......................................................................................................... iv ACKNOWLEDGEMENTS ................................................................................... v VITA .......................................................................................................................... vi LIST OF TA BLES................................................................................................... ix LIST OF FIGURES................................................................................................. xi INTRODUCTION................................................................................................... 1 CHAPTERS 1. Expression of the cbbLcbbS and cbbM Genes and Distinct Organization of theebb Calvin Cycle Structural Genes of Rhodobacter capsulatus ...................... 4 Introduction ....................................................................................... 4 Materials and M ethods .................................................................... 7 Results............................................................................................... 13 Discussion ......................................................................................... 27 Acknowledgments ............................................................................ 30 2. Organization and Nucleotide Sequence of the Rhodobacter capsulatus cbbLcbbS and Neighboring Genes. Phylogenetic Analysis Suggests That These Genes Were Acquired by a Horizontal Gene Transfer ........................................... 31 Introduction ....................................................................................... 31 Materials and M ethods .................................................................... 33 Results............................................................................................... 39 Discussion ......................................................................................... 60 Acknowledgments ............................................................................. 63 vii 3. Studies of the Regulation of the ebb Structural Genes of Rhodobacter capsulatus ................................................................................................................. 64 Introduction ...................................................................................... 64 Materials and M ethods .................................................................... 68 Results............................................................................................... 80 Discussion ............................................................................................. 130 Acknowledgments ................................................................................ 134 4. Aerobic ChemoUthoautotrophic Growth of Rhodobacter capsulatus strain SB 1003 and a Spontaneous Mutant of Rhodobacter sphaeroides strain HR.... 135 Introduction ...................................................................................... 135 Materials and M ethods .................................................................... 138 Results............................................................................................... 141 Discussion ........................................................................................ 159 Acknowledgments ............................................................................ 162 5. SUM M ARY....................................................................................................... 163 REFERENCES........................................................................................................