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Ann Arbor, MI 48106 MOLECULAR AND BIOCHEMICAL STUDIES OF RUBISCO ACTIVATION IN ANABAENA SPECIES DISSERTATION Presented in Partial Fullfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lih-Ann Li, B.S. 41 ♦ ♦ 41 4t The Ohio State University 1994 Dissertation Committee: Approved by C.J. Daniels K.E. Kendrick W.R. Strohl Adviser Department of Microbiology F.R, Tabita Copyright by Lih-Ann Li 1994 To My Parents ii ACKNOWLEDGEMENTS I thank Dr. F. Robert Tabita for his guidance and training during the course of my graduate study. I also thank my lab fellows, Janet Gibson, Te-Jin Chaw, James Dubbs, Deane Falcone, Robert Ramagc and Katherine Tcrlesky for contributing their thoughts and technical assistance to this work. I would like to express appreciation to my dissertation committee members for their time and evaluation. I thank Dn. J. Ehlai, P. Wolk, S. Nierzwicki-Bauer, S. Golden, B. Diner, K. Ohki and S.E. Steve ns, Jr. for sending us cyanobacteria! strains and Dr. D. Bryant for pointing out the potential close relationship of chloroplasts to heterocystous cyanobacteria, I am grateful to |Drs, W.L. Ogren, G.W. Snyder and J.M. Wemeke for the spinach rca clone and the aiitiserum to spinach RubisCO activase and to Drs. J. Ehlai, P. Wolk and R. Haselkam for the Anabaena conjugation system and rbcLr£cS*containing plasmids. I am also grateful to Drs. R. Sayre, T.C. Huang and R.J. Mural for the psbA, nifH and p ic probes, respectively, to Dr. Y. Kawata for pKY206, to Dr. Darzins for pNOT19 anl pMOB3, and to Dr. A. Portis for CA1P. Finally, I wish to express sincere gratit ide to my parents, brother and sisters for their continuous encouragement and support. VITA July 1, 1963 Bom in Taitung, Taiwan, Republic of China, 1985 B.S., Chinese Culture University, Taipei, Republic of China. 1986-1988 Graduate Research Associate, University of Texas, Austin, Texas, USA. 1989-1994 Graduate Research Associate, Ohio State University, Columbus, Ohio, USA. PUBLICATIONS Li L-A, Gibson JL and Tabita FR (1993) The Rubisco activase ( rca) gene is located downstream from rbcS in Anabaena sp. strain CA and is detected in other Anabaena/Nostoc strains. Plant Mol. Biol. 31: 753-764. Tabita FR, Gibson JL, Falcone DL, Wang X, Li L-A, Read BA, Terlesky KC and Paoli GC (1993) Current studies on the molecular biology and biochemistry of C02 fixation in phototrophic bacteria. In: Murrell C and Kelly PD (eds.) Microbial Growth on Ct Compounds pp.469-479. Intercept Scientific Publication, Andover, UK. FIELD OF STUDY Major Field: Microbiology. TABLE OF CONTENTS ACKNOWLEDGEMENTS.....................................................................................iii VITA....................................................................................................................... iv LIST OF TABLES..................................................................................................vii LIST OF FIGURES................................................................................................viii CHAPTER PAGE I. Overall Introduction GENERAL BACKGROUND ON CYANOBACTERIA 1 CALVIN REDUCTIVE PENTOSE PHOSPHATE CYCLE 2 RUBISCO....................................................................................... 5 PRK.................................................................................................11 ORGANIZATION OF CALVIN CYCLE GENES...................... 12 SCOPE OF THIS DISSERTATION..............................................14 II The RubisCO Activase (rca) Gene Is Located Downstream from rbcS in Anabaena sp. Strain CA and Is Detected in Other Anabaena/Nostoc Strains INTRODUCTION.......................................................................... 16 MATERIALS AND METHODS................................................... 18 RESULTS....................................................................................... 23 DISCUSSION................................................................................ 35 v TABLE OF CONTENTS (continued) CHAPTER PAGE III Transcription Control of Ribulose Bisphosphate Carboxylase/Oxygenase Activasc and Adjacent Genes in Anabaena Species INTRODUCTION..........................................................................41 MATERIALS AND METHODS................................................... 43 RESULTS....................................................................................... 47 DISCUSSION................................................................................ 66 IV Regulation of Anabaena RubisCO in vitro and in a RubisCO Activase Mutant INTRODUCTION..........................................................................73 MATERIALS AND METHODS................................................... 78 RESULTS....................................................................................... 81 DISCUSSION................................................................................ 101 V Cyanobacterial Genes Encoding Phosphoribulokinase and Ribulose Bisphosphate Carboxylase/Oxygenase Are Not Closely Linked INTRODUCTION..........................................................................108 MATERIALS AND METHODS................................................... 110 RESULTS AND DISCUSSION.....................................................112 VI Concluding Remarks ...................................................................................123 UST OF REFERENCES 129 LIST OF TABLES TABLE PAGE 2.1 Bacterial strains and plasmids (used in CHAPTER II) ............................ 19 4.1 Solubility and RubisCO activity of Anabaena sp. strain CA rbcL and rbcS gene products in Escherichia coli..............................................85 LIST OF FIGURES FIGURE PAGE 1.1 Calvin reductive pentose phosphate cycle and phosphorcspiratory oxidation of ribulose 1,5-bisphosphate (RuBP) ..........................................3 1.2 Hypothetical mechanism of activase-mediated RubisCO activation 9 2.1 Southern hybridization localizes the rca and rbcS genes from Anabaena sp. strain CA within the same 4.3 kb EcoRI fragment 25 2.2 Restriction map of the fragment containing the Anabaena sp. strain CA rbcL, rbcS and rca genes ..................................................................... 26 2.3 Nucleotide and deduced amino acid sequence of the rca gene from Anabaena sp. strain CA ..............................................................................28 2.4 Comparison of RubisCO activase sequences ...............................................31 2.5 SDS gel electrophoresis (A) and Western immunoblot analysis (B) of Anabaena sp. strain CA RubisCO activase expressed in E. coli......... 34 2.6 Southern hybridization analysis of genomic DNA isolated from different cyanobacteria .............................................................................. 36 3.1 Genomic organization of C02-fixation genes in Anabaena strains 48 3.2 Nucleotide sequence and deduced amino acid sequences of the region between rbcL and rca in Anabaena sp. strain CA ....................... 50 3.3 Potential transcriptional terminators for Anabaena rbcLXS and rca 54 3.4 Northern blot hybridization analysis of gene expression in Anabaena sp. strain CA ..............................................................................57 3.5 Stability analysis of Anabaena sp. strain CA mRNA.............................. 60 viii 3.6 Influence of fructose metabolism on rbcL and rca transcription’ in Anabaena variabilis .....................................................................................62 3.7 Primer extension analysis of the S' end of the Anabaena sp. strain CA rca mRNA and role of the 154 bp S' noncoding sequence in recombinant RubisCO activase synthesis ..................................................63 4.1 Isomerization