CIRCADIAN RHYTHMS IN Synechococcus elongatus PCC 7942: INSIGHTS INTO THE REGULATORY MECHANISMS OF THE CYANOBACTERIAL CLOCK SYSTEM A Dissertation by SHANNON ROSE MACKEY Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2006 Major Subject: Microbiology CIRCADIAN RHYTHMS IN Synechococcus elongatus PCC 7942: INSIGHTS INTO THE REGULATORY MECHANISMS OF THE CYANOBACTERIAL CLOCK SYSTEM A Dissertation by SHANNON ROSE MACKEY Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Susan S. Golden Committee Members, Deborah Bell-Pedersen David Earnest Ryland Young Head of Department, Vincent M. Cassone August 2006 Major Subject: Microbiology iii ABSTRACT Circadian Rhythms in Synechococcus elongatus PCC 7942: Insights into the Regulatory Mechanisms of the Cyanobacterial Clock System. (August 2006) Shannon Rose Mackey, B.A., The University of Texas at Austin Chair of Advisory Committee: Dr. Susan S. Golden Circadian rhythms of behavior have been well characterized in organisms including mammals, plants, insects, fungi, and photosynthetic bacteria. Cyanobacteria, such as the unicellular Synechococcus elongatus PCC 7942, display near 24-h circadian rhythms of gene expression. These rhythms persist in the absence of external cues, can be reset by the same stimuli to which they entrain, and are relatively insensitive to changes in ambient temperature within their physiological range. Key components have been identified as belonging to the central oscillator that comprises the timekeeping units, output pathways that relay temporal information to clock-controlled processes, and input pathways that synchronize the oscillator with local time. The emerging model of the cyanobacterial clock depicts the internal timekeeping elements KaiA, KaiB, and KaiC interacting with one another to form a large, multimeric complex that assembles and disassembles over the course of a day. Information is sent into and out of the oscillator via signal transduction pathways that include proteins involved in bacterial two- component systems. The research presented in this dissertation explores the regulatory mechanisms that exist at each level of the clock system. New components were iv identified that interact with an important protein in the input pathway; these new players are involved in clock-associated phenomena, such as resetting the internal oscillation to external stimuli and maintaining proper circadian periodicity, as well as the process of cell division. The model formerly associated with the temporal, transcriptional regulation of the kai genes was redefined to reflect the unique properties of the prokaryotic oscillator. The differential output of the clock was examined by studying the circadian regulation of the psbA gene family. Overall, these data provide insight into the complex molecular events that occur to create a circadian timing circuit in S. elongatus. v ACKNOWLEDGMENTS I would like to thank my committee chair and friend, Dr. Susan Golden, for her endless support and guidance throughout this process. Her careful mentoring and undoubting faith provided the backdrop to my academic performance in both research and teaching. In addition to my chair, the members of my committee, Drs. Deb Bell-Pedersen, Dave Earnest, and Ry Young, provided great interest and insights into my research and future scientific career. Their encouragement was, and will continue to be, greatly appreciated. The many friends and colleagues I have made during my time at Texas A&M have made the experience enjoyable and unforgettable. I would especially like to thank Drs. Jayna Ditty, Eugenia Clerico, and Stan Williams for their infinite supply of humor and life lessons. And most importantly, I thank Mom, Dad, Jenny, and Peter for their immeasurable patience with, and pride in, each hurdle I jump. vi NOMENCLATURE 6xHis six adjacent histidine residues 12:12 LD 12 h light, 12 h dark Ap ampicillin ATP adenosine triphosphate BG-11M modified BG-11 medium C-6xHis six adjacent carboxy-terminal histidine residues C-terminus carboxy-terminus CI first of the tandem repeats of KaiC CII second of the tandem repeats of KaiC CikA circadian input kinase CikA-I CikA-interactor CikA~P phosphorylated CikA Cm chloramphenicol CpmA circadian phase modifier cps counts per second DAPI 4’, 6-diamidino-2-phenylindole DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone DNA deoxyribonucleic acid EMS ethyl methane sulfate FFT-NLLS fast Fourier transform–nonlinear least square FRP free-running period vii Gm gentamycin GTP guanosine triphosphate HPK histidine protein kinase Hyp-1 hypothetical protein 1 Hyp-2 hypothetical protein 2 I&A Import and Analysis IPTG isopropyl-beta-D-thiogalactopyranoside kb kilobase kDa kiloDalton Km kanamycin LD light/dark LdpA light-dependent period LL constant light mRNA messenger ribonucleic acid N-6xHis six adjacent amino-terminal histidine residues N-terminus amino terminus NHT-1 aminotransferase Ni-NTA nickel-nitriloacetic acid NS1 neutral site I NS2 neutral site II OD optical density ORF open reading frame viii Pex period extender PSII photosystem II PsR pseudo-receiver RR response regulator SasA Synechococcus adaptive sensor Sm streptomycin Sp spectinomycin Spk serine-threonine protein kinase WT wild type ix TABLE OF CONTENTS Page ABSTRACT................................................................................................... iii ACKNOWLEDGMENTS .............................................................................. v NOMENCLATURE ....................................................................................... vi TABLE OF CONTENTS................................................................................ ix LIST OF FIGURES........................................................................................ xii LIST OF TABLES.......................................................................................... xiv CHAPTER I INTRODUCTION ............................................................................. 1 The circadian clock.................................................................... 1 A cyanobacterial model system for circadian rhythms................ 5 The Kai oscillator: keeping track of time.................................... 9 The input pathways: keeping in sync with the environment........ 17 The output pathways: regulating cellular processes .................... 22 The periodosome ....................................................................... 27 II DETECTION OF RHYTHMIC BIOLUMINESCENCE FROM LUCIFERASE REPORTERS IN CYANOBACTERIA ..................... 31 Introduction ............................................................................... 31 Description of luxAB, luxCDE, and luc vectors .......................... 32 Transformation, chromosomal segregation, and clonal propagation................................................................................ 34 TopCount measurement ............................................................. 37 Plate preparation ................................................................. 37 Sample preparation ............................................................. 38 TopCount protocol and interpretation of results................... 38 III STABILITY OF THE CIRCADIAN CLOCK UNDER DIRECTED ANTI-PHASE EXPRESSION OF THE kai GENES ...... 45 Introduction ............................................................................... 45 x CHAPTER Page Methods..................................................................................... 47 Bacterial strains and plasmids ............................................. 47 Media and growth conditions .............................................. 47 DNA manipulations and sequencing ................................... 50 Construction of kai mutant strains....................................... 50 Construction of a neutral site I Gateway vector ................... 52 Construction of ectopic kai alleles....................................... 52 Measurement and analysis of in vivo bioluminescence ........ 54 Whole-cell extract preparation and immunoblot analyses .... 55 Results....................................................................................... 58 Construction and characterization of kai gene mutant strains...................................................................... 58 Complementation of ∇kaiA with class 2 PpurF::kaiA restores WT circadian rhythms............................................ 61 PpurF::kaiBC can restore WT rhythms to a ∆kaiBC strain.. 63 Pervasive effects of PpurF::kaiBC expression on both class 1 and class 2 reporters......................................... 65 Discussion ................................................................................. 67 IV DIFFERENTIAL CIRCADIAN REGULATION OF psbA GENES IN Synechococcus elongatus PCC 7942................................ 70 Introduction ............................................................................... 70 Methods..................................................................................... 73 Bacterial strains and growth conditions ............................... 73 Measurement and analysis of in vivo bioluminescence ........ 73 Results....................................................................................... 75 Discussion ................................................................................