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University of Alberta Autoregulation of RNA helicase operon expression by Albert Remus R. Rosana A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Microbiology and Biotechnology Department of Biological Sciences ©Albert Remus R. Rosana Fall 2013 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Abstract The cyanobacterium Synechocystis sp. PCC 6803 encodes for an RNA helicase, crhR, whose expression is regulated by the redox status of the electron transport chain and further enhanced by temperature downshift. In this study, the effect of crhR inactivation was investigated in response to temperature alteration. The inactivation of CrhR has drastic morphological and physiological effects particularly under cold stress, including a rapid cessation of photosynthesis, impaired cell growth, decrease in viability, cell size and DNA content and accumulation of structural abnormalities. Extensive transcript and protein analyses between wild type and mutant cells revealed that CrhR activity is linked to its autoregulation of expression involving complex network acting at the transcriptional and post-transcriptional levels. Furthermore, autoregulation is extended to the rimO-crhR operon where processing and degradation contributes to differential accumulation of the cistrons. Lastly, the potential association of CrhR in a degradosome-polysome complex in the thylakoid membrane is proposed. Acknowledgement I would like to extend by deepest appreciation and sincerest gratitude to the many individuals who contributed to the successful completion of this chapter in my life. These individuals deserved my humble thanks that made this thesis possible. Dr. George W. Owttrim, my mentor who gave me the opportunity to conduct research under his tutelage. His unreserved guidance, support and encouragement allowed me to grow both professionally and personally as a scientist. Dr. Danuta Chamot for imparting invaluable techniques especially the paranoia of RNA research. Dana’s constant guidance on molecular biology techniques made this journey navigable. My supervisory committee, Dr. Lisa Y. Stein and Dr. Michael Deyholos for their valuable suggestions and advice. Oxana Tarassova, Reem Skeik and Denise Whitford made this whole journey so enjoyable. The camaraderie we all shared, the scientific craziness, the exhausting laughter, the unforgettable travels, sumptuous food trips and of course the love for ‘gambling’ will always be a part of my life that I will cherish. My family, Papa, Mama, Tin-tin and Mommy Ella who always supported me in all my endeavour. For believing in me especially when I doubt myself, for loving me and encouraging me to finish the battle. I love you all. And finally to the Lord God, who made everything possible. Thank you very much. Table of Contents Page Chapter 1: Introduction 1 1.1 Cyanobacteria as model organism 2 1.2 DEAD-box RNA helicases and abiotic stress response 3 1.3 CrhR RNA helicase biology 1.3.1 Regulation of CrhR RNA helicase 5 1.3.2 Catalytic function of CrhR RNA helicase 7 1.3.3 crhR inactivation studies 7 1.4 Cyanobacterial operons 9 1.5 Thesis objectives 11 1.6 References 14 Chapter 2: Inactivation of a low temperature-induced RNA helicase in Synechocystis sp. PCC 6803: Physiological and morphological consequences 21 2.1 Introduction 22 2.2 Materials and methods 24 2.2.1 Bacterial strains and culture conditions 24 2.2.2 Photosynthetic oxygen evolution 25 2.2.3 Mass spectrometric measurements of photosynthetic CO2 uptake 26 2.2.4 Intracellular Ci concentration 26 2.2.5 Cell morphology 26 2.2.6 Electron microscopy 27 2.3 Results 28 2.3.1 crhR inactivation 28 2.3.2 Cell growth 29 2.3.3 Photosynthetic oxygen evolution 33 2.3.4 Ci transport and accumulation 33 2.3.5 Photosynthetic electron transport 37 2.3.6 Photosynthetic pigment composition 38 2.3.7 Cellular morphology 41 2.3.8 Ultrastructure analysis 43 2.4 Discussion 46 2.5 References 51 Chapter 3: Autoregulation of RNA helicase expression in response to temperature stress in Synechocystis sp. PCC 6803 55 3.1 Introduction 56 3.2 Materials and Methods 58 3.2.1 Bacterial strains and growth conditions 58 3.2.2 RNA manipulation 59 3.2.3 Protein manipulation 59 3.2.4 ImageJ analysis 60 3.2.5 Supplementary Methods 60 3.2.5.1 RNA Quantitation 60 3.2.5.2 Protein quantitation 62 3.3 Results 64 3.3.1 Induction of crhR expression in response to temperature and light-dark stress 64 3.3.2 Time course of crhR transcript accumulation 65 3.3.3 Time course of CrhR protein accumulation 70 3.3.4 crhR transcript and CrhR protein half-life 70 3.3.5 Temperature gradient induction of crhR transcript and protein accumulation 73 3.3.6 Time course of crhR transcript and protein accumulation at 10oC 76 3.4 Discussion 82 3.5 References 88 Chapter 4: CrhR RNA helicase regulates the expression of rimO-crhR operon in response to cold stress 92 4.1 Introduction 93 4.2 Materials and methods 95 4.2.1 Strains and growth conditions 95 4.2.2 RNA manipulation 96 4.2.3 Protein manipulation 97 4.2.4 5’RACE-RCA, inverse PCR and DNA sequencing 97 4.2.5 His-CrhR expressing strains 98 4.3 Results 101 4.3.1 The slr0082-slr0083 operon in Synechocystis sp. PCC 6803 101 4.3.2 CrhR inactivation results in deregulated expression of the slr0082- slr0083 operon 104 4.3.3 Transient accumulation of slr0082 mRNA in response to cold stress is CrhR-dependent 106 4.3.4 slr0082 transcript half-life is not affected by temperature downshift or crhR mutation 108 4.3.5 His-CrhR expressing strains resulted in transient accumulation of slr0082 mRNA in response to cold stress 110 4.3.6 Accumulation of an internal stable RNA from the open reading frame of slr0082 113 4.3.7 5’ UTR of slr0082 is constitutively accumulated at all temperatures 116 4.3.8 5’ RACE-RCA analysis 117 4.4 Discussion 120 4.5 References 128 Chapter 5: Summary and Conclusion 134 5.1 CrhR RNA helicase and the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 135 5.2 CrhR autoregulation of gene expression 137 5.3 CrhR association in RNA metabolism: processing, maturation and degradation of the slr0082-slr0083 operon 139 5.4 References 146 Chapter 6: Appendix 152 6.1 Translational control of CrhR expression 153 6.2 Phylogenetic analysis of rimO-crhR operon 153 6.3 Localization of CrhR in Synechocystis sp. PCC 6803: co-sedimentation with the polysome and thylakoid membrane 157 6.3.1 CrhR RNA helicase co-sediments with the cyanobacterial polysome complex 157 6.3.2 Truncated CrhR impairs CrhR cellular localization 160 6.3.3 CrhR RNA helicase localized in the thylakoid region of Synechocystis 160 6.4 CrhR Protein Interactome: Effectors of transcription, processing and translation 164 6.5 CrhR and RNA-folding predictions 168 6.6 References 172 List of Tables Page Table 2.1 Oxygen exchange in WT and ΔcrhR cells 39 Table 2.2 Cellular features of WT and ΔcrhR cells 42 Table 3.S1 Quantification of crhR transcript hybridization detected in the ΔcrhR mutant shown in Figure 3.7B 81 Table 4.1Bacterial strains, plasmids and oligonucleotides utilized in this study 100 Table 6.1 Predicted CrhR protein interactome using the STRING ver 9.05 167 List of Figures Page Figure 1.1. Organization of the photosynthetic electron transport chain 6 Figure 2.1 crhR mutagenesis. 30 Figure 2.2 Growth and viability. 32 Figure 2.3 Photosynthetic oxygen evolution, Ci uptake and accumulation. 34 Figure 2.4 Photosynthetic pigment composition. 40 Figure 2.5 Ultrastructure analysis (SEM) 44 Figure 2.6 Ultrastructure analysis (TEM) 45 Figure 3.1 crhR expression in response to abiotic stress. 66 Figure 3.2 Time course of crhR transcript accumulation. 68 Figure 3.3 Time course of CrhR protein accumulation. 71 Figure 3.4 crhR transcript half-life. 72 Figure 3.5 CrhR protein half-life. 74 Figure 3.6 Temperature gradient of crhR expression. 75 Figure 3.7 Time course of crhR expression at 10°C. 77 Figure 3.S1 Ethidium bromide stained gel corresponding to the crhR induction time course at 10oC. 80 Figure 3.8 Schematic summary of crhR expression and regulation. 83 Figure 4.1 Genomic organization of the Synechocystis slr00082-slr0083 operon and CrhR protein accumulation 102 Figure 4.2 slr0082 transcript accumulation 105 Figure 4.3 Time course of slr0082 transcript accumulation 107 Figure 4.4 slr0082 transcript half-life 109 Figure 4.5 Time course of cold induced slr0082 transcript accumulation in His-CrhR expressing strains. 111 Figure 4.6 Effect of His-CrhR expression on growth and CrhR accumulation. 112 Figure 4.7 Temperature gradient of slr0082 expression – individual culture. 114 Figure 4.8 Temperature gradient of slr0082 expression – single culture. 115 Figure 4.9 Temperature gradient accumulation of the slr0082 5’ UTR. 118 Figure 4.10 Time course of slr0082 5’UTR accumulation at 10oC.

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