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Chapter 1: Beyond the Classic Heat Shock Response: Novel Thermal Stress Regulators in the Thermophilic Archaea

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Chapter 1: Beyond the Classic Heat Shock Response: Novel Thermal Stress Regulators in the Thermophilic Archaea ABSTRACT COOPER, CHARLOTTE RENÉE. VapBC Toxin-Antitoxin Loci in the Extreme Thermoacidophile Sulfolobus solfataricus: Regulation of and Functional Biochemical Roles during Thermal Stress Response. (Under the direction of Dr. Robert M. Kelly.) The heat shock response is universal across all domains of life and includes conserved mechanisms for protein refolding and protein degradation necessary for organisms to survive thermal and other stresses. In bacteria, chaperones, such as DnaK, DnaJ, GroEL, GroES, and GrpE, have been well characterized. However, the majority of archaea lack homologues for such chaperones, though most archaeal genomes encode a “thermosome” that is functionally similar to GroEL. Archaea also lack many of the RNA management tools found in eukaryotes and bacteria, such as RNA interference, siRNA, the bacterial Rho transcription termination factor, and bacterial degradasomes. Recently, it has been proposed that toxin- antitoxin (TA) loci could fill important roles in stress response, especially in the archaea. TA loci are ubiquitous in prokaryotic genomes and abundant in the archaea, especially in the thermophilic archaea. Functional genomics analysis of model extreme thermoacidophile Sulfolobus solfataricus during heat shock (80oC to 90oC) revealed dynamic changes in novel heat shock regulators and in the chromosomally encoded VapBC family TA loci. Several of these genes were targeted for disruption and deletion mutations in S. solfataricus strain PBL2025. When transcriptional regulator tetR (SSO2506) was disrupted, the importance of the Sulfolobus heat shock regulator (Shr, SSO1589) was further implicated in the thermal stress response. When the most highly transcribed VapC-22 toxin (SSO3078) was disrupted, there were several cognate and non-cognate VapB antitoxins responding to heat shock. This suggested that VapC-22 plays a role in regulating the abundance of active versus silenced toxins in the cell by mediating the levels of antitoxins present. Finally, when the most heat shock responsive TA loci (VapBC-6, SSO1494 and SSO1493) was deleted, the VapBC-6 deficient mutant was thermally labile. Furthermore, the VapC-6 deficient mutant was more like the VapC-22 mutant, as there was an abundance of VapB antitoxins present, even before heat shock, including cognate VapB-6. This implicates VapC-6 in VapB regulations as well. Careful analysis of the genome lead to the discovery that a VapC toxin overlaps with the γ subunit of the translation initiation factor in S. solfataricus, and that the cognate VapB is predicted to be part of aIF2γ. The fact that this construct is common in many archaeal genomes led to a study of this particular toxin’s form and function. The S. solfataricus VapCγ and VapC-18 toxins were demonstrated to be ribonucleases, though VapC-18 is dimeric and VapCγ is monomeric. Deletion of VapCγ from S. solfataricus led to a non-viable phenotype. Recombinant VapC-18 was shown to specifically “fish” its cognate antitoxin (VapB-18) from recombinant E. coli cell extracts, forming either a heterohexameric or heterooctameric complex. While the VapCγ did not bind to subunit γ or the aIF2 heterotrimeric complex in the same manner as VapBC-18, this was consistent with the structural details of the aIF2 complex. It is likely that if VapCγ and aIF2 interact at all, it will be unique from typical TA interactions. A unique role was proposed for VapCγ in RNA management in archaea, in which it is involved in freeing aIF2 bound to non-initiator tRNAs that have been esterfied by methionine or complexes bound to the 5’ end of mRNA transcripts. The results of this study further emphasize the importance of TA loci as post- transcriptional regulators. It also sheds light on the complex, and still unresolved, thermal stress response network in archaea that involves interplay between transcriptional and post- transcriptional regulators and translation initiation machinery. VapBC Toxin-Antitoxin Loci in the Extreme Thermoacidophile Sulfolobus solfataricus: Regulation of and Functional Biochemical Roles during Thermal Stress Response by Charlotte Renée Cooper A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemical Engineering Raleigh, North Carolina March 2011 APPROVED BY: _________________________ _________________________ David F. Ollis Balaji M. Rao _________________________ _________________________ Fred J. Fuller Robert M. Kelly Chair of Advisory Committee DEDICATION To my mother, for believing in me and my dreams (no matter how crazy) and always showing me that anything is possible. ii BIOGRAPHY Charlotte Renée Cooper was born and raised in the small southern town of Easley, SC. Throughout most of her childhood she could be found buried in a book or banging on the piano. In high school, Charlotte fell in love with Chemistry, particularly the labs. It was here that a wise teacher, Dr. Johnson, persuaded her to think about majoring in Chemical Engineering. In 2001, she left for the University of South Carolina where she relished her years as a Gamecock. Before she knew it, she had earned her Bachelors of Science in Engineering in Chemical Engineering. While at the University of South Carolina, she worked on many research projects including fuel cell studies and Alzheimer’s Disease. Her love of research prompted her to apply to graduate schools. After graduation in 2005, she headed northward to North Carolina State University where she has investigated the role of stress responsive genes in model extreme thermoacidophile Sulfolobus solfataricus. Charlotte will next be joining Novozymes as a Fermentation Engineer. iii ACKNOWLEDGMENTS They say it takes a village to raise a child, and it also takes one to prepare a dissertation. I would like to thank Dr. Kelly for allowing me the opportunity to study under his guidance and our collaborator Dr. Blum who made much of this work possible. I would also like to thank fellow members of the Kelly group who have offered me so much. First, I would like to thank Sabrina Tachdjian who was a wonderful mentor and patient teacher. In addition, I would like to thank Derrick Lewis and Kate Auernik for their endless discussions and masterful ideas; but most of all I would like to thank them for their genuine friendship. Finally, I would like to recognize the contributions of Kelly group members past and present. I cannot begin to describe the immeasurable thanks I owe to my family. To my mother, I thank you for your unwavering support and for being my #1 cheerleader. I’m glad Verizon has the family plan or else I would be really poor! And to my future husband Chris, thank you for keeping me sane through to the hectic end and helping me see the big picture; you are truly the love of my life. To the rest of my family (those I was given and those that I’ve chosen), I love you all. Finally, I would like to thank a dear friend and mentor Dr. George Roberts. Dr. Roberts was the first person I talked to from NC State after my acceptance, my favorite dance partner at the Cardinal Club, and an invaluable support through many tough problems I encountered in graduate school. Dr. Roberts, you are missed but never forgotten. iv TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................x LIST OF FIGURES .................................................................................................................xi Chapter 1: Beyond the classic heat shock response: novel thermal stress regulators in the thermophilic archaea..................................................................................................................1 Introduction................................................................................................................2 Thermal Stress Response - Heat Shock .....................................................................2 Heat Shock in Bacteria and Archaea .............................................................4 Beyond the classic heat shock response in Bacteria ......................................5 Novel thermal stress response elements from the thermophilic Archaea......6 Toxin-Antitoxin Loci...............................................................................................10 The ccdAB family ........................................................................................12 The relBE family..........................................................................................12 The phd-doc family......................................................................................14 The higBA family.........................................................................................14 The mazEF (chp) family ..............................................................................15 The parDE family........................................................................................16 The hicAB family .........................................................................................17 The hipBA family.........................................................................................17 The ω−ε−ζ family.......................................................................................17 v The vapBC family........................................................................................18 TA loci as novel thermal stress regulators in Archaea ............................................20
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