Oxidative Stress Response in Archaea: Elucidation of Oxidant Sensing and Tolerance Mechanisms

179 Pages·2017·5.52 MB·English

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Graduate Theses and Dissertations
5-2016
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Matthew Edward Jennings
University of Arkansas, Fayetteville
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Jennings, M. E. (2016). Oxidative Stress Response in Archaea: Elucidation of Oxidant Sensing and
Tolerance Mechanisms in Methanosarcina acetivorans. Graduate Theses and Dissertations Retrieved
from https://scholarworks.uark.edu/etd/1514
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Oxidative Stress Response in Archaea: Elucidation of Oxidant Sensing
and Tolerance Mechanisms in Methanosarcina acetivorans

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy in Biology

by

Matthew Edward Jennings
University of Scranton
Bachelor of Science in Biology, 2005
Villanova University
Master of Science in Biology, 2010

May 2016
University of Arkansas

This dissertation is approved for recommendation to the Graduate Council.

___________________________________
Dr. Daniel Lessner
Dissertation Director

___________________________________ ___________________________________
Dr. Ralph Henry Dr. Suresh Thallapuranam
Committee Member Committee Member

___________________________________ ___________________________________
Dr. Inés Pinto Dr. David Ivey
Committee Member Committee Member
© 2016 by Matthew Edward Jennings
All Rights Reserved
ABSTRACT
Methanogens are archaea possessing a conserved metabolic pathway which produces
methane. Many of the enzymes in the methanogenesis pathway are Fe-S proteins, meaning
methanogens are sensitive to conditions which disrupt Fe-S clusters. Molecular oxygen is
capable of disrupting Fe-S clusters through oxidation of the iron atoms. Furthermore, reduced
iron can facilitate the production of reactive oxygen species (ROS), meaning methanogens must
possess antioxidant mechanisms. Detection and eradication of ROS is important for all cells, due
to the potentially fatal consequences of unchecked oxidation. This dissertation presents two
separate projects investigating mechanisms the model methanogen Methanosarcina acetivorans
possess for dealing with ROS. One project investigated the roles two [4Fe-4S] clusters present
in RNA polymerase (RNAP) subunit D play in assembly and activity of RNAP; to determine if a
mechanism exists for linking sensitivity of the clusters to oxygen to RNAP function. My data
shows that both clusters and the cluster binding domain play an important role in assembly of
RNAP downstream of D-L heterodimer formation, preventing optimal assembly of at least
subunits B’ and A’’ when the clusters are absent. Cluster one plays a more critical role in this
process compared to cluster two. Coupled with experimental evidence that the clusters are
oxygen sensitive, this provides support for the hypothesis that the clusters regulate RNAP
assembly in response to redox state of the cell. The second project investigated two putative
catalase genes present in the M. acetivorans genome. Experimental evidence showed neither
catalase was functional. Engineering of a M. acetivorans strain to express functional catalase
from Escherichia coli increased the tolerance of M. acetivorans to H O , but not oxygen during
2 2
growth in standard conditions. Catalase does not appear to be an important component in the
oxidative stress response of M. acetivorans.
ACKNOWLEDGEMENTS
All science is collaboration, and this dissertation was finished with help from many
different people. William Metcalf and his lab at the University of Illinois Urbana-Champaign
developed the genetic system for Methanosarcina acetivorans, which I used liberally in my
experiments. Liz Karr and her lab at the University of Oklahoma provided the protocol for the
non-specific transcription assays.
Jim Wilson was my advisor at Villanova University where I pursued my M.S. degree. He
encouraged me to continue my education, and if the experience doing research in his lab wasn’t
pleasant, I doubt I would have continued to get my doctorate. I learned many good lab practices
from him, and I was able to hit the ground running here in Arkansas because of his guidance.
My fellow graduate students provided a sounding board for scientific questions, technical
advice for experiments, and emotional support for the times when I questioned my sanity in
continuing to pursue a doctorate. I want to thank Tom Deere, Addison McCarver, and Ryan
Sheehan, in particular, for their help throughout the years. In retrospect, having other grad
students join the lab was the best thing that could have happened, even with the extra noise and
smells.
Faith Lessner deserves my eternal gratitude for providing additional hands for
experiments when I was overwhelmed, technical advice for when I was stymied, delicious food
for eating, and harsh criticism for when I did dumb things both in and out of the lab. This
dissertation would not have come together without her help and support, and I appreciate
everything that she did for me.
Finally, this dissertation owes much to the enthusiasm of my advisor, Dan Lessner. It was
at his suggestion that I work on both projects, which turned out to be very fruitful from a
publication standpoint. It was his excitement which drove me on, even which I was sick of all
my experiments failing. Observing other graduate students who have come and gone in our
department has shown me that Dan was able to a walk a fine line between letting me figure
things out on my own without making me feel like I was abandoned, and I always thought he
cared deeply about the projects. I will be the first Ph.D. student that graduates from his lab, and I
know I won’t be the last.
DEDICATION
Committing to a Ph.D. is not something you should do lightly; it’s a huge undertaking,
and, in retrospect, I can definitely say it is not for everyone. However, my parents, Ken and
Rosemary Jennings, always told me that I was a smart boy and that I could do anything if I put
my mind to it. They encouraged me to go for it, comforted me when things were tough and it
looked like I would never finish, and constantly offered financial assistance even though I kept
telling them I was fine (I still accepted the money in holiday greeting cards though). They
supported me even though they don’t really understand what I do, but they know I’ll have some
extra letters after my name when I’m done. So, Mom and Dad, six years in Arkansas and I
finally have the degree. Thanks in no small part to you.
My sister, Lauren Jennings, was able to commiserate with me over my graduate student
woes in ways my parents couldn’t. I was able to vent to her about my parents when they started
getting on my case about when I was planning on finishing, what my plans were following
graduation, and how much money I’d be making. Thanks for being a sympathetic ear sis, and I
seem to have one upped you in the degree department.
TABLE OF CONTENTS

INTRODUCTION ........................................................................................................................ 1
References ................................................................................................................................. 18
Figures....................................................................................................................................... 23
Chapter I ...................................................................................................................................... 30
Preface....................................................................................................................................... 31
Abstract ..................................................................................................................................... 32
Introduction ............................................................................................................................... 33
Materials and Methods .............................................................................................................. 35
Results ....................................................................................................................................... 41
Discussion ................................................................................................................................. 53
Conclusions ............................................................................................................................... 58
Acknowledgements ................................................................................................................... 59
References ................................................................................................................................. 59
Figures and Tables .................................................................................................................... 64
Chapter II .................................................................................................................................... 78
Abstract ..................................................................................................................................... 79
Introduction ............................................................................................................................... 80
Materials and Methods .............................................................................................................. 83
Results ....................................................................................................................................... 90
Discussion ................................................................................................................................. 98
Acknowledgements ................................................................................................................. 103
References ............................................................................................................................... 103
Figures and Tables .................................................................................................................. 106
Appendix ................................................................................................................................. 132
Chapter III ................................................................................................................................. 133
Abstract ................................................................................................................................... 134
Introduction ............................................................................................................................. 135
Materials and Methods ............................................................................................................ 137
Results ..................................................................................................................................... 140
Discussion ............................................................................................................................... 147
Conclusions ............................................................................................................................. 150
Acknowledgements ................................................................................................................. 151
References ............................................................................................................................... 152
Figures and Tables .................................................................................................................. 155
Appendix ................................................................................................................................. 164
CONCLUSIONS ....................................................................................................................... 165
LIST OF PUBLISHED PAPERS

Chapter I - Lessner FH, Jennings ME, Hirata A, Duin EC, Lessner DJ. Subunit D of RNA
Polymerase from Methanosarcina acetivorans Contains Two Oxygen-labile [4Fe-4S] Clusters:
Implications for Oxidant-Dependent Regulation of Transcription. Journal of Biological
Chemistry. 2012 287(22):18510-18523. (http://www.jbc.org/content/287/22/18510.full)
Chapter II – Jennings ME, Lessner FH, Karr EA, Lessner DJ. The [4Fe-4S] clusters of subunit D
are key determinants in the post D-L heterodimer assembly of RNA polymerase in
Methanosarcina acetivorans. Molecular Microbiology. Submitted 2016.
Chapter III - Jennings ME, Schaff CW, Horne AJ, Lessner FH, Lessner DJ. Expression of a
bacterial catalase in a strictly anaerobic methanogen significantly increases tolerance to
hydrogen peroxide but not oxygen. Microbiology. 2014, 160(Pt 2):270-278.
(http://mic.sgmjournals.org/content/160/Pt_2/270.long)