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Open Access Dissertations Theses and Dissertations
Winter 2015
Transcriptional stress response in foodborne
pathogens
Aaron M. Pleitner
Purdue University
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Pleitner, Aaron M., "Transcriptional stress response in foodborne pathogens" (2015).Open Access Dissertations. 537.
https://docs.lib.purdue.edu/open_access_dissertations/537
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Graduate School Form 30
Updated 1/15/2015
PURDUE UNIVERSITY
GRADUATE SCHOOL
Thesis/Dissertation Acceptance
This is to certify that the thesis/dissertation prepared
Aaron M. Pleitner
By
Entitled
Transcriptional Stress Response in Foodborne Pathogens
Doctor of Philosophy
For the degree of
Is approved by the final examining committee:
Haley F. Oliver
Chair
Arun Bhunia
Paul Ebner
Scott Hood
To the best of my knowledge and as understood by the student in the Thesis/Dissertation
Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32),
this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of
Integrity in Research” and the use of copyright material.
Haley F. Oliver
Approved by Major Professor(s):
Mario Ferruzzi 2/24/2015
Approved by:
Head of the Departmental Graduate Program Date
i
TRANSCRIPTIONAL STRESS RESPONSE IN FOODBORNE PATHOGENS
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Aaron M. Pleitner
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
May 2015
Purdue University
West Lafayette, Indiana
ii
“Enjoy when you can, endure when you must.” – Johann Wolfgang von Goethe
To my wife, Brenna,
who always stands by me and keeps things in perspective. You encourage me to
consistently pursue greatness.
iii
ACKNOWLEDGEMENTS
I have been extremely fortunate to have had very influential advisors throughout
my graduate career. Drs. Lynn McMullen and Michael Gänzle provided me my initial
foothold into science and academia. You took a very young scientist and lent me your
patience and trust to develop and pursue my academic mind. Dr. Haley Oliver, I am
grateful for the opportunities you provided me while at Purdue. In addition, you have
shown me more about the depths and intricacies of the human mind and spirit than I
could have ever fathomed.
All of my work and endeavors in and outside of science are for my wife, Brenna.
You make me want to be the best, most successful and kind person that I possibly can be.
The patience and support you provided me while I moved back to Purdue and as we’ve
moved forward are appreciated with my whole heart.
I would also like to thank my funding sources, without this support my work
would not have been possible. The Industry Fellows program provided me support for
my first year and an incredible opportunity. The Showalter Trust and Showalter Fund for
Assistant Professors provided support for all MRSA research and analysis. For the
pedagogy studies, equipment support was provided Purdue University College of
Agriculture Institutional Instruction grants and graduate staff was supported by the
USDA NIFA National Education Program in Food Safety 2011-67005-30010.
To Simmon Hofstetter and Januana Teixeira, you have acted as mentors and
models for being passionate, professional and brilliant in science and life. The effect
you’ve had on me in and outside the lab will last a lifetime. To Katie Satchwell and
Andrea Balutis, you give me hope for the future, your youthful passion gives me comfort.
I credit Joshue Krabbe with showing me the methodical way to approach goals and
projects. I am indebted to the friends I made at Purdue who pursued science and drove
iv
success alongside me: Sydney Moser, Tristan Lipke, Simran Kaur, Stacey Hirt, Alyssa
Beatty, Amudhan, Ponrajan, Clara Vásquez-Mejía, Jen Kahn, Shanleigh Thomson,
Ashley Broady, Krystin Marrs, Titiksha Dikshit, and Gregory Weber. I will always be
your friend and have a fondness in my heart for you all. I was lucky enough to have
passionate and brilliant undergraduates to work with: Jake Eichinger, Natalie Donovan,
Peter Cook and Carmen Wickware. I appreciate the willingness to listen to me while I
explained my science to you and the aid you provided within the lab. Jake, I will always
fondly remember listening to a specific genre of music with you in the lab.
Thank you, for everything
v
TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................................. xi
LIST OF FIGURES ......................................................................................................... xiv
CHAPTER 1. INTRODUCTION .................................................................................... 1
1.1 Introduction ............................................................................................................... 1
1.2 Foodborne Disease and Outbreaks ............................................................................ 1
1.2.1 Listeria monocytogenes ................................................................................... 3
1.2.2 Staphylococcus aureus ..................................................................................... 3
1.3 Stress Response and Adaptation ............................................................................... 5
1.3.1 Stress Response Regulation ............................................................................. 5
1.4 Antibiotic Resistance ................................................................................................. 7
1.5 Strategies to Manage Environmental Pathogen Presence ......................................... 8
1.6 Molecular Methods for Identifying and Characterizing Foodborne Pathogens ........ 9
1.6.1 Polymerase Chain Reaction ........................................................................... 10
1.6.2 Pulsed-Field Gel Electrophoresis .................................................................. 12
1.6.3 Whole Genome Sequencing .......................................................................... 12
1.7 Transcriptional Analysis ......................................................................................... 13
1.7.1 Transcription and Gene Annotation ............................................................... 14
1.7.2 Microarray Analysis ...................................................................................... 14
1.7.3 Quantitative Polymerase Chain Reaction ...................................................... 15
1.7.4 RNA-Sequencing ........................................................................................... 15
1.8 Food Microbiology and Safety Pedagogy ............................................................... 17
1.9 Aims of This Work .................................................................................................. 18
1.9.1 Response of L. monocytogenes to Chlorine Dioxide ..................................... 18
vi
Page
1.9.2 Genomic and Transcriptomic Assessment of MRSA .................................... 19
1.9.3 Measurement of the Major Stressosome in MRSA ....................................... 19
1.9.4 Integration of Molecular Techniques into Food Microbiology Curricula ..... 20
CHAPTER 2. TRANSCRIPTIONAL AND PHENOTYPIC RESPONSES OF
LISTERIA MONOCYTOGENES TO CHLORINE DIOXIDE .......................................... 21
2.1 Introduction ............................................................................................................. 22
2.2 Materials and Methods ............................................................................................ 24
2.2.1 Bacterial Strains and Storage ......................................................................... 24
2.2.2 Chlorine Dioxide Effect on Log Phase Cell Survival .................................... 24
2.2.3 RNA Extraction ............................................................................................. 25
2.2.4 cDNA Synthesis and Microarray Hybridization............................................ 26
2.2.5 Microarray Statistical Analysis ...................................................................... 27
2.2.6 TaqMan quantitative Reverse Transcriptase-PCR (qRT-PCR) ..................... 28
2.3 Results ..................................................................................................................... 29
2.3.1 Effect of [ClO ] on L. monocytogenes 10403S Survival ............................... 29
2
2.3.2 Microarray Analyses Identified 340 Differentially Expressed Genes upon
Exposure to ClO ....................................................................................................... 30
2
2.3.3 Six JCVI Role Categories Were Either Over- and Underepresented among
Significantly Upregulated Genes ............................................................................... 36
2.3.4 Significantly Downregulated Genes Were Over- or Underrepresented in 7
JCVI Role Categories ................................................................................................ 37
2.3.5 Genes in Two Major Regulatory Networks Are Differentially Expressed
under ClO Stress ....................................................................................................... 39
2
2.3.6 TaqMan qRT-PCR Confirms Select Differentially Expressed Genes upon
Exposure to ClO ....................................................................................................... 40
2
2.3.7 σB and CtsR Contribute L. monocytogenes ClO Survival ............................. 41
2
2.4 Discussion ............................................................................................................... 41
2.4.1 Chaperone Proteins Play a Role in Oxidative Stress Management ............... 42
vii
Page
2.4.2 Altered Activity of Cell Division Genes, Universal Stress Response and SOS
Response Is Evident following ClO Exposure ......................................................... 43
2
2.4.3 Genes Involved with Redox Reactions Help Manage ClO Oxidative
2
Stress ....................................................................................................................... 44
2.4.4 σB and CtsR Contribute to L. monocytogenes ClO Survival ........................ 45
2
CHAPTER 3. GENOMIC AND TRANSCRIPTOMIC ASSESSMENT OF
METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS ...................................... 47
3.1 Introduction ............................................................................................................. 48
3.2 Materials and Methods ............................................................................................ 50
3.2.1 Bacterial Strain and Culture Conditions ........................................................ 50
3.2.2 Genomic DNA Extraction ............................................................................. 51
3.2.3 Illumina Library Preparation, Whole Genome Sequencing and Analysis ..... 51
3.2.4 Antibiotic Exposure ....................................................................................... 52
3.2.5 RNA Extraction ............................................................................................. 52
3.2.6 Library Preparation and Illumina RNA-Sequencing ..................................... 53
3.2.7 Differential Gene Expression ........................................................................ 54
3.3 Results ..................................................................................................................... 55
3.3.1 WGS Assembly Results in Partial Closure and Reveals Novel ncRNAs ...... 55
3.3.2 Analysis Packages for Differential Expression Identify Similar Genes and
ncRNAs with Slight Variations ................................................................................. 57
3.3.3 A Comparison of Growth Phase Reveals 1692 Genes and 322 ncRNAs as
Differentially Expressed ............................................................................................ 61
3.3.4 Ten IMG Functional Role Categories Were either Over- or Underrepresented
during Stationary Phase ............................................................................................. 84
3.3.5 Exposure to Tetracycline Results in the Differential Expression of 1641
Genes and 241 ncRNAs ............................................................................................. 86
3.3.6 Ten IMG Functional Role Categories Were Over- or Underrepresented
following Tetracycline Exposure for both Up- and Downregulated Genes ............ 106
