LISTERIA MONOCYTOGENES ADJUSTS ITS MEMBRANE FLUIDITY, ATPase ACTIVITY AND ...

180 Pages·2009·1.39 MB·English

LISTERIA MONOCYTOGENES ADJUSTS ITS MEMBRANE FLUIDITY, ATPase
ACTIVITY AND atpE TRANSCRIPTION LEVELS IN RESPONSE TO COLD AND
ACID STRESS.

by
MOHAMED Z. BADAOUI NAJJAR
A Dissertation submitted to the
Graduate School-New Brunswick
Rutgers, The State University of New Jersey
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Graduate Program in Food Science
written under the direction of
Professor Thomas J. Montville
and approved by
________________________
________________________
________________________
________________________
New Brunswick, New Jersey
[May, 2009]
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ABSTRACT OF THE DISSERTATION
Listeria monocytogenes adjusts its membrane fluidity, ATPase activity and atpE
transcription levels in response to cold and acid stress.

By MOHAMED Z. BADAOUI NAJJAR

Dissertation Director:
Dr. Thomas J. Montville

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a disease
associated with a high mortality rate. The organism responds to a variety of stresses by
activating stress-response pathways and adjusting its membrane fluidity by altering its
fatty acid composition. We hypothesize that the F F ATPase plays a central role in
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L. monocytogenes’ response to multiple stresses. Wild-type and mutant cells were used to
investigate the response to cold, acid, and nisin. The branched-chain fatty acid deficient,
cold-sensitive mutant strain, cld1, showed significantly higher membrane rigidity (r =
0.175) compared to its wild-type 10403s (r = 0.121) when grown at 30 °C, but not at 15
°C (r = 0.124 and 0.116, respectively). Strain cld1 adjusted its membrane fluidity to cold
stress. The F F ATPase activity in strain cld1 was 3-fold higher than that of wild-type
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strain 10403s (0.0267 vs 0.0097 µmole Pi/min.mg protein, respectively) when cells were
grown at 30 °C. Supplementing cld1’s growth medium with the precursor to branched
fatty acid, 2-methylbutyrate, restored its fluidity but not the F F ATPase activity to wild-
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type levels. The acid tolerance response examined differences in initial acid-sensitivity
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and development of tolerance to lactic acid. Strain cld1 had decreased viability when
directly exposed to pH 3.5 (2.64 log CFU/ml), but gained increased viability at pH 3.5
after exposure to pH 5.5 (8.62 log CFU/ml) compared to the wild-type. Finally, a genetic
approach examined the F F ATPase c-subunit (atpE) expression in strains 10403s and
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cld1 using real-time PCR. Strain cld1 showed a 10-fold lower atpE mRNA transcript
compared to 10403s. When examining the generated data, we observed that strain cld1
has higher F F ATPase activity, higher initial acid sensitivity, increased protection due
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to the acid tolerance response, and lower c-subunit mRNA compared to strain 10403s in
addition to a rigid membrane. These results support the hypothesis that adjustments took
place at the level of the F F ATPase. The various data were pooled in a model in which
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the mutant strain cld1 has a smaller c-subunit carousel compared to its wild-type 10403s,
and highlights the important role of the ATPase in microbial stress response.

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Dedication

Thank god for his endless blessing
To my family, aunts and uncle for your love, care and support
I am forever in your debt.
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Acknowledgements

First and foremost, I am deeply grateful to Dr. Montville for the chance he has
given me to work on such an interesting and versatile project. Dr. Montville is the
energetic, sharp and uncompromising advisor that everybody wants to work with. He
encouraged an independent thought process, but was always available to discuss my
results and offer constructive feedback. He has a true and unaltered approach to science, a
witty sense of humor and a genuine interest in the wellbeing of his students. Dr.
Montville believes that the student advisor relationship should go beyond the science. He
offered me valuable professional development hints, suggested new approaches and
identified various opportunities that added to my academic, intellectual, and personal
growth. This experience was the biggest contribution to my academic, scientific and
personal growth, and I hope that many more will get that opportunity. Indeed, it was a
privilege to work with such a distinguished and accomplished professor and scientist.
A special thanks to Dr. Michael Chikindas for offering me a chance to collaborate
with him and expand my knowledge in areas related to my work. Dr. Chikindas is always
available to provide helpful advice and a new perspective for every problem. He wraps
science in an interesting and humorous, yet witty, context, making you look forward to
his discussions. It was due to his efforts and recommendations that I received my TA
funding for the first two years of my Ph.D. and for that, he has my deepest gratitude. In
addition, his sense of humor, and versatile scientific discussions were things to look
forward to every morning.
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I want to thank Dr. Richard Ludescher for his assistance in spectrofluorometry.
His tips and feedback in that area were essential to the success of the study. He was also a
driving factor to improve the quality of the research and was even involved in suggesting
new methods that strengthened the work.
I am also grateful to Dr. Bassam Annous from the USDA, Eastern Regional
Research Center for accepting to serve on my committee. Dr. Annous’s versatile research
background in addition to his expertise in Listeria’s membrane fluidity was a strong
addition to the committee and generated helpful feedback that strengthened the study. Dr.
Annous has generously agreed to share many tips and hints on competitions and the job
search. His expert advice broadened my perspective and was invaluable in improving my
applications.
I also want to thank Dr. George Carman and Dr. Gil Soo Han for use of their
equipments and their valuable comments and suggestions throughout my research. Dr.
Carman generously shared his expertise and comments to improve the quality of the
research.
I also want to thank Dr. Ruth Wirawan for her help with the DNA, RNA and real-
time PCR.
To my labmates and fellow graduate students Jason, Ruth, Allison, Katia, Shadi,
Jonathan, Sally, Renee, Linda and others, thank you for sharing the joys and hectic life in
graduate education. Your friendship and company enriched my cultural experience and
made every reunion something to look forward to.
I want to thank the wonderful staff at the department of food science, Paulette,
Jackie, Karen, Debbie, Yakov, Miriam, Patricia, Millie and others for their generous
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assistance throughout my studies. Their pleasant attitude, knowledge in university
processes and timely delivery deserves high praise.
Last but certainly not least, I want to thank my parents Rachid and Amira, my
sister Hala, Aunt Sabah, Aunt Samira and Uncle Samir for all their love, care and support
throughout the years. I would not have made it this far without you.

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Table of Contents

Abstract of the dissertation..................................................................................... ii
Dedication................................................................................................................. iv
Acknowledgments.................................................................................................... v
Table of contents......................................................................................................viii
List of Tables............................................................................................................xiii
List of illustrations...................................................................................................xiv
List of Abbreviations...............................................................................................xvii
Chapter I – Literature Review.................................................................................... 1
I.1. Listeria monocytogenes....................................................................................... 1
I.2. Disease history and outbreaks............................................................................. 2
I.2.a. History.............................................................................................................. 2
I.2.b. Disease.............................................................................................................. 2
I.2.c. Outbreaks.......................................................................................................... 3
I.3. Innovative techniques for Listeria monocytogenes control................................. 5
I.3.a Active Packaging............................................................................................... 5
I.3.b Multiple Hurdle Technology............................................................................. 6
I.4. L. monocytogenes cellular energetics.................................................................. 7
I.5. The F F ATPase................................................................................................. 7
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I.6. Factors influencing ATPase structure and activity.............................................. 9
I.7. Stress Resistance of Listeria monocytogenes...................................................... 10
I.7.a. Acid................................................................................................................... 10
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I.7.b. Salt.................................................................................................................... 11
I.7.c. Antimicrobials.................................................................................................. 11
I.8. Nisin: background, activity and resistance.......................................................... 12
I.9. Membrane fluidity............................................................................................... 13
I.10. Fluidity adjustment in response to temperature stress....................................... 14
I.11. Fluorescence and membrane fluidity................................................................. 16
I.12. Fluidity of liposomes and vegetative cells........................................................ 17
Chapter II – Hypothesis............................................................................................. 19
Chapter III – Materials and Methods......................................................................... 20
III.1. Culture and culture conditions.......................................................................... 20
III.2. Lipid extraction................................................................................................. 20
III.3. Liposome construction...................................................................................... 21
III.4. Steady-state fluorescent anisotropy measurement............................................ 22
III.5. Effect of DPH on Listeria whole-cell viability................................................. 23
III.6. Whole-cell anisotropy....................................................................................... 23
III.7. Measurement of cell adjustment to temperature shift....................................... 24
III.8. De novo protein synthesis inhibition................................................................. 25
III.9. Supplementation of L. monocytogenes with 2-methylbutyrate......................... 25
III.10. Minimal growth pH of L. monocytogenes ..................................................... 26
III.11. Acid tolerance response.................................................................................. 26
III.12. Nisin sensitivity of L. monocytogenes............................................................ 27
III.13. Nisin sensitivity of acid tolerant cells (ATR+)................................................ 28
III.14. Changing the membrane fluidity by fatty acid addition................................. 28
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III.15. Preparation of membrane vesicles.................................................................. 29
III.16. Bradford protein quantification....................................................................... 29
III.17. F F ATPase activity determination................................................................ 30
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III.18. Chromosomal DNA extraction....................................................................... 30
III.19. Agarose gel electrophoresis............................................................................ 31
III.20. RNA extraction............................................................................................... 31
III.21. RNA treatment for gel electrophoresis........................................................... 32
III.22. RNA concentration determination.................................................................. 33
III.23. DNase treatment and standardization of RNA................................................ 33
III.24. Reverse transcription...................................................................................... 33
III.25. cDNA testing by PCR..................................................................................... 34
III.26. Real time PCR................................................................................................. 34
Chapter IV – Acid tolerance response induces resistance to nisin and alters
membrane fluidity in Listeria monocytogenes........................................................... 36
IV.1. Resistance of L. monocytogenes to lactic acid.................................................. 37
IV.2. Listeria acid tolerance response........................................................................ 37
IV.3. Effect of 2-methylbutyrate on L. monocytogenes acid tolerance..................... 39
IV.4. Effect of nisin on L. monocytogenes................................................................. 39
IV.5. Effect of nisin on acid tolerant L. monocytogenes (ATR+).............................. 40
IV.6. Anisotropy measurement of ATR+ Listeria...................................................... 41
IV.7. Anisotropy measurements of nisin-resistant cells in the presence of nisin...... 41
Chapter V – Changes in Listeria monocytogenes Membrane Fluidity in Response
to temperature Stress.................................................................................................. 43
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