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Differential proteome analysis of selected lactic acid bacteria, stress response and database construction [Elektronische Ressource] / Oliver Drews

161 pages
Ajouté le : 01 janvier 2005
Lecture(s) : 64
Signaler un abus

Technische Universität München

FG Proteomik



Differential proteome analysis of
selected lactic acid bacteria,
stress response and database construction


Oliver Drews


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften (Dr. rer. nat.)

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. A. Delgado
Prüfer der Dissertation: 1. apl. Prof. Dr. A. Görg
2. Univ.-Prof. Dr. K.-H. Schleifer
3. Univ.-Prof. Dr. Dr. h.c. (Zonguldak Univ./Türkei) H. Parlar

Die Dissertation wurde am 29.11.2004 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 25.01.2005 angenommen. Acknowledgements

The work presented in this thesis was done under the guidance of Prof. Dr.
Görg at the FG Proteomics of the Technische Universität München, Germany, in the
period between April 2000 and November 2003. I would like to thank my supervisor
Prof. Dr. Görg for excellent research facilities, her confidence and the freedom to
pursue the projects with own ideas, helpful discussions and unrestricted support in all
concerns. This work was supported by the Deutsche Forschungsgemeinschaft (FOR
358/1).
I especially thank Prof. Dr. Parlar and Prof Dr. Schleifer for kindly accepting to
co-examine this thesis, as well as Prof. Dr. Delgado for accepting the position as
chairman in my examination and his valuable discussions in the high pressure
research group.
My further thanks go to Dr. Walter Weiss for productive collaboration and
helpful discussions on proteomics and manuscripts, and Dr. Gerold Reil of the
Institute for Chemisch-Technische Analyse und Chemische Lebensmitteltechnologie,
who kindly identified proteins by MALDI-TOF MS.
In addition, at this point, I express my gratitude to Dr. Robin Wait of the Imperial
College in London for his excellent support in protein identification by tandem mass
spectrometry.
I thank Prof. Dr. Vogel for providing his microbiological view of high pressure
and the high pressure apparatus. Thanks also to Maher Korakli and Adriana Molina-
Höppner for sharing distinctive high pressure moments and working with me under
pressure.
Burghardt Scheibe, Kay Junghanns, Reiner Westermeier and all other
Amersham guys, who are now GE guys, deserve my gratitude for equipping me with
the latest Amersham gadgets for proteomics and giving me nice diversions
(especially from Elsendorf), whenever they crossed the lab.
Special thanks go also to Daikun Preuss (Daikun Solutions) for his
ingeniousness in realization of the online access to the proteome database. Similar
thanks go to (Gel-)Carsten Lück, our computer specialist, for untiringly listening to
questions like: Have you ever had the problem with Windows blablabla…?
Thanks also to Christian Obermaier, Günter Boguth and Robert Wildgruber for
being there in the very beginning and providing a hot start in 2D electrophoresis, as
well as all other former and present lab members for sharing jokes and having fun.
As always, also my chaos team Darius and Simon deserve my credits for the
latest PhD gossip and adventurous tours outside the lab.
My mom, Wilfried and my grandma deserve my deepest gratitude for providing
save haven and unconditional support whenever I needed them. I will give you tips
on your future graduate studies too, mom!
Finally and most importantly, Maren Engelhardt receives the most thanks of all
for countless hours of proofreading manuscripts, reminding me to get some sleep late
at night, keeping me healthy with tiger food and running during stressful periods, and
all the other little things with which she enriches my life. All the best for 10 years of
Otter relationship!
IIPublications

Wildgruber R., Reil G., Drews O., Parlar H., Görg A.: Web-based two-dimensional
database of Saccharomyces cerevisiae proteins using immobilized pH gradients from
pH 6 to pH 12 and MALDI-TOF MS. Proteomics. 2002 Jun; 2(6):727-32.

Drews O., Weiss W., Reil G., Parlar H., Wait R., Görg A.: High pressure effects step-
wise altered protein expression in Lactobacillus sanfranciscensis. Proteomics. 2002
Jun; 2(6):765-74.

Görg A., Drews O., Weiss, W.: Separation of Proteins Using Two–dimensional Gel
Electrophoresis. Chapter 16 in: Purifying Proteins for Proteomics: A Laboratory
Manual (Richard J. Simpson, ed.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 2004: p. 391-430.

Drews O., Reil G., Parlar H., Görg A.: Setting up standards and a reference map for
the alkaline proteome of the Gram-positive bacterium Lactococcus lactis. Proteomics.
2004 May; 4(5):1293-1304.

Drews O. and Görg A.: DynaProt 2D: an advanced proteomic database for dynamic
online access to proteomes and two-dimensional electrophoresis gels. Nucleic Acids
Res. 2005 Jan; 33: in press

Drews O., Reil G., Parlar H., Görg A.: Analysis of the heat shock response of
Lactococcus lactis with advanced proteomic tools reveals altered abundance of
proteins implicated in the purine metabolism. in preparation
Görg A.: High pressure induced alterations in the
proteome of Lactococcus lactis point out harmful effects at sublethal pressures. in
preparation

Poster Presentations

O. Drews, W. Weiss, R.F. Vogel, A. Görg: Stressproteine von Milchsäurebakterien
nach subletaler Hochdruckbehandlung. Hochdruck-Workshop, 2001, Freising,
Germany

O. Drews, W. Weiss, G. Reil; H. Parlar, R. Wait and A. Görg: Lactic Acid Bacteria
Under Pressure; P-71, Proteomic Forum, International Meeting on Proteome
Analysis, 2001, Munich, Germany

G. Reil, O. Drews, R. Wildgruber, B. Boguth, H. Parlar, A. Görg: 2D Reference Map
of alkaline E. coli proteins using Immobilized pH Gradients up to pH 12 and MALDI-
TOF MS; P-112, Proteomic Forum, International Meeting on Proteome Analysis,
2001, Munich, Germany

R. Wildgruber, G. Reil, O. Drews, H. Parlar, A. Görg,; Web-based 2D-Database of
Saccharomyces cerevisae proteins using Immobilized pH Gradients from pH 6 to pH
12 and MALDI-TOF MS; P-113, Proteomic Forum, International Meeting on
Proteome Analysis, 2001, Munich, Germany

IIIO. Drews, R. Wait, G. Reil, H. Parlar, W. Weiss, A. Görg: Lactobacillus
ndsanfranciscensis under Pressure; I-32, 102 General Meeting of the American
Society for Microbiology, 2002, Salt Lake City, UT, USA

G. Reil, O. Drews, R. Wildgruber, H. Parlar, A. Görg; 2D Reference maps of alkaline
E. coli and S. cerevisiae proteins using immobilized pH grafients up to pH 12 and
MALDI-TOF MS; P-96, From Genome To Proteome: Functional Proteomics, 5th
Siena Meeting, 2002, Siena, Italy

O. Drews, W. Weiss, R. Wait and A. Görg: Stress Response of Lactic Acid Bacteria -
A closer view; P-108, From Genome To Proteome: Functional Proteomics, 5th Siena
Meeting, 2002, Siena, Italy

A. Köpf, O.Drews, W. Weiss and A. Görg: Sample Prefractionation with Sephadex-
IEF; P-5, Proteomic Forum, International Meeting on Proteome Analysis, 2003,
Munich, Germany

L. Thoenes, O. Drews, A. Görg and W. Weiss: Protein Carbamylation – An Actual
Problem in 2-D Electrophoresis?; P-9, Proteomic Forum, International Meeting on
Proteome Analysis, 2003, Munich, Germany

O. Drews and A. Görg: Current 2D Electrophoresis based Technologies in Stress
Response Assessment: Fluorescence- (DIGE) & Radioactive Pulse Labeling; P-113,
Proteomic Forum, International Meeting on Proteome Analysis, 2003, Munich,
Germany

O. Drews, G. Reil, H. Parlar and A. Görg: Mapping of Minimal Differences in the
Alkaline Proteome of Two Lactococcal Strains by Difference Gel Electrophoresis; P-
166, Proteomic Forum, International Meeting on Proteome Analysis, 2003, Munich,
Germany

C. Lück, O. Drews, G. Reil; H. Parlar and A. Görg: Towards a Database of Very
Alkaline Corynebacterium glutamicum Proteins; P-193, Proteomic Forum,
International Meeting on Proteome Analysis, 2003, Munich, Germany

M. Thiel, O. Drews, A Köpf, H. Behrendt, A. Görg, C. Traidl-Hoffmann: Proteomic
Approach for the Analysis of Allergen Carriers; 16. Mainzer Allergie-Workshop, 2004,
Mainz, Germany

Oral Presentations

O. Drews, A. Köpf, C. Lück, G. Reil, W. Weiss, A. Görg: Challenges and routines of
2-D electrophoresis. Perfection in view?; Journadas sobre Proteómica, 05.02.03,
Cordoba, Spain

O. Drews, A. Görg: 2D Electrophoresis: From Basics to Challenges.; From 2D to
MALDI-TOF, 24.06.03, Alghero, Italy
IVContents
1 INTRODUCTION.........................................................................................1
1.1 Aim of the study..................................................................................................................... 2
1.2 Proteome analysis................................................................................................................. 4
1.2.1 2D electrophoresis with immobilized pH gradients (IPG-DALT) .......................................4
1.2.2 Focus on alkaline proteins in proteomics .........................................................................5
1.2.3 Advanced protein detection and quantification with fluorescent dyes...............................6
1.2.4 Difference gel electrophoresis (DIGE)..............................................................................6
1.2.5 Radioactive labeling of proteins for analyses with 2D electrophoresis .............................8
1.2.6 Mass spectrometry in proteomics.....................................................................................9
1.2.7 Databases dedicated to 2D electrophoresis...................................................................10
1.3 Lactococcus lactis and Lactobacillus sanfranciscensis: two lactic acid bacteria....... 12
1.3.1 Lactococcus lactis in proteomics....................................................................................13
1.4 Stress response of microorganisms ................................................................................ 14
1.4.1 Heat shock response with focus on Lactococcus lactis..................................................14
1.4.2 High pressure effects on microorganisms ......................................................................16
1.4.3 Pressure resistant phenotypes.......................................................................................17
1.4.4 High pressure stress analyses with 2D electrophoresis .................................................18
2 MATERIALS AND METHODS20
2.1 Bacterial strains, growth conditions and media.............................................................. 20
2.1.1 Growth media.................................................................................................................20
2.1.2 Bacterial strains and growth conditions ..........................................................................22
2.2 Stress treatments ................................................................................................................ 22
2.2.1 Heat treatment................................................................................................................22
2.2.2 High pressure treatment.................................................................................................23
2.3 Protein extraction ................................................................................................................ 24
2.4 2D electrophoresis with immobilized pH gradients (IPG-DALT) ................................... 25
2.4.1 Preparation of immobilized pH gradient (IPG) gels ........................................................26
2.5 Protein gel staining techniques......................................................................................... 28
2.5.1 Silver staining.......28
2.5.2 Coomassie Brilliant Blue staining ...................................................................................28
TM2.5.3 SYPRO RUBY staining ...............................................................................................28
352.6 S pulse labeling in proteomics ........................................................................................ 29
352.6.1 S pulse labeling of proteins in vivo...............................................................................29
352.6.2 2D electrophoresis of S labeled proteins .....................................................................29
352.6.3 Phosphor imaging of S labeled proteins after 2D electrophoresis ...............................30
TM2.6.4 Image analysis after phosphor imaging, silver, CBB or SYPRO RUBY staining using
ImageMaster 2D.............................................................................................................30
2.7 Difference gel electrophoresis (DIGE) .............................................................................. 32
2.7.1 Protein extraction and solubilization for DIGE ................................................................33
2.7.2 Fluorescence labeling of proteins for DIGE....................................................................34
2.7.3 Mixing of the labeled samples for DIGE .........................................................................34
2.7.4 2D electrophoresis with up to three samples per gel for DIGE.......................................35
2.7.5 Image acquisition of DIGE gels ......................................................................................35
2.7.6 Image analysis after DIGE with the DeCyder software package ....................................36
2.8 Protein identification........................................................................................................... 38
2.8.1 Peptide mass fingerprinting MALDI-TOF MS .................................................................39
2.8.2 Peptide fragmentation LC-MS/MS..................................................................................40
V2.9 Resources and algorithms for the in silico analysis of L. lactis ................................... 41
2.10 Resources, algorithms and software used for the dynamic online database........... 41
3 RESULTS...............................................................................................43
3.1 Detection of pressure dependent proteins in Lactobacillus sanfranciscensis........... 46
3.1.1 Characterization of pressure dependent proteins by MALDI-TOF MS ...........................52
3.1.2 Characpressure dependent spots by LC-MS/MS........................................52
3.2 Analysis of the heat shock response of L. lactis with advanced proteomic tools....... 55
3.2.1 45 min heat shock analyzed with DIGE..........................................................................55
3.2.2 20 min heat shock a62
353.2.3 20 min heat shock analyzed with S pulse labeling.......................................................64
3.3 Analysis of high pressure effects on L. lactis at protein level....................................... 71
3.3.1 Analysis of high pressure response by DIGE .................................................................71
353.3.2 Analysis of protein expression after pressure stress by S pulse labeling.....................79
3.4 Establishing alkaline reference maps for L. lactis .......................................................... 89
3.4.1 Analyzing the alkaline in silico proteome of L. lactis89
3.4.2 Optimization of IEF conditions for the alkaline pH range................................................91
3.4.3 Mapping the alkaline proteome ......................................................................................93
3.5 Features of the dynamic online database for the proteome of L. lactis..................... 100
4 DISCUSSION ........................................................................................105
4.1 High hydrostatic pressure effects step-wise altered protein expression in
Lactobacillus sanfranciscensis....................................................................................... 105
4.2 Heat shock analysis of L. lactis reveals several proteins previously not reported ..... 109
4.2.1 Differential expression of purine metabolism related proteins at heat shock...................110
4.2.2 Further proteins induced at heat shock and previously not reported............................112
4.3 High pressure induced changes in the proteome of L. lactis...................................... 114
4.4 The alkaline proteome of L. lactis ................................................................................... 119
4.5 The dynamic online database for proteomes sets new standards for online 2D
databases........................................................................................................................... 123
5 SUMMARY ...........................................................................................125
6 ZUSAMMENFASSUNG ............................................................................129
7 REFERENCES.......................................................................................133
8 TABLE OF FIGURES...............................................................................146
9 APPENDIX............................................................................................147
VIAbbreviations:

2D two-dimensional
AA acrylamide
APS ammoniumpersulfate
Bis bisacrylamide
%C percentage of crosslinker in acrylamide solution
CAI codon adaptation index
CBB Coomassie Brilliant Blue
CID Collision-induced Dissociation
CIRCE controlled inverted repeat of chaperone expression
Clp caseinolytic protease
CSP cold shock protein
Cy cyanine
Cys cysteine
Da Dalton
DIGE difference gel electrophoresis
DMAA dimethyl-acrylamide
DNA desoxyribonucleic acid
DTT dithiothreitol
ESI electropspray ionization
e expectation value (in MALDI-TOF MS analysis)
GRAVY grand average of hydropathicity
h hour
HSP heat shock protein
HTML hypertext markup language
IEF isoelectric focusing
IPG immobilized pH gradient
IPG-DALT two-dimensional electrophoresis with immobilized pH gradient
IPS internal pooled standard
LC liquid chromatography
Lys lysine
mA milliampere
MALDI matrix assisted laser desorption ionization
VIIMet methionine
MOPS 3-[N-Morpholino]propanesulfonic acid
MPa megapascal
Mr relative molecular mass
MS mass spectrometry
MS/MS tandem mass spectrometry
NEPHGE non equilibrium pH gradient gel electrophoresis
NHS N-hydroxy succinimidyl
NR non redundant
OD optical density
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PHP PreHypertextProcessor
pl isoelectric point
PIP pressure-induced protein
PMF peptide mass fingerprint
PRPP 5-phosphoribosyl-1-pyrophosphate
Q-TOF quadrupole-time of flight
RNA ribonucleic acid
SDS sodium dodecylsulfate
SQL structured query language
%T percentage of acrylamide in total
TEMED N,N,N',N'-tetramethylethylendiamine
TOF time of flight
Tris tris(hydroxymethyl)-aminomethane
V volt
v/v volume per volume
W watt
w/v weight per volume

VIIIIntroduction
1 Introduction

On the first Siena 2D electrophoresis meeting in 1994, M. Wilkins publicly
introduced the term proteome, by which he described the protein complement of the
genome. Since the genome of an organism is almost completely static, while the
proteome is highly dynamic, the analogy of genome and proteome is only superficial.
The proteome varies in the way that i) not all proteins are expressed at the same
time, ii) proteins are expressed in different amounts, iii) several forms of one protein
occur due to post translational modifications, and iv) different cell types in multi-
cellular organisms express specialized sets of proteins. The dynamics of protein
expression is again highly dependent on the cellular state and the environment.
Exactly these characteristics enable by analysis of the proteome molecular insights
into cellular processes like stress response or adaptation.
Heat shock response in bacteria is a commonly accepted mechanism, by which
they adapt to the extreme condition and cope with deleterious effects such as protein
denaturation [1]. Although general schemes in heat shock response are observed,
regulation and scope of such a response can be different. E. coli and B. subtilis serve
as model organisms for Gram-negative and –positive bacteria, respectively. Much is
known about the heat shock response of these model bacteria, but knowledge of the
response for example in lactic acid bacteria is rather limited to the general scheme
(reviewed in [2-4]). The influence of heat treatment on lactic acid bacteria is not less
interesting, since these bacteria encounter heat treatments in food processing on a
regular basis. Lactococcus lactis gained model status among the lactic acid bacteria
and thus, is probably the best investigated example of them with respect to heat
shock [3, 5-7]. Though, besides prominent heat shock proteins the scope of heat
shock response in L. lactis is unknown, not to mention the regulation. For example,
Bno counterpart for the alternate sigma factor σ , which regulates class two stress
genes in the model organism B. subtilis, was identified in the genome of L. lactis [8].
Analyses of the heat shock response of L. lactis with current tools in proteomics
provide a more comprehensive view of the scope of this response and promise in
combination with the availability of a fully sequenced genome high identification
rates.
High hydrostatic pressure is used in food processing as non thermal
preservation technique [9-11]. Advantages of this technique are that flavor and
1Introduction
vitamins are less affected by high pressure than by heat treatment [10, 12].
Microorganisms are generally inactivated by high pressure treatment, though the
pressure level for successful inactivation varies largely among different bacteria [13,
14]. In addition, certain environmental factors as well as physiological conditions
increase the pressure resistance of bacteria [15-18]. The elucidation of deleterious
effects of high pressure to bacteria, their adaptation and the potential to acquire
resistance has just begun. Reports of unexpected pressure resistance in certain
species demonstrate the great need in comprehension of such mechanisms to
ensure safe food quality [19, 20]. Analysis of high pressure effects on bacteria at
proteome level is highly potential to identify cellular mechanisms, which are activated
in response to pressure stress. Few analyses at proteome level of high pressure
response in bacteria have been published. Altered protein levels of heat or cold
shock proteins have been observed, which indicate similar responses as under heat
or cold shock [16, 21]. Indeed, high pressure causes similar effects as, for instance,
heat shock causes protein denaturation and thus might explain an analog cell
response [22]. Thus, the first analyses show promising results and encourage further
investigations of high pressure response at proteome level.

1.1 Aim of the study

The analysis of heat shock response in L. lactis at proteome level was so far
analyzed by radioactive pulse labeling [5-7] and in these cases, protein identification
from 2D gels was moderately successful (reviewed in [23]). In the meantime, access
to the complete genomic sequence of L. lactis [8] in combination with higher
sensitivity in mass spectrometry improves the success rate in spot identification of 2D
gels in L. lactis [24]. Furthermore, improved technologies in 2D electrophoresis are
available, such as 2D electrophoresis with immobilized pH gradients (IPG-Dalt) and
difference gel electrophoresis (DIGE) (reviewed in [25]). Therefore, one aim of the
study was the analysis of the heat shock response of L. lactis with advanced
proteomic tools to identify hitherto undetected and unidentified proteins involved in
heat shock response. In particular, total protein expression was analyzed after heat
shock with the DIGE technique as well as temporal protein expression under heat
shock conditions analyzed with radioactive pulse labeling. DIGE is a relatively new
technique in the field of proteomics. Therefore, the suitability of this technique for
2

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