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1

Proteomic signatures of Bacillus subtilis





Dissertation
in fulfilment of the academic grade
doctor rerum naturalium (Dr. rer. nat.)

at the Faculty of Mathematics and Natural Sciences
Ernst-Moritz-Arndt-University Greifswald



Le Thi Tam
born on 08.01.1979 in Bacninh, Vietnam





Greifswald, Germany, 2006
2



























Dekan:

1. Gutachter 1:
2. Gutachter 2:
3. Gutachter 3:

Tag der Promotion: 3
Contents

Contents 3
Abbreviations 5
Summary of thesis 7
Chapter 1: Introduction 9
1. Proteomic approachs and definition of proteomic signatures 10
2. B. subtilis as model organism for functional genomics 12
3. Proteome maps in Gram-positive bacteria 13
4. Regulation and function of stress responses 14
14 4.1. Heat shock response
4.2. Salt stress response 17
4.3. Oxidative stress response 19
4.4. Antibiotic response 21
5. Regulation and function of the starvation responses in B. subtilis 23
5.1. Glucose starvation response 24
5.2. Phosphate starvation response 26
5.3. Nitrogen starvation response 27
5.4. Tryptophan starvation response 29
5.5. The RelA-dependent stringent response 31
5.6. The CodY-dependent starvation response 32
H5.7. The σ -dependent general starvation response 32
6. Degradation of aromatic compounds in microorganism 33
7. Scopes of thesis 36
Chapter 2: A comprehensive proteome map of growing Bacillus subtilis cells 39
Chapter 3: 69 Proteome signatures for stress and starvation in Bacillus subtilis
as revealed by a 2D gel image color coding approach
Chapter 4: Global gene expression profiling of Bacillus subtilis in response to 91
ammonium and tryptophan starvation as revealed by
transcriptome and proteome analysis
Chapter 5: Differential gene expression in response to phenol and catechol 131
reveal different metabolic activities for the degradation of aromatic
compounds in Bacillus subtilis
Chapter 6: Proteomic signature catalog of B. subtilis in response to stress, 161
aromatic substances and nutrient starvation
Chapter 7: General discussion 221
1. The vegetative proteome map of B. subtilis 222
2. Proteome signatures of B. subtilis in response to stress, starvation and 223
xenobiotics
2.1. The catalog of proteome signatures of B. subtilis 223 4
2.2. Proteome signatures of B. subtilis in response to stress and xenobiotics 224
225 2.3. Proteome signatures of B. subtilisse to starvation
3. The response of B. subtilis to ammonium and tryptophan starvation 226
4. The response of B. subtilis to the aromatic compounds phenol and 227
catechol
References 230
List of publications 252
Curriculum vitae 253
Acknowledgements 254
5
ABBREVIATIONS FULL NAMES
2D two-dimensional
2DE two-dimensional electrophoresis
2D-PAGE two-dimensional polyacrylamide gel electrophoresis
ACN acetonitril
ATP adenosine-5’-triphosphate
B. subtilis Bacillus subtilis
BMM Belitsky minimal medium
BOC Belitsky minimal medium without citrate
CBB Coomassie Brilliant Blue
cDNA complementary deoxyribonucleic acid
CFU colony forming unit
CHAPS 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propane sulfonate
DNA deoxyribonucleic acid
DTT dithiolthreitol
E. coli Escherichia coli
EDTA ethylenediamine tetra acetic acid
rEm erythromycine resistance
g gravity
GTP guanosine triphosphate
h hour
IEF isoelectric focusing
incl. including
IPG non-linear immobilized pH gradients
IPTG isopropyl- β-D-thiogalactoside
kb kilo bases
kDa kilo Daltons
L liter
LB Luria Bertani broth medium
MIC minimal inhibitory concentration
min minute
M molecular weight r
MS mass spectrometry
nm nanometer
OD optical density
ORF open reading frame
PCR polymerase chain reaction 6
pI isoelectric point
PMSF phenylmethylsulphonylfluoride
RNA ribonucleic acid
rpm rounds per minute
s second
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
TFA trifluoroacetic acid
U unit
V voltage
v/v volume per volume
w/v weight per volume





7
Summary of thesis

The proteome obtained by the high resolution 2D protein electrophoresis reflects the
physiological state of a cell. From a physiological point of view, there are two main
proteomes in microorganisms- the proteomes of growing and non-growing cells. The goals of
this PhD thesis were (1) to establish the vegetative proteome map of B. subtilis; (2) to define
the proteome signatures of B. subtilis in response to stress and starvation and aromatic
substances towards the comprehensive proteome map of non-growing B. subtilis cells; (3) to
study the response of B. subtilis to ammonium and tryptophan starvation using
transcriptomics; (4) to investigate and characterize the global response of B. subtilis to
phenol and catechol stress.
In the vegetative proteome of B. subtilis, 745 proteins were identified using the 2D
gel-base approach. These include more than 40% of the predicted vegetative proteome in
the standard pH range 4-7 and most of the proteins involved in the central metabolic
pathways. This vegetative proteome map was complemented by the proteome map of B.
subtilis in response to heat, salt, hydrogen peroxide and paraquat stress, after ammonium,
tryptophan, glucose and phosphate starvation as well as in response to the aromatic
compounds phenol, catechol, salicylic acid, 2-methylhydroquinone, 6-brom-2-vinyl-chroman-
4-on. In total, 224 induced marker proteins have been identified using the 2D gel-based
approach including 122 stress-induced and 155 starvation-induced marker proteins. Of
these, 89 marker proteins are not expressed in the vegetative proteome map. Fused
proteome map and a color coding approach have been used to define stress-specific
regulons that are involved in specific adaptation functions (HrcA for heat, PerR and Fur for
oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition,
starvation-specific regulons are defined involved in the uptake or utilization of alternative
L Lnutrient sources (TnrA, σ /BkdR for ammonium; TRAP for tryptophan; CcpA, CcpN, σ /AcoR
for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome
L B H F Esignatures include the CtsR, Spx, σ /RocR, σ , σ , CodY, σ and σ regulons. Among these,
the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-
Hinduced protein damages. Finally, a subset of σ -dependent proteins (Spo0A, YvyD, YtxH,
YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as
general starvation proteins that indicate the transition to stationary phase caused by
starvation.
The global gene expression profile of B. subtilis was monitored in response to
ammonium and tryptophan starvation using the transcriptome approach. The results 8
demonstrated that both starvation conditions induced specific, overlapping and general
Lresponses. The TnrA and GlnR regulons as well as σ -dependent bkd- and roc- operons are
most strongly and specifically induced after ammonium starvation which are involved in the
uptake and utilization of ammonium and alternative nitrogen sources such as arginine,
proline and branched chain amino acids. In addition, the induction of several carbon
catabolite controlled genes (e.g. acsA, citB) as well as α-acetolactate synthase/
decarboxylase (alsSD) involved in acetoin biosynthesis and rather specific for ammonium
starvation. The specific response to tryptophan starvation includes the TRAP-regulated
tryptophan biosynthesis genes, a few RelA-dependent genes (e.g. adeC, ald) as well as
spo0E. Furthermore, we recognized overlapping responses between ammonium and

Htryptophan starvation (e.g. dat, maeN) as well as the common induction of the CodY and σ
general starvation regulons and the RelA-dependent stringent response. Several genes
encoding proteins of so far unknown functions are induced in response to ammonium and/or
tryptophan starvation which gained novel insights into the ammonium and tryptophan
starvation responses of B. subtilis.
Finally, the global expression profile of B. subtilis was investigated in response to
Bphenol and catechol using transcriptome analyses. Phenol induced the HrcA, σ and CtsR
heat shock regulons as well as the Spx disulfide stress regulon. Catechol caused the
activation of the HrcA and CtsR heat shock regulons and a thiol- specific oxidative stress
Bresponse involving the Spx, PerR and Fur regulons but no induction of the σ regulon. The
most surprising result was that several catabolite controlled genes are derepressed by
catechol, even if glucose is taken up under these conditions. This derepression of the carbon
catabolite control was dependent on the glucose concentration in the medium, since glucose
excess increased the derepression of the CcpA-dependent lichenin utilization licBCAH
operon and the ribose metabolism rbsRKDACB operon by catechol. Growth and viability
experiments with catechol as a sole carbon source suggested that B. subtilis 168 is not able
to utilize catechol as a carbon-energy source. In addition, the microarray results revealed the
very strong induction of the yfiDE operon by catechol of which the yfiE gene shares
similarities to glyoxalases/bleomycin resistance proteins/extradiol dioxygenases. Using
recombinant His -YfiE , we demonstrate that YfiE shows catechol-2,3-dioxygenase activity 6 Bs
in the presence of catechol since the metabolite 2-hydroxymuconic semialdehyde was
measured . Furthermore, both genes of the yfiDE operon are essential for the growth and
viability of B. subtilis in the presence of catechol. Thus, our studies revealed that the
catechol-2,3-dioxygenase YfiE is the key enzyme of a meta cleavage pathway in B. subtilis
involved in the catabolism of catechol. 9






Chapter 1

Introduction


















10
1. Proteomic approaches and definition of proteomic signatures
The genome sequence of an organism predicts the number of coding sequences and
represents only the “blue-print of life”, not “life itself”. The proteome consisting of all
expressed proteins at a certain time and under a certain condition brings the genetic
information of the DNA sequence to the life. In contrast to the DNA sequence, the proteome
provides informations about the expression levels, stabilization, localization, interaction and
post-translational modifications of the proteins (e.g. phosphorylation, glycosylation).
Therefore, the proteome analysis is an essential approach for the study of functional
genomics and the regulation in biological systems.
Transcriptomic and proteomic approaches in response to changes in the environment
including mutants in central regulatory genes are the major tools for functional genomics.
The transcriptome is defined as the transcriptional expression profile in the cell under a
certain condition [Velculescu et al., 1997] and the proteome refers to the complete protein set
expressed in the cell under a certain condition. In 1975, O’Farrell and Klose introduced the
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique [O’Farrell 1975;
Klose 1975]. Using this technique, a complex mixture of proteins can be separated because
each single protein will migrate to it’s unique position in the 2D-gel determined by its charge
and molecular mass. Later, this technique was introduced into bacterial physiology by
Neidhardt and VanBogelen [Neidhardt and VanBogelen, 2000] who addressed crucial
physiological questions by proteomics such as the heat stress response or the phosphate
starvation response of E. coli. This new approach was extremely attractive for studies on cell
physiology because proteomics could visualize cellular events not seen before by more
conventional techniques.
However, the standard 2D gel-based proteome analysis has also certain limitations.
For example, only a restricted protein amount can be separated in a 2D gel. Additionally,
there are problems to separate hydrophobic transmembrane or alkaline proteins using the
2D gel-based approach [Choe and Lee, 2003]. Many new gel-based and alternative gel-free
proteomic techniques have been developed to address these limitations. For example, for
proteome analysis of membrane bound proteins in B. subtilis, Bunai and coworker have
developed a membrane-protein-enrichment protocol using stepwise extraction of membrane
proteins with mixtures of detergents [Bunai et al., 2004]. This purified membrane fraction was
separated by three 2D gel-based techniques (IPGs, 16-BAC-PAGE and blue native PAGE).
The 2D gel-based approach combined with the fluorescence thiol modification assay is a
further proteome approach that allows to monitor the thiol state of cytoplasmic proteins and
the oxidized state of sulfhydryl groups [Hochgräfe et al., 2005]. In addition, the ProQ
Diamond Phosphoprotein Stain provides an excellent tool to study protein phosphorylation in
a 2D gel. These advances in the proteome analysis improved significantly the study of post-

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