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 4.1. Heat shock response 14 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 5.7. The σH-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: Proteome signatures for stress and starvation in Bacillus subtilis 69 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 2.3. Proteome signatures of B. subtilis in response to starvation 225 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 Emr 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 Mr molecular weight 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 nutrient sources (TnrA, σL/BkdR for ammonium; TRAP for tryptophan; CcpA, CcpN, σL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, σL/RocR, σB, σH, CodY, σF and σE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress- induced protein damages. Finally, a subset of σH-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 responses. The TnrA and GlnR regulons as well as σL-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 tryptophan starvation (e.g. dat, maeN) as well as the common induction of the CodY and σH 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 phenol and catechol using transcriptome analyses. Phenol induced the HrcA, σB 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 response involving the Spx, PerR and Fur regulons but no induction of the σB 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 His6-YfiEBs, we demonstrate that YfiE shows catechol-2,3-dioxygenase activity 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