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Bacillus Subtilis Regulatory mechanisms of the disulfide stress response and the role of the bacillithiol redox buffer in Gram-positive bacteria ! ! " I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Bui Khanh Chi geboren am 15.08.1983 in Hanoi, Vietnam Greifswald, den 16. November 2012" " !" " ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Dekan: Prof. Dr. Klaus Fesser 1. Gutachter: Priv.-Doz. Dr. Haike Antelmann 2. Gutachter: Prof. Dr. Henry A. Claiborne 3. Gutachter: Tag der Promotion : 01 – 02 – 2013 ! "! ! Table of Contents Chapter 1 Introduction and general conclusion 4 Chapter 2 S-bacillithiolation protects against hypochlorite stress in Bacillus 35 subtilis as revealed by transcriptomics and redox proteomics. Chapter 3 S-bacillithiolation protects conserved and essential proteins against 57 hypochlorite stress in Firmicutes bacteria. Chapter 4 Structural insights into the redox-switch mechanism of the 81 MarR/DUF24-family regulator HypR. Chapter 5 The redox-sensing regulator YodB senses quinones and diamide via 97 a thiol-disulfide switch in Bacillus subtilis. Chapter 6 The paralogous MarR/DUF24-family repressors YodB and CatR 109 (YvaP) control expression of the catechol dioxygenase CatE in Bacillus subtilis. Zusammenfassung 121 Eidesstattliche Versicherung 125 Publications and poster presentations 127 Acknowledgements 132 ! #! ! ! ! "#$%&'(!)! ! ! ! ! ! ! ! ! ! ! ! ! Chapter 1 Introduction and general conclusion Bui Khanh Chi 1. The biological function of Cysteine and its thiol group ......................................... 5 2. Functions of low molecular weight thiols in bacteria ............................................. 5 2.1. Biosynthesis and functions of glutathione and role of S-glutathionylation .......... 6 2.2. Biosynthesis and functions of mycothiol and role of MSH-dependent enzymes . 9 2.3. Biosynthesis and functions of bacillithiol and role of S-bacillithiolations ......... 11 3. Reactive oxygen, nitrogen and electrophilic species (ROS, RNS, RES) ............. 14 3.1. Sources of ROS, RNS and RES ......................................................................... 14 3.2. Reaction of ROS, RNS, RES with protein thiols ............................................... 16 3.3. Reaction of ROS with Iron-sulfur clusters and DNA damage ........................... 17 4. Bacterial defence mechanisms against ROS and RES ......................................... 18 5. Thiol-based redox sensors for ROS and RES ....................................................... 19 5.1. OxyR as thiol-based peroxide redox-sensor of E. coli ....................................... 20 5.2. PerR as metal-based peroxide sensor of B. subtilis ............................................ 21 5.3. OhrR as MarR-family thiol-based redox sensor of organic hydroperoxides ..... 22 5.4. MarR/DUF24-family thiol-based sensors of RES ............................................. 24 5.5. Spx as thiol-based redox sensor for disulfide stress ........................................... 27 6. Conclusion and future perspectives ....................................................................... 28 References ........................................................................................................................ 29 ! "! ! ! ! "#$%&'(!)! ! ! Introduction and general conclusion 1. The biological function of Cysteine and its thiol group The amino acid Cysteine (Cys) with its functional thiol group is the most rare amino acid in proteins and plays a key role to determine the structure and functions of proteins. Oxidation of cysteines to disulfides occurs mainly in the extracellular environment or periplasm and links polypeptides to stabilize the protein structure [1]. The biosynthesis of Cys occurs from sulfate in the sulfate assimilation pathway in most bacteria. Cys is used as precursor for the biosynthesis of many sulfur-containing compounds, including methionine, Fe-S clusters, thiamine cofactors, coenzyme A and low molecular weight (LMW) thiols. In most proteins, the thiol group has a pKa above 8 and is present in its protonated form [2]. However, the thiol group of redox-sensitive Cys residues is often highly reactive and present in its de-protonated thiolate anion form (R-S- ) at low pKa values. The reactivity of the thiol group can be determined by surrounding basic amino acids that can lower its pKa value at physiological pH values in the cytoplasm. Furthermore, hydrogen bonds with positively charged amino acids stabilize the thiolate anion. The activities of many redox-sensing enzymes or regulators depends on the reactivity of the thiol group of Cys residues that are mentioned in detail later [2]. Protein thiols are among the most susceptible oxidation-sensitive targets and can be reversibly and irreversibly post-translational modified that has regulatory and metabolic consequences for cellular physiology. 2. Functions of low molecular weight thiols in bacteria Low molecular weight thiols are thiol-containing, non-proteinogenous compounds that are usually smaller than 1 kDa. LMW thiols are often present in millimolar concentrations in the cellular cytoplasm and function to maintain the reduced state of the cytoplasm #$%!&'( This reduced state ensures proper protein functions and helps to avoid damages of cellular components that occur during cellular metabolism or stressful conditions [5]. Life evolved from transition of anaerobic to aerobic conditions and LMW thiols represent a major biological adaptation to oxidative stress conditions as consequence of the aerobic life [6]. The best studied LMW thiol-redox buffer is the tripeptide glutathione (GSH) (Figure 1), predominantly found in eukaryotes and most Gram-negative bacteria [4, 6]. Most Gram-positive bacteria do not produce GSH. The Actinomycetes produce mycothiol "! ! ! ! "#$%&'(!)! ! ! (MSH) as their LMW thiol-redox buffer (Figure 1) and MSH-deficient mutants are very sensitive to thiol-reactive species and antibiotics that affect the redox balance [7, 8]. In Bacillus megaterium, Bacillus cereus, and Staphylococcus aureus Coenzyme A (CoASH) serves as an abundant LMW thiol [9]. Many Firmicutes bacteria, including Bacillus and Staphylococcus species have recently been discovered to utilise bacillithiol (BSH) as their major LMW thiol-redox buffer (Figure 1) [10]. Alternative LMW thiols include also the betaine-histidine derivative ergothioneine present in Actinomycetes and Fungi. For example, ergothioneine was shown to compensate for MSH-deficiency in Mycobacterium smegmatis [11]. There are also several glutathione-derivatives, such as trypanothione (TSH2) that is the major redox buffer of the protozoa Leishmania and Trypanosoma or the glutathionylspermidine detected in E. coli during the stationary phase [6]. Here, the functions and biosynthesis pathways for the major bacterial redox buffers GSH, MSH and BSH are summarized in the following parts including novel results of our studies about S- bacillithiolations in Firmicutes bacteria. ! ! ! ! ! ! ! ! ! ! ! ! ! ! Figure 1: Structures of low molecular weight (LMW) thiols. Major LMW thiols are glutathione (GSH) in eukaryotes and Gram-negative bacteria, mycothiol (MSH) in Actinomycetes and bacillithiol (BSH) in Firmicutes. Coenzyme A (CoASH) also serves as a LMW thiol-redox buffer in some bacteria and Archaea. The schematic is derived from Antelmann & Hamilton, 2012 [12]. 2.1. Biosynthesis and functions of glutathione and role of S-glutathionylation The best studied redox buffer is the tripeptide y-glutamylcysteinyl-glycine or glutathione (GSH) present in eukaryotes, in Escherichia coli (in concentrations at 3.5 - 6.6 mM), in Gram-negative !- and "-Proteobacteria, and few Firmicutes bacteria, such as "! ! ! ! "#$%&'(!)! ! ! Streptococcus agalactiae and Listeria monocytogenes [4, 6, 13]. GSH acts as a reducing agent, antioxidant, enzyme cofactor and it mediates protein protection by S- glutathionylations. GSH functions in detoxification of xenobiotics, antibiotics, reactive oxygen and nitrogen species (ROS and RNS) and maintains protein thiols in its reduced state [4]. GSH detoxifies xenobiotics, toxic electrophiles and antibiotics by conjugation either spontaneously or by the catalytic activity of GSH-S-transferases. The conjugates are usually excreted from the cell. GSH also serves also as a reservoir for cysteine [14]. The biosynthesis of GSH occurs in two steps (Figure 2A). The !-glutamate cysteine ligase catalyzes the formation of !-glutamylcysteine from L-glutamate and L- cysteine. In the second step, ligation of glycine to !-glutamylcysteine is catalyzed by glutathione synthase. The !-glutamyltranspeptidase is able to hydrolyse the gamma carbon peptide bond of GSH and then transfers the !-glutamyl moiety to amino acids [15]. In Listeria species, Streptococcus agalatiae and other Gram-positive bacteria, a bifunctional fusion protein encoded by gshAB exhibits both !-glutamate cysteine ligase 2 H. Antelmann and C.and J. Hamilton glutathione᭿ synthase activity [16]. !"#$%&'( !"#$%&'( !"#$%&'( !"#$%&'( !"#$%&'( !"#$%&'( Figure 2: Biosynthesis of GSH (A) and summary of redox regulation of protein disulfides by the TrxAB and Grx/GSH/Gor systems in E. coli (B). A) Biosynthesis of GSH by !-glutamylcysteine ligase (GshA) and glutathione synthetase (GshB). The schematic is derived from Fahey (2012) [6]. B) In the GSH-utilising Gram-negative bacterium E.
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