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Global Characterization of Manganese Limitation in Bacillus Subtilis

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Global Characterization of Manganese Limitation in Bacillus Subtilis Genome-wide responses and regulatory mechanisms to thiol-specific electrophiles in Bacillus subtilis Inauguraldissertation zur Erlangung des akademischen Grades doktor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald Vorgelegt von Nguyen Thi Thu Huyen geboren am 30.08.1979 in Haiduong-Vietnam Greifswald, 2008 Dekan: Prof. Dr. Klaus Fesser 1. Gutachter 1: Prof. Dr. Michael Hecker 2. Gutachter 2: Prof. Dr. Jan Maarten van Dijl Tag der Promotion: 16. 10. 2008 i CONTENTS Summary of the thesis........................................................................................................................................... ii Part 1: Genome-wide responses and regulatory mechanisms to thiol-specific electrophiles in Bacillus subtilis..................................................................................................................................................................1 INTRODUCTION................................................................................................................................................2 1. The role of low molecular-weight thiols in the oxidative stress response....................................................2 2. Induction of the thiol-specific stress response by quinone-like electrophiles and diamide in B. subtilis.....3 3. Regulation of the thiol-specific stress response in B. subtilis.......................................................................5 3.1. Spx-an activator of genes involved in the maintenance of the thiol-disulfide balance..........................6 3.2. CymR-the repressor of the cysteine metabolism regulon......................................................................7 3.3. HrcA and CtsR-heat-shock-specific regulators .....................................................................................8 3.4. PerR-the main regulator of the oxidative stress response......................................................................8 3.5. The MarR-type regulators ...................................................................................................................10 4. The response of bacteria to the electrophile methylglyoxal (MG) .............................................................11 5. The response of bacteria to the electrophile formaldehyde (FA) ...............................................................16 6. The aim of this part of the thesis ................................................................................................................21 Chapter 1: Regulation of quinone detoxification by the thiol stress sensing DUF24/MarR-like repressor, YodB in Bacillus subtilis....................................................................................................................................23 Chapter 2: The MarR/DUF24-type repressor CatR (YvaP) controls the thiol-stress inducible catechol dioxygenase YfiE (CatE) in Bacillus subtilis.....................................................................................................41 Chapter 3: Genome wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol- dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the novel MerR-family regulator YraB (AdhR).......................................................................................................................................62 GENERAL DISCUSSION.................................................................................................................................96 1. The role of the MarR/DUF24 regulators as sensors for electrophilic quinones and diamide in B. subtilis96 2. The MG and FA stimulons of B. subtilis..................................................................................................106 Part 2: Genome-wide responses to manganese limitation in Bacillus subtilis ...............................................116 SUMMARY.....................................................................................................................................................117 INTRODUCTION............................................................................................................................................118 1. Manganese (Mn) transport .......................................................................................................................118 2. Cellular functions of Mn ..........................................................................................................................119 2.1. Function of Mn in the oxidative stress response ...............................................................................121 2.2. Role of Mn during B. subtilis sporulation and germination ..............................................................122 2.3. Role of Mn in gene regulation...........................................................................................................124 2.3.1. MntR as regulator of Mn homeostasis........................................................................................124 2.3.2. Fur-like metalloregulators ..........................................................................................................125 3. The aim of this part of the thesis ..............................................................................................................128 MATERIALS AND METHODS .....................................................................................................................129 1. Bacterial strain and medium.....................................................................................................................129 2. Growth conditions....................................................................................................................................129 3. Scanning electron microscopy (SEM)......................................................................................................129 4. 2-D PAGE ................................................................................................................................................130 4.1. Protein extraction ..............................................................................................................................130 4.2. 2-D PAGE and quantitative image analysis ......................................................................................130 5. DNA microarray.......................................................................................................................................131 RESULTS AND DISCUSSION ......................................................................................................................132 1. The impact of Mn on growth behavior.....................................................................................................132 2. Morphological, proteomic and transcriptomic studies of B. subtilis in response to Mn limitation ..........132 2.1. Morphology of B. subtilis in response to Mn limitation....................................................................132 2.2. Proteome and transcriptome analyses of B. subtilis in response to Mn limitation ............................134 References...........................................................................................................................................................154 List of publications.............................................................................................................................................170 Curriculum vitae ................................................................................................................................................171 Acknowledgements.............................................................................................................................................172 Nguyen Thi Thu Huyen ii Summary Summary of the thesis The soil-dwelling bacterium Bacillus subtilis is regarded as model organism for functional genomic research of low GC Gram-positive bacteria. Several stress and starvation- responsive stimulons have been extensively studied in B. subtilis using transcriptomic and proteomic approaches. However, there was only little information about the response of B. subtilis to toxic phenolic compounds, which are often found in the soil. The group of Haike Antelmann has monitored the expression profile of B. subtilis after exposure to phenol, salicylic acid, catechol, chromanon and MHQ. Interestingly, proteome and transcriptome analyses showed a strong overlap in the expression profile after exposure to catechol, MHQ and the thiol-reactive electrophile diamide. Later on it was shown that catechol and MHQ are auto-oxidized to electrophilic quinones which target thiols via the thiol-(S)-alkylation reaction leading to aggregation and depletion of LMW thiols and thiol-containing proteins. The response to electrophilic quinones and diamide is governed by a complex network of transcription factors, including Spx, CtsR, PerR, CymR and the novel MarR-type repressors MhqR (YkvE), YodB and YvaP. The regulatory mechanisms of these novel thiol- stress sensors YodB and YvaP are studied as part of this thesis in collaboration with the group of Peter Zuber (Oregon). YodB negatively regulates the expression of the nitroreductase YodC and the azoreductase YocJ (AzoR1) after exposure to electrophilic quinones and diamide. The azoreductase AzoR1 is a paralog of AzoR2 that is under
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