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Sulfite Dehydrogenases in Organotrophic Bacteria : Enzymes

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Sulfite Dehydrogenases in Organotrophic Bacteria : Enzymes Sulfite dehydrogenases in organotrophic bacteria: enzymes, genes and regulation. Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz Fachbereich Biologie vorgelegt von Sabine Lehmann Tag der mündlichen Prüfung: 10. April 2013 1. Referent: Prof. Dr. Bernhard Schink 2. Referent: Prof. Dr. Andrew W. B. Johnston So eine Arbeit wird eigentlich nie fertig, man muss sie für fertig erklären, wenn man nach Zeit und Umständen das möglichste getan hat. (Johann Wolfgang von Goethe, Italienische Reise, 1787) DANKSAGUNG An dieser Stelle möchte ich mich herzlich bei folgenden Personen bedanken: . Prof. Dr. Alasdair M. Cook (Universität Konstanz, Deutschland), der mir dieses Thema und seine Laboratorien zur Verfügung stellte, . Prof. Dr. Bernhard Schink (Universität Konstanz, Deutschland), für seine spontane und engagierte Übernahme der Betreuung, . Prof. Dr. Andrew W. B. Johnston (University of East Anglia, UK), für seine herzliche und bereitwillige Aufnahme in seiner Arbeitsgruppe, seiner engagierten Unter- stützung, sowie für die Übernahme des Koreferates, . Prof. Dr. Frithjof C. Küpper (University of Aberdeen, UK), für seine große Hilfsbereitschaft bei der vorliegenden Arbeit und geplanter Manuskripte, als auch für die mentale Unterstützung während der letzten Jahre! Desweiteren möchte ich herzlichst Dr. David Schleheck für die Übernahme des Koreferates der mündlichen Prüfung sowie Prof. Dr. Alexander Bürkle, für die Übernahme des Prüfungsvorsitzes sowie für seine vielen hilfreichen Ratschläge danken! Ein herzliches Dankeschön geht an alle beteiligten Arbeitsgruppen der Universität Konstanz, der UEA und des SAMS, ganz besonders möchte ich dabei folgenden Personen danken: . Dr. David Schleheck und Karin Denger, für die kritische Durchsicht dieser Arbeit, der durch und durch sehr engagierten Hilfsbereitschaft bei Problemen, den zahlreichen wissenschaftlichen Diskussionen und für die aufbauenden Worte, . Michael Weiss, Dr. Jutta Maier, Dr. Sonja Weinitschke, Manuel Serif, Ann-Kartrin Felux und Anna Kesberg für die freundschaftliche Atmosphäre im und über das Labor hinaus, . Dr. Andrew R. J. Curson, Dr. Jonathan D. Todd, Emily Fowler, Dr. Rob Green, Dr. Mark Kirkwood für die herzliche Aufnahme in Ihrem Labor, Ihrer tatkräftigen Unter- stützung, als auch den außeruniversitären Unternehmungen, . Dr. Adam Hughes, Dr. David Green, Dr. Richard Abell, Elaine Azzopardi und Leah Morrison, für Ihre Hilfbereitschaft und Offenheit sowie den nicht nur wissenschaftlichen Diskussionen! Herzlich danken möchte ich weiterhin: . Dr. Ulrike Kappler (University of Queensland, Australia) und Prof. Dr. Peter Kroneck (Universität Konstanz), für die sehr inspirierenden und sehr wertvollen Diskussionen, . den Arbeitsgruppen Schink, Mendgen, Kroth und Welte für die Mitbenutzung der Chemikalien und Gerätschaften sowie Dr. Klaus Hollemeyer (Universität des Saarlandes), für die MALDI-TOF-MS Messungen. Zu guter letzt, möchte ich folgenden Personen für Ihre unermüdliche mentale Unterstützung, Ihrer bedingungslosen Liebe und Ihrer aufopfernden Geduld danken: . meinen Freunden, ganz besonders Nina Kaczmarek, Martin Dauner, Jutta & Markus, Janaka Suwandaratne, Maria Hof-Glatz und Stefan Fennrich, . meiner Familie Jörg, Kerstin, Stephanie und Philipp Lehmann, meiner Oma Karla Schneider und meinem Opa Dieter Lehmann, die mir als sehr großes Vorbild dienen, mir immer wieder mit wertvollen Ratschlägen zur Seite stehen und vor allem in stürmischen Zeiten mein Fels in der Brandung sind! TABLE OF CONTENTS Summary……………………………………………………………………………………………………………………..…..1 Zusammenfassung………….…………………………………………………………………………………………………3 1. Introduction .............................................................................................................. 5 1.1. Organosulfonates-an overview ................................................................................... 5 1.1.1. Natural and xenobiotic organosulfonates .................................................................. 5 1.1.2. Microbial aerobic dissimilation of organosulfonates ............................................... 10 1.1.2.1. Degradation pathways and desulfonating enzymes ................................................ 10 1.1.2.2. Sulfite: an useful but fateful product ....................................................................... 14 1.2. Microbial mechanisms for sulfite elimination .......................................................... 16 1.2.1. Sulfite exporters ........................................................................................................ 17 1.2.2. Sulfite reduction ........................................................................................................ 18 1.2.3. Sulfite oxidation ........................................................................................................ 19 1.3. Sulfite-oxidizing enzymes (SOEs) ............................................................................... 20 1.3.1. Phylogeny .................................................................................................................. 21 1.3.2. The molybdopterin cofactor (Moco) ......................................................................... 24 1.3.3. Characterized SOEs .................................................................................................... 26 1.3.3.1. Eukaryotic sulfite oxidases ....................................................................................... 28 1.3.3.2. Prokaryotic sulfite dehydrogenases (SDHs) ............................................................. 29 1.4. Aims of this study ...................................................................................................... 33 2. Materials and Methods ........................................................................................... 35 2.1. Materials .................................................................................................................... 35 2.1.1. Chemicals, kits, and equipment ................................................................................ 35 2.1.2. Organisms, and growth media .................................................................................. 36 2.1.3. Plasmids and oligonucleotides .................................................................................. 38 2.2. Cultivation, harvesting and preparation of cell extracts ........................................... 41 2.3. Molecular methods ................................................................................................... 42 2.3.1. In vitro and in vivo genetic manipulation ................................................................. 42 2.3.2. Southern blotting ...................................................................................................... 43 2.3.3. Transcriptional analysis ............................................................................................. 43 2.3.4. Protein expression ..................................................................................................... 44 2.4. Biochemical methods ................................................................................................ 45 2.4.1. Solubilization of membrane-bound sulfite-oxidizing activity ................................... 45 2.4.2. Enzyme purification ................................................................................................... 45 2.4.3. Enzyme assay ............................................................................................................. 46 2.5. Analytical methods .................................................................................................... 47 2.6. Bioinformatics ........................................................................................................... 48 3. Results and Discussion ............................................................................................. 50 3.1. A sulfite-stress inducible 11 kDa protein in R. pomeroyi DSS-3 ................................ 50 3.1.1. Physiology of growth of R. pomeroyi DSS-3 with taurine and cysteate .................... 50 3.1.2. Sulfite dehydrogenase (SDH) activities in R. pomeroyi DSS-3 ................................... 52 3.1.3. Purification and identification of an 11 kDa protein with SDH activity in cysteate- grown cells ................................................................................................................. 53 3.1.4. Biochemical characterization of the 11 kDa protein ................................................. 54 3.1.5. Characterization of the 11 kDa protein (SPO3124) gene cluster .............................. 56 3.1.6. Regulation of the SPO3124 gene cluster ................................................................... 57 3.1.7. The effect of sulfite on growth of Ruegeria pomeroyi DSS-3.................................... 60 3.1.8. Discussion .................................................................................................................. 61 3.2. The missing majority of sulfite-oxidizing enzymes (SOEs) in sulfonate-degrading bacteria ...................................................................................................................... 64 3.2.1. Genomic and proteomic approaches to identify new genes and proteins involved in sulfite oxidation ......................................................................................................... 64 3.2.2. Attempts to measure
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