Characterization of the key enzymes involved in anaerobic degradation of phthalate A dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.) at the University of Konstanz Faculty of Sciences Department of Biology Submitted by: Madan Junghare (M.Sc.) born on 3rd Nov. 1985 in Washim (MH), India Konstanz, 2016 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-395942 Tag der mündlichen Prüfung: 2. Februar 2017 1. Gutachter: Prof. Dr. rer. nat. Bernhard Schink 2. Gutachter: Prof. Dr. Dieter Spiteller 3. Gutachter: Prof. Dr. Valentin Wittmann ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere thanks to my supervisor Professor Bernhard Schink for giving me the opportunity to complete my Ph.D dissertation in his lab at the University of Konstanz on such an interesting project. You have provided me valuable guidance, support, and encouragements during my doctoral research, whenever it was needed. Secondly, I would like to thank my co-supervisor Professor Dieter Spiteller for his guidance, nurturing and offering me to use his lab facility for my research. You provided priceless aid for my research project. I wouldn’t have succeeded without your help. I really appreciate everything. Thanks to Professor Valentin Whitmann for acting as a third member of my thesis committee at the Graduate School of Chemical Biology. I am very thankful to Dr. David Schleheck for allowing me to use his proteomics lab facility for doing my research work. Thank you for your helpful discussion, suggestions, and for enriching my scientific knowledge during this period. I would like to thank everyone else in the Schink group for all their kindness, assistance, friendship and providing a great working environment, as well as enjoying coffee and cake time together. It has been a largely very enjoyable time. Vielen Vielen Danke nochmal an Alle! Many thanks to the friendly team at the Welcome Center, University of Konstanz for their support, advice and friendly cooperation in all bureaucratic matter. I like to acknowledge DAAD for providing me a PhD scholarship to begin my doctoral research and giving me an opportunity to study in Germany. A special thanks to my incredible family in India for their endless support, love and for all of the sacrifices that you’ve made on my behalf. You might not have known anything about my research, but you were always there for me. I dedicate this work to my father and mama, I know you would have been very happy and proud of me. Special thanks to my wife for her support, care and love. And also thank you for reading my thesis, feedback and for spending sleepless nights during the thesis writing. Lots of love to my sweet daughter Grishma. I hope my research work has advanced the knowledge in the field of anaerobic phthalate degradation. Enjoy! Table of contents Dissertation summary……………………………………………………………………………………….……………………………………………07 Dissertation Zusammenfassung….…………….…………………………………………………………...………………………………………09 Chapter 1. General introduction 1. Aromatic compounds in nature….…………………..………………………………………..………………………………………………...12 1.1 Aromaticity and stability 1.2 Occurrence and sources 2. Microbial degradation of aromatic compounds in nature………………………………………………….………………….…....13 2.1 Aerobic degradation 2.2 Anaerobic degradation 3. Phthalate esters, applications and biodegradation…………….……………………………..…………………………………………19 3.1 Phthalate esters as ‘plasticizers’ 3.2 ‘Man-made’ phthalate as pollutant 4. Microbial degradation of phthalate esters………………………………………………………………..…………………………………22 4.1 Importance of phthalate esters degradation 4.2 Initial steps in phthalate ester degradation 4.3 Aerobic phthalate degradation 4.4 Anaerobic phthalate degradation 5. Aims of the dissertation and objectives………………………………………………………………………………………….…………...28 6. Research co-authored but not included in the dissertation…………………………………….…………………………………..29 Chapter 2. Draft genome sequence of a nitrate-reducing, o-phthalate-degrading bacterium, Azoarcus sp. strain PA01 Abstract…………………………………………………………………..………………………………………………………………………………………31 Introduction…………………………………………………………………………..………………………………………………………………………..31 Organism information……………………………….………………………………..…………………………………………………………………..32 Classification and features Chemotaxonomy Genome sequencing and information……………………………………………………………………………………………………………..36 Genome project history Growth conditions and genomic DNA preparation Genome sequencing and assembly Genome annotation Genome properties………………………………………………………………………………………………………………………………………….38 Insight from the genome sequence……………………………………..…………………………………………………………………….......41 I Chapter 3. Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01 Abstract……………………………………………………………………………………………………………………….……………………………….…45 Introduction……………………………………………………………………………………………..……………………………………………………..45 Results………………………………………………………………………………………………………………………………………………..…………..47 Anaerobic growth with o-phthalate and physiological characteristics Differential proteome analysis of o-phthalate-grown cells versus benzoate-grown cells Characterization of the gene cluster involved in anaerobic o-phthalate degradation Enzyme activity measurements with cell-free extracts of Azoarcus sp. strain PA01 Discussion........…………………………………………………………………….…………………………………………………………….……………59 Experimental procedures…………………………………………………….………………………..………………………………………………..63 Bacterial strain and growth conditions Bacterial growth analysis Preparation of cell-free extracts Differential proteome analysis Protein identification by mass spectrometry Identification of gene clusters and phylogenetic analysis Determination of enzyme activities in cell-free extracts Synthesis of o-phthalyl-CoAs LC-MS/MS analysis of Coenzyme A esters Supporting information……………………………………………………………………………………………………………………………………70 Chapter 4. Cloning and over-expressions of genes involved in anaerobic o-phthalate decarboxylation to benzoate by the Azoarcus sp. strain PA01 Abstract……………………………………………………………………………………..……………………………………………………………………77 Introduction……………………………………………………..……………………………………………………………………………………………..77 Materials and methods…………………………………..……………………………………………………………………………………………….80 Materials and reagents Strains and cultivation conditions Gene amplifications, plasmid construction and sequencing Optimization of protein expression and purifications Molecular weight determination by SDS-PAGE Decarboxylase activity assays and cofactor identification Native protein PAGE analysis Sequence alignment and structure homology modelling Results………………………………………………………………………………………….………………….……………………………………………..86 Gene amplifications, cloning of PhtDa and PhtDb II 5 | P a g e Heterologous expression, purification and protein identification Enzyme activity, substrate specificity and cofactor requirement Phylogenetic position of PhtDa and PhtDb Multiple sequence alignment and homology-based structure modelling Native PAGE and protein identification by MS analysis Discussion……………………………………………………………………………………………………………..…………………………………………98 Chapter 5: Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-stain-negative, benzoate-degrading, sulfate-reducing bacterium isolated from a wastewater treatment plant Abstract…………………………………………………………………………………………………………….…………………..........................103 Introduction………………………………………………………………………….………………………………….……………………………………104 Material and methods………………………………………..………………………………..……………………………………………………….104 Results and discussion……………….………………………………………………………….………………………………………………………107 Description of Desulfoprunum gen. nov. Description of Desulfoprunum benzoelyticum gen. nov., sp. nov. Chapter 6: Enrichment cultures degrading phthalate isomers under nitrate-reducing, sulfate-reducing and fermenting conditions Introduction…………………………………………………………………………………………………………………..………………………………115 Phthalate isomer degradation under denitrifying conditions Phthalate degradation under sulfate-reducing/fermenting conditions Partial characterization of enrichment culture KOPA Chapter 7: General discussion and concluding remarks General discussion…...…………………………………………………………………………………………………………………………………..121 Activation of o-phthalate by Azoarcus sp. strain PA01 Decarboxylation of activated o-phthalate by a two enzyme component system Concluding remarks and general outlook…………...………………………………………………............................................125 Personal record of achievements……………………………………………..…………………………………………………….126 List of publications……………………………………………………….………………….…………………………...127 Workshops and training courses attended……………………………….………………………………………………….128 References………………………………………………..………………………………..……………………………….129 Abbreviations………………………………………………………………….………………………………….…………146 III 6 | P a g e Dissertation summary Anaerobic phthalate degradation was assumed to proceed through initial decarboxylation of phthalate (ortho) to benzoate. However, the intermediates and enzymes involved in anaerobic phthalate decarboxylation were largely unknown. The aim of this dissertation was to investigate the biochemistry of anaerobic phthalate degradation, especially the steps and enzymes involved in the decarboxylation of phthalate to benzoate. In particular, the study focused on the enrichment and isolation of anaerobic phthalate-degrading bacteria under nitrate-reducing, sulfate-reducing and fermenting conditions. Strain PA01 was isolated and purified from an enrichment culture that degrades phthalate coupled to nitrate reduction. 16S rRNA