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Chapter 1: Introduction 6 | Page Introduction Linking Metabolic Capacity and Molecular Biology of Methylocystis sp. Strain SC2 by a Newly Developed Proteomics Workflow Dissertation „Kumulativ“ Zur Erlangung des Grades eines Doktor der Naturwissenschaften (Dr. rer.nat.) des Fachbereichs Biologie der Philipps-Universität Marburg Vorgelegt von Anna Hakobyan Aus Gyumri, Armenien Marburg | 2020 Originaldokument gespeichert auf dem Publikationsserver der Philipps-Universität Marburg http://archiv.ub.uni-marburg.de Dieses Werk bzw. Inhalt steht unter einer Creative Commons Namensnennung Keine kommerzielle Nutzung Weitergabe unter gleichen Bedingungen 3.0 Deutschland Lizenz. Die vollständige Lizenz finden Sie unter: http://creativecommons.org/licenses/by-nc-sa/3.0/de/ Die vorliegende Dissertation wurde von November 2015 bis Dezember 2019 am Max- Planck-Institut für terrestrische Mikrobiologie in Marburg unter Leitung von Herrn PD Dr. Werner Liesack angefertigt. Vom Fachbereich Biologie der Philipps-Universität Marburg (Hochschulkennziffer 1180) als Dissertation angenommen am 13. Dezember, 2019 Erstgutachter(in): Herr PD Dr. Werner Liesack Zweitgutachter(in): Herr Prof. Dr. Lennart Randau Weitere Mitglieder der Prüfungskommission: Herr Prof. Dr. Andreas Brune Herr Prof. Dr. Uwe G. Maier Tag der Disputation: 17. April, 2020 Die in dieser Dissertation beschriebenen Ergebnisse sind in folgenden Publikationen veröffentlicht bzw. zur Veröffentlichung vorgesehen: Hakobyan A.#, Zhu J.#, Glatter T., Liesack, W. (under review) Hydrogen utilization by Methylocyctis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs. (#equal contribution) Hakobyan A., Schneider M.B., Liesack W., Glatter T. (2019) Efficient tandem LysC/trypsin digestion in detergent conditions. Proteomics, 19(20), e1900136. Bordel S., Rodríguez Y., Hakobyan A., Rodríguez E., Lebrero R., Muñoz R. (2019) Genome scale metabolic modeling reveals the metabolic potential of three Type II methanotrophs of the genus Methylocystis. Metabol Eng, 54, 191 Hakobyan A., Liesack W.*, Glatter T.* (2018) Crude-MS Strategy for in-depth proteome analysis of the methane-oxidizing Methylocystis sp. strain SC2. J Proteome Res, 17 (9), 3086 (*corresponding authors) Erklärung Ich versichere, dass meine Dissertation mit dem Titel „Linking metabolic capacity and molecular biology of Methylocystis sp. strain SC2 by a newly developed proteomics workflow” selbstständig ohne unerlaubte Hilfe angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfsmittel bedient habe. Diese Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner anderen Hochschule eingereicht und hat keinen sonstigen Prüfungszwecken gedient. Marburg, den 23. April, 2020 Anna Hakobyan Dedicated to my son, my husband, and my parents "I was taught that the way of progress was neither swift nor easy." - Marie Curie Table of Contents SUMMARY 1 ZUSAMMENFASSUNG 3 1 INTRODUCTION 5 1.1. Global methane budget 6 1.2. Methane-oxidizing microorganisms or methanotrophs 9 1.2.1. Aerobic methanotrophs 9 1.2.2. Biochemistry of aerobic methanotrophs 13 1.2.3. “High-affinity” and “low-affinity” methanotrophs 16 1.2.4. Methanotrophs in hypoxic and anaerobic environments 18 1.2.5. Facultative methanotrophy 21 1.3. Molecular biology methods to study methane-oxidizing bacteria 24 1.3.1. Proteomics as a new tool for methanotroph research 26 1.4. Our model organism Methylocystis sp. strain SC2 34 1.5. Objectives of the study 38 1.6. References 41 CRUDE-MS STRATEGY for IN-DEPTH PROTEOME ANALYSIS of the METHANE - 2 OXIDIZING METHYLOCYSTIS sp. STRAIN SC2 49 2.1. Abstract 50 2.2. Introduction 50 2.3. Materials and methods 53 2.3.1. Cell culture conditions 53 2.3.2. Sample preparation and protein solubilization 54 2.3.3. Protein digestion 55 2.3.4. LC–MS/MS analyses, peptide/protein identification, and LFQ 56 2.3.5. Development of PRM assays for targeted MS 57 2.3.6. Additional data processing and visualization 58 2.4. Results and discussion 59 2.4.1. Elucidating efficient protein solubilization conditions for strain SC2 59 2.4.2. Proteome coverage and quantification accuracy 63 2.4.3. Enrichment of membrane-related proteins in all crude-MS 65 Table of Contents 2.4.4. Targeted quantification and intensity-based absolute quantification 67 2.4.5. Recovery of methane oxidation and C1 assimilation proteins 70 2.4.6. Assessment of the newly developed proteomics workflow 72 2.4.7. Strain SC2: A highly specialized proteome compared to E. coli 77 2.5. Conclusions 80 2.6. Acknowledgements 81 2.7. Supporting information 81 2.7.1. Supporting figures 81 2.9. References 90 3 EFFICIENT TANDEM LYS-C/TRYPSIN DIGESTION in DETERGENT CONDITIONS 97 3.1. Abstract 98 3.2. Introduction 98 3.3. Results and discussion 99 3.5. Raw files and associated data deposition 106 3.6. Supporting information 106 3.6.1. Supporting text 107 3.6.2. Supporting figures 110 3.7. References 114 HYDROGEN UTILIZATION by METHYLOCYCTIS sp. STRAIN SC2 EXPANDS the 4 KNOWN METABOLIC VERSATILITY of TYPE IIA METHANOTROPHS 115 4.1. Abstract 116 4.2. Introduction 116 4.3. Materials and methods 119 4.3.1. Culture conditions for hydrogen treatments 119 4.3.2. Determination of cell dry weight per OD600 121 4.3.3. Culture conditions for proteomics and RNA extraction 121 4.3.4. Extraction, solubilization and digestion of proteins 121 4.3.5. LC-MS/MS analyses, peptide/protein identification and LFQ 122 4.3.6. Prediction of protein-protein interactions 123 4.3.7. RNA extraction 123 4.3.8. Real-time quantitative PCR and primer design 123 4.3.9. Raw files and associated data deposition 124 Table of Contents 4.4. Theory/calculations 125 4.4.1. Thermodynamic calculations for cell carbon synthesis 125 4.5. Results 126 4.5.1. Impact of the CH4/O2 ratio on H2 utilization by strain SC2 126 4.5.2. Impact of H2 on SC2 growth during long-term incubation 129 4.5.3. Effects of H2 availability on the SC2 proteome 129 4.5.4. Predicted protein-protein interactions 132 4.5.5. Differential SC2 gene expression in response to H2 availability 133 4.6. Discussion 135 4.6.1. Metabolic adaptation of strain SC2 to H2 availability 135 4.6.2. Differential regulation of key enzymes in response to H2 136 4.6.3. Proposed model of electron flow and energy conservation in strain SC2 140 4.7. Conclusions 141 4.8. Acknowledgements 142 4.9. Supporting information 142 4.9.1. Supporting figures 143 4.9.2. Supporting tables 146 4.10. References 149 GENOME SCALE METABOLIC MODELING REVEALS the METABOLIC POTENTIAL 5 of THREE TYPE II METHANOTROPHS of the GENUS METHYLOCYSTIS 153 5.1. Abstract 154 5.2. Introduction 154 5.3. Materials and methods 157 5.3.1. Reconstruction of GSMMs 157 5.3.2. Strain, chemicals and culture conditions 158 5.3.3. Analytical methods 159 5.4. Results 159 5.4.1. Genome scale metabolic models 159 5.4.2. Model validation 162 5.5. Discussion 173 5.6. Acknowledgements 175 5.7. Supporting information 175 5.7.1. Supporting figures 176 Table of Contents 5.7. References 177 6 DISCUSSION and OUTLOOK 179 6.1. Discussion 180 6.2. Outlook and concluding remarks 187 6.3. References 190 ACKNOWLEDGEMENTS 1933 S u m m a r y P a g e | 1 Summary Microbial methane oxidation is one of the fundamental processes in global methane cycle. Methane-oxidizing bacteria, or methanotrophs, are the major biological sink for the methane produced from anthropogenic and natural sources. Our model organism, Methylocystis sp. strain SC2, is one of the best-studied representatives of alphaproteobacterial (type IIa) methanotrophs. Proteobacterial methanotrophs possess a unique cell architecture characterized by intracytoplasmic membranes (ICMs). The cellular amount of the ICMs is increasing with methanotrophic activity. The presence of ICMs makes molecular biology approaches, but in particular global proteomics, highly challenging. In this study, we therefore aimed to develop an efficient proteomics workflow for strain SC2 and to apply this state-of-the-art tool for investigation of the strain SC2 response to environmental factors. To successfully develop the proteomics workflow, we particularly focused on an efficient solubilization and digestion of the integral membrane proteins of strain SC2 for further downstream analysis. We introduced the so-called crude-MS proteomics workflow, upon assessing and optimizing all the major steps in the proteomics workflow, including cell lysis, protein solubilization, and protein digestion. Our new SC2 proteomics workflow greatly increased not only the protein quantification accuracy (mean coefficient of variation 3.2 %) but also the proteome coverage to 62%, with up to 10-fold increase in the detection intensity of membrane-associated proteins. Previous studies have shown that the LysC/trypsin tandem digestion resulted in higher coverage of fully cleaved tryptic peptides than a trypsin-only digestion. Therefore, the development of our optimized proteomics workflow involved the application of the LysC/trypsin tandem digestion in detergent environment to increase the SC2 proteome coverage. Prior to publication of our crude-MS approach, all systematic assessments of LysC/trypsin proteolysis were conducted in chaotropic environments, like urea. As a spin-off, we therefore initiated a follow-up study to compare the efficiency of the LysC/trypsin tandem digestion in detergent environments (e.g., SDC, SLS) relative to chaotropic environments. The study revealed that the LysC/trypsin tandem digestion could be efficiently carried out not only in chaotropic environments but also in MS-compatible detergent environments. In fact, the 2 | P a g e S u m m a r y LysC/trypsin tandem digestion in both environments resulted in a higher coverage of fully cleaved peptides than the trypsin-only digestion. After successful development of the crude-MS proteomics workflow, we used this high-throughput method to assess the molecular response of strain SC2 to the availability of hydrogen as a potentially alternative energy source.
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