Osmotic stress response in the industrially important bacterium Gluconobacter oxydans Dissertation to obtain the degree of Doctorate (Dr. rer. nat.) from the Faculty of Mathematics and Natural Sciences of the Rheinische Friedrich-Wilhelms University of Bonn, Germany submitted by Nageena Zahid from Lahore, Pakistan Bonn, November 2016 Finalized with the permission from the Faculty of Mathematics and Natural Sciences of the Rheinische Friedrich-Wilhelms University of Bonn, Germany First Referee: Prof. Dr. Uwe Deppenmeier Second Referee: Prof. Dr. Erwin A. Galinski Day of Promotion: 07.02.2017 Year of Publication: 2017 List of parts of this thesis that have already been published. Zahid N., Schweiger P., Galinski E., Deppenmeier U. (2015). Identification of mannitol as compatible solute in Gluconobacter oxydans. Appl Microbiol Biotechnol 99, 5511- 5521. Zahid N. and Deppenmeier U. (2016). Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans. Appl Microbiol Biotechnol 100, 9967-9978. For my Son TABLE OF CONTENTS 1. INTRODUCTION 1 1.1. Acetic acid bacteria 1 1.2. The genus Gluconobacter 2 1.2.1. Respiratory chain of G. oxydans 3 1.2.2. Intracellular carbohydrate metabolism in G. oxydans 6 1.2.3. Types and function of sugars and polyols metabolizing enzymes in Gluconobacter 7 1.2.4. Biotechnological applications of G. oxydans and its limitations 8 1.3. Aims of the work 10 2. MATERIALS AND METHODS 12 2.1. Chemicals and Enzymes 12 2.2. Bacterial strains, plasmids, primers 12 2.2.1. Bacterial strains 12 2.2.2. Oligonucleotides and Plasmids 13 2.3. Antibiotic stock solutions 18 2.4. Microbiology methods 18 2.4.1. Media and culture conditions 18 2.4.2. Measurement of osmolalities of growth media 22 2.4.3. Measurement of growth parameters 22 2.4.4. Preparation of stock cultures 23 2.5. Molecular biology methods 23 2.5.1. Isolation and purification of DNA 23 2.5.2. Isolation and purification of plasmid DNA 23 2.5.3. Isolation and purification of RNA 24 2.5.3.1. RNA extraction using the Trizol reagent method 24 2.5.3.2. RNA extraction with the Ribopure-Bacterial Kit 24 2.5.4. Spectrophotometric quantifications of DNA and RNA samples 25 2.5.5. Restriction digestion of DNA 25 2.5.6. Ligation 26 v 2.5.7. Polymerase chain reaction (PCR) 26 2.5.8. Reverse transcription quantitative PCR (RT-qPCR) 27 2.5.9. Agarose gel electrophoresis 28 2.5.10. Denaturing agarose gel electrophoresis 29 2.5.11. Staining of agarose gels 29 2.5.12. Evaluation of RNA integrity with Bioanalyzer 29 2.5.13. DNA sequencing 30 2.5.14. Illumina Next Generation Sequencing (NGS) and data analysis 30 2.5.15. Transformation of E. coli and G. oxydans 32 2.5.16. Generation of G. oxydans strains carrying in-frame deletions 33 2.5.17. Plasmid-based expression of genes 35 2.6. Biochemical methods 35 2.6.1. Protein overproduction and purification 35 2.6.1.1. Heterologous overproduction of proteins in E. coli 35 2.6.1.2. Heterologous overproduction of proteins in G. oxydans 36 2.6.1.3. Cell disruption and extraction of crude cell extract 36 2.6.1.4. Protein purification by Strep-Tactin Affinity chromatography 36 2.6.2. Extraction of cell cytoplasm 37 2.6.3. Quantification of protein concentration 37 2.6.4. PolyAcrylamide Gel Electrophoresis (PAGE) 38 2.6.4.1. Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis 38 2.6.4.2. Native PAGE 38 2.6.5. Silver staining 39 2.6.6. Immunoblot 39 2.6.7. Activity staining 40 2.6.8. Determination of enzymatic activities 41 2.6.8.1. Measurement of activities of NAD(P)H dependent oxidoreductases 41 2.6.8.2. Glucose isomerase: enzyme assay with auxillary enzymes 43 2.7. Analytical methods 44 2.7.1. Sample preparation for the determination of intracellular solutes 44 2.7.2. High Performance Liquid Chromatography 45 2.7.3. Photometric determination of glucose and fructose concentration 46 2.7.4. 13C-Nuclear magnetic resonance spectroscopy 46 vi 2.7.5. Microscopy 47 2.8. Internet tools used for bioinformatic analysis 47 3. RESULTS 49 3.1 Osmotic stress responses in G. oxydans 50 3.1.1. Choice of substrate and osmolyte 50 3.1.2. Expression studies of osmotically regulated genes in G. oxydans 52 3.1.3. Analysis of osmotic stress responses in G. oxydans through genome-wide transcriptome analysis 54 3.1.3.1. Quality testing of RNA samples 55 3.1.3.2. Differential gene expression under osmotic stress analyzed by transcriptome sequencing 57 3.1.3.3. Validation of the transcriptome data and selection of candidate genes 63 3.1.3.4. Generation of G. oxydans strains carrying in-frame deletions for gox1118 and gox1119 64 3.1.3.5. Characterization of Gox1849: an uncharacterized oxidoreductase 68 3.2. Mannitol as a major intracellular metabolite and osmolyte in G. oxydans 70 3.2.1. 13C-NMR spectroscopy of total cellular metabolites from G. oxydans 70 3.2.2. Effect of mannitol on cellular catalytic activity 72 3.2.3. De novo synthesis of mannitol in G. oxydans under reduced water activity 74 3.2.4. Effect of carbon sources and osmolytes on mannitol accumulation 79 3.2.5. Effect of exogenous mannitol on growth and morphology of osmotically stressed cells 80 3.2.6. Effect of polyols on growth and substrate oxidation rates of G. oxydans 83 3.3. Enzymatic routes for the biosynthesis of mannitol in G. oxydans 84 3.3.1. Identification and bioinformatic analysis of the mannitol dehydrogenases from G. oxydans 85 3.3.2. Characterization of the mannitol dehydrogenases from G. oxydans 87 3.3.3. Transcript abundance of genes coding for D-fructose reductases in G. oxydans 91 3.3.4. Characterization of fructose reductase deletion mutants 92 3.3.4.1. Effect of the deletion of fructose reductases on growth of G. oxydans 92 3.3.4.2. Intracellular mannitol formation and activity of cytoplasmic fructose reductases 97 vii 3.3.4.3. Effect of the deletion of fructose reductases on cellular catalytic efficiency 101 3.4. Metabolic engineering of G. oxydans for enhanced osmotolerance 102 3.4.1. Overproduction of D-fructose reductase (Gox1432) in G. oxydans 103 3.4.2. Heterologous overproduction of glucose isomerases in G. oxydans 104 4. DISCUSSION 108 4.1. Osmotic stress responses in G. oxydans 111 4.1.2. Response of G. oxydans to osmotic stress at transcriptional level 113 4.1.3. Genome-wide transcriptome analysis of osmotically stressed cells of G. oxydans 113 4.2. Mannitol as a major intracellular metabolite and osmolyte in G. oxydans 116 4.2.1. De novo synthesis of mannitol in G. oxydans under reduced water activity 118 4.2.2. Osmodependent accumulation of mannitol in G. oxydans 119 4.2.3. Effect of carbon sources and osmolytes on mannitol accumulation in G.oxydans 120 4.2.4. Protective effect of mannitol on cell physiology 121 4.2.5. Effect of polyols on growth and substrate oxidation rates of G. oxydans 125 4.3. Biosynthesis of mannitol in G. oxydans 126 4.4. Characterization of the relative contribution of Gox1432 and Gox0849 in cellular osmoprotection 131 4.5. Characterization of fructose reductase deletion mutants 133 4.6. Metabolic engineering of G. oxydans for enhanced osmotolerance 134 4.6.1. Overproduction of D-fructose reductase (Gox1432) 135 4.6.2. Heterologous overproduction of glucose isomerases in G. oxydans 136 4.7. Gox1432: key player for osmotolerance of G. oxydans 138 5. SUMMARY 140 6. REFERENCES 142 7. CURRICULUM VITAE 165 8. ACKNOWLEDGEMENTS 167 viii ABBREVIATION LIST ACN Acetonitrile ADP Adenosine diphosphate α Alpha Amp Ampicillin APS Ammonium persulfate ATP Adenosine triphosphate BLAST Basic Local Alignment Search Tool bp Base pair β Beta cDNA Complementary DNA cdw Cell dry weight oC Degree centigrade δ Chemical shift ddH2O Double distilled water DMSO Dimethyl sulfoxide DNase Deoxyribonuclease dNTP Desoxyribonucleotide triphosphate DSMZ German Collection of Microorganisms and Cell cultures e.g., exempli gratia (For example) EDP Entner-Doudoroff pathway EDTA Ethylenediaminetetraacetic acid EMP Embden-Meyerhof-Parnas glycolytic pathway et al. et alii (and others) FAD Flavin adenine dinucleotide 5-FC 5-fluorocytosine For Forward g Gravitational acceleration (9.8 m/s2) GI Glucose isomerase Gox Gluconobacter oxydans h Hour H2O2 Hydrogen peroxide H2Odest Destillata (Distilled water) H2SO4 Sulfuric acid HABA 4-hydroxyazobenzen-2-carbonic acid HCl Hydrogen chloride HEPES 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid HPLC High performance liquid chromatography Km Kanamycin kb Kilobase Kcat Turnover number kDa Kilodalton KEGG Kyoto Encyclopedia of Genes and Genomes kg Kilogram KM Michaelis Menten constant K-phosphate Potassium phosphate LB Lysogeny broth M Molar (mol L-1) mbar Millibar ix Mbp Megabase pair MDH Mannitol dehydrogenase MgCl2 Magnesium chloride MgSO4 Magnesium sulphate µ Micro min Minute MOPS 3-(N-morpholino)-propansulfonic acid mRNA Messenger RNA ms Millisecond NaCl Sodium chloride NAD+ Nicotinamide adenine dinucleotide NADP+ Nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology information ng Nanogram NGS Next generation sequencing NMR Nuclear magnetic resonance OD Optical density Osm Number of osmoles ox. PPP Oxidative pentose phosphate pathway P Phosphate PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction PEG Polyethylene glycol % Percent (grams per 100 mL or mL per 100 mL) pg Picogram Pi Inorganic phosphate PPP Pentose phosphate pathway PQQ Pyrroloquinoline quinone R2 The Pearson correlation coefficient of determination rev Reverse rpm Revolutions per minute RNA-Seq RNA sequencing RPKM Reads Per Kilobase of transcript per Million mapped reads rRNA Ribosomal RNA RT-qPCR Real Time quantitative reverse transcription PCR SDS Sodium dodecyl sulphate SOC Super optimal broth with catabolite repression sp.