Exploring the diversity and antimicrobial potential of predatory bacteria from Indonesian mangroves Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n Von Senlie Octaviana Aus Salatiga / Indonesien 1. Referent: Professor Dr. Michael Steinert 2. Referent: Privatdozent. Dr. Joachim Wink eingereicht am: 22.02.2021 mündliche Prüfung (Disputation) am: 05.05.2021 Druckjahr 2021 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Senlie Octaviana, Tjandrawati Mozef, and Joachim Wink. Taxonomy and antimicrobial activity of gliding bacterium from Indonesian Mangroves. Submitted to Hayati Journal of Biosciences (2020). Senlie Octaviana, Gian Primahana, Tjandrawati Mozef, Luiz G. A. Borges, Dietmar H. Pieper and Joachim Wink. Antimicrobial Potential of Myxobacteria from Indonesian Mangroves. Submitted to Current Microbiology Journal (2021). Tagungsbeiträge Diversity and antimicrobial potential of myxobacteria from Indonesian Mangroves (2020). Retreat of the Helmholtz Centre for Infection Research Graduate School, Braunschweig, Germany (Mündlicher Vortrag). Posterbeiträge Myxobacteria from Coastal Area in Indonesia as a source of novel anti-infective. June 2018. HIPS-Symposium, Saarbrücken, Germany. A gliding bacterium, closely related to Ohtaekwangia kribbensis as a potential source of anti-infective. June 2019. HIPS-Symposium, Saarbrücken, Germany. Table of Contents Pages Table of Contents .................................................................................................. iii List of Figures ...................................................................................................... vii List of Tables ......................................................................................................... ix Abbreviations ......................................................................................................... x ABSTRACT .......................................................................................................... xi ABSTRAKT ......................................................................................................... xii CHAPTER 1. INTRODUCTION ......................................................................... 1 1.1. New Diseases: CHallenges and Solutions ...................................................... 1 1.2. Microbial Natural Products ........................................................................... 3 1.2.1. Myxobacteria ................................................................................................ 3 1.2.2. Other gliding bacteria .................................................................................. 6 1.3. PolypHasic ApproacH Leads to a Robust Classification System ................ 7 1.4. Metagenomics Analysis of Myxobacterial Communities ............................ 9 1.5. Mangrove Microbial RicHness ..................................................................... 10 1.6. Outline of work ............................................................................................. 11 1.7. Aim of the study ............................................................................................ 12 1.8. Sample collection .......................................................................................... 13 CHAPTER 2. MATERIAL and METHODS .................................................... 15 2.1. Material ...................................................................................................... 15 2.1.1. Organisms ................................................................................................... 15 2.1.2. CHemicals ................................................................................................... 18 2.1.3. Equipment .................................................................................................. 22 2.2. Methods 22 Evaluation of myxobacterial diversity by Illumina Miseq Sequencing .......... 22 2.2.1. DNA extraction ........................................................................................... 22 2.2.2. Amplicon library preparation ................................................................... 23 2.2.3. Data analysis ............................................................................................... 23 Potential of myxobacteria from Indonesia mangroves as antimicrobial agents 24 2.2.4. Isolation of Myxobacteria .......................................................................... 24 2.2.5. Identification of pure cultures by 16S rRNA gene sequencing .............. 24 iii 2.2.6. Preparation of crude extract .................................................................... 25 2.2.7. Preparation of pathogenic microorganisms ............................................ 25 2.2.8. Screening for antimicrobial activity and identified of active compound 26 Multilocus sequences analysis (MLSA) of myxobacteria from mangrove .... 27 2.2.9. Myxococcales strains ............................................................................... 27 2.2.10. Cultivation and DNA extraction ............................................................ 27 2.2.11. Amplification and Sequencing ............................................................... 27 2.2.12. Data analysis ............................................................................................ 28 Ohtaekwangia sp. nov., a gliding bacterium isolated from Indonesian mangroves and the related strains from HZI strain collection ........... 28 2.2.13. Isolation and Identification of Gliding Bacteria ................................... 28 2.2.14. Preparation of cultures and type strains ............................................... 28 2.2.15. MorpHological analysis ........................................................................... 29 2.2.15.1. MorpHology ................................................................................. 29 2.2.15.2. Gram Staining ............................................................................. 29 2.2.15.3. PHysiological analysis .................................................................. 29 2.2.15.3.1. Medium variation ................................................................. 29 2.2.15.3.2. Detection of optimal temperature for growth .................... 29 2.2.15.3.3. Detection of optimal pH for growth .................................... 30 2.2.15.3.4. NaCl Tolerances ................................................................... 30 2.2.15.3.5. Anaerobic Tolerances .......................................................... 30 2.2.15.3.6. Catalase and Oxidase Test ................................................... 30 2.2.15.3.7. Flexirubin Pigment Test ...................................................... 31 2.2.15.3.8. APIZYM and APICAMPY Test ......................................... 31 2.2.15.3.9. Gen III Microplate Biolog System ...................................... 31 2.2.15.3.10. Antibiotic Resistances .......................................................... 31 2.2.15.4. CHemotaxonomy ....................................................................... 32 2.2.15.4.1. Polar lipid Analysis .............................................................. 32 2.2.15.4.2. Fatty Acid Methyl Ester (FAME) ....................................... 33 CHAPTER 3. RESULTS .................................................................................... 34 3.1. Evaluation of myxobacterial diversity by Illumina Miseq Sequencing .. 34 3.2. Potential of myxobacteria from Indonesia mangroves as antimicrobial agents 36 3.2.1. Cultivation of myxobacteria ..................................................................... 36 iv 3.2.2. Antimicrobial activities from cultivated of myxobacteria ...................... 40 3.3. Multilocus sequences analysis (MLSA) of myxobacteria from mangrove 44 3.3.1. Type strains ................................................................................................. 44 3.3.2. Myxobacterial isolates ............................................................................... 46 3.3.3. PHylogenetic tree analysis .......................................................................... 49 3.3.4. Intraspecies and interspecies sequences similarities ............................... 51 3.4. Ohtaekwangia sp. nov., gliding bacterium isolated from Indonesian mangroves and the related strains from HZI strain collection ................ 53 3.4.1. Strain isolation ............................................................................................ 53 3.4.2. CHaracteristics of strain 313MSO ............................................................ 54 3.4.3. Antimicrobial activity ................................................................................ 58 3.4.4. Related Strains from HZI Culture Collection ......................................... 60 3.4.4.1. Isolation and Ecology ..................................................................... 60 3.4.4.2. CHaracteristics of genome ............................................................. 61 3.4.4.3. CHaracteristic of pHysiology and cHemotaxonomy ..................... 62 3.4.4.4. Protologue ......................................................................................