Actinobacteria and Myxobacteria isolated from freshwater snails and other uncommon Iranian habitats, their taxonomy and secondary metabolism 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 Nasim Safaei aus Teheran / Iran 1. Referent: Professor Dr. Michael Steinert 2. Referent: Privatdozent Dr. Joachim M. Wink eingereicht am: 24.02.2021 mündliche Prüfung (Disputation) am: 20.04.2021 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 Safaei, N. Mast, Y. Steinert, M. Huber, K. Bunk, B. Wink, J. (2020). Angucycline-like aromatic polyketide from a novel Streptomyces species reveals freshwater snail Physa acuta as underexplored reservoir for antibiotic-producing actinomycetes. J Antibiotics. DOI: 10.3390/ antibiotics10010022 Safaei, N. Nouioui, I. Mast, Y. Zaburannyi, N. Rohde, M. Schumann, P. Müller, R. Wink.J (2021) Kibdelosporangium persicum sp. nov., a new member of the Actinomycetes from a hot desert in Iran. Int J Syst Evol Microbiol (IJSEM). DOI: 10.1099/ijsem.0.004625 Tagungsbeiträge Actinobacteria and myxobacteria isolated from freshwater snails (Talk in 11th Annual Retreat, HZI, 2020) Posterbeiträge Myxobacteria and Actinomycetes isolated from freshwater snails and other uncommon Iranian habitats, their taxonomy and secondary metabolites (11th and 12th International PhD Symposium, HZI Braunschweig, 2018 and 2019) Taxonomy of a novel Actinobacteria isolated from Iranian soil sample (8th and 9th International PhD Symposium, HIPS Saarbrücken, 2018 and 2019) Poly-phasic taxonomy of a novel Acinobacteria isolated from soil sample (9th Annual Retreat, Quedlinburg, 2018) Acknowledgements “May the gratitude in my heart, kiss all the universe.” Hafez Firstly, I would like to express my deepest gratitude to my supervisor PD. Dr. Joachim Wink, who believed in me and supported me through this journey with his calmness, kindness and wisdom. Secondly, I would like to deeply thank Prof. Dr. Michael Steinert, who without his guidance and support, this work would not have been possible. My sincere thanks also goes to Prof. Dr. Rolf Müller, for giving me the opportunity to work although for a short time in their fascinating group. I am thankful to Dr. Emilia Oueis, Dr. Daniel Krug and Dr. Lena Keller. With a deep sense of gratefulness, I wish to thank Prof. Dr. Yvonne Mast and Dr. Imen Nouioui for their kind help and scholarly advice. I really enjoyed their collaboration. I would like to extend my gratitude to all the members of MISG group, who are my second family. We shared many memories together. I would like to deeply thank Klaus Peter Conrad, Stephanie Schulz, Birte Trunkwalter, Aileen Gollasch, Wera Collisi and Jolanta Lulla for the best technical support. I feel blessed to have great coworkers and thank all of them. In particular, I am thankful to Senlie Octaviana and Dr. Chandra Risdian. With a deep sense of gratefulness, I wish to thank Prof. Dr. Marc Stadler for proofreading the manuscript of the thesis. In addition, my lovely friends who helped me for proofreading and gave me emotional support. I’m grateful to Dr. Abhishek Bakuli, Daniela Gornyk, Michael Grützner, Iman Kianfar, Dr. Zahra Khosravi, Dr. Ziba Najmi, Sara Zamani, Ahlem Askri, Paul van Vulpen, Khadija Hassan, Dr. Azam Moradi, Dr. Mohammad Yaghoobi and special thanks to Dr. Gholamhossein Ebrahimipour. Last but not least, this thesis dedicated to my lovely family. No words can express my gratitude to them. My lovely parents and my beautiful sisters, Narges, Nastaran and Negar, I want you to know that all I have been through in life and whatever lead me to where I am is because of you. Your unconditional love and support made this journey possible to me. Dad and Mom you are the first supervisors and the best friends of mine. Wish you all the best. Table of contents 1 Introduction .......................................................................................... 1 1.1 Antibiotics and natural products ..................................................................... 1 1.2 Dereplication ................................................................................................... 4 1.3 Good producers of natural products ................................................................ 5 1.3.1 Myxobacteria ........................................................................................... 5 1.3.2 Actinobacteria .......................................................................................... 7 1.4 Polyphasic taxonomy ...................................................................................... 8 1.4.1 Phenotype ................................................................................................. 9 1.4.1.1 Morphology ............................................................................................................... 9 1.4.1.2 Biochemistry ............................................................................................................ 10 Chemotaxonomy .......................................................................................................... 10 Enzymology ................................................................................................................. 11 1.4.1.3 Physiology ............................................................................................................... 11 1.4.2 Genotype ................................................................................................ 11 1.4.2.1 16S rRNA gene ........................................................................................................ 11 1.4.2.2 DNA-DNA hybridization (DDH) ............................................................................ 12 1.4.2.3 Average Nucleotide Identity (ANI) ......................................................................... 13 1.4.2.4 G+C content ............................................................................................................. 13 1.4.2.5 Whole-genome sequencing ...................................................................................... 13 1.4.2.6 MALDI-TOF ........................................................................................................... 14 1.4.3 Ecology .................................................................................................. 14 1.4.3.1 Uncommon habitats ................................................................................................. 15 Medicinal plants ........................................................................................................... 15 Mangrove forest ........................................................................................................... 15 Desert ........................................................................................................................... 16 Freshwater snail ........................................................................................................... 17 1.4.3.2 Selective isolation methods ..................................................................................... 18 Rare Actinomycetes ..................................................................................................... 19 1.5 Aim of study .................................................................................................. 20 2 Material and methods ......................................................................... 21 2.1 Media ............................................................................................................. 21 2.2 Sampling date and locations .......................................................................... 21 2.2.1 Soil ......................................................................................................... 21 2.2.2 Snail ....................................................................................................... 22 I 2.3 Isolation and purification .............................................................................. 23 2.3.1 Myxobacteria ......................................................................................... 23 2.3.2 Actinobacteria ........................................................................................ 23 2.3.3 Myxobacteria and Actinobacteria from snails ....................................... 24 2.4 Identification ................................................................................................. 24 DNA extraction ................................................................................................................. 24 PCR amplification ............................................................................................................ 25 Purification of PCR products ............................................................................................ 25 Phylogenetic analyses ....................................................................................................... 26 2.5 Screening for secondary metabolites............................................................. 26 Harvesting the metabolite cultures .................................................................................... 26 Antimicrobial