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Community of Soft Coral Alcyonium Digitatum Associated Bacteria and Their Antimicrobial Activities

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Community of Soft Coral Alcyonium Digitatum Associated Bacteria and Their Antimicrobial Activities Community of soft coral Alcyonium digitatum associated bacteria and their antimicrobial activities Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel Vorgelegt von Pham Thi Mien Kiel 2014 Referent: Prof. Dr. Johannes F. Imhoff Koreferent: PD Dr. Florian Weinberger Tag der mündlichen Prüfung: 17.11.2014 Zum Druck genehmigt: 17.11.2014 gez. Prof. Dr. Johannes F. Imhoff i The present work was carried out at the GEOMAR Helmholtz Centre for Ocean Research Kiel Christian-Albrechts-University of Kiel from October 2010 to September 2014 under the supervision of Prof. Dr. Johannes F. Imhoff ii Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit unter Einhaltung der Regeln guter wissenschaftlicher Praxis der Deutschen Forschungsgesellschaft verfasst habe, und dass sie nach Form und Inhalt meine eigene Arbeit ist. Diese Arbeit, auch Teile davon, wurden noch nicht veröffentlicht oder zur Veröffentlichung eingereicht. Sie wurde keiner anderen Stelle im Rahmen eines Prüfungsverfahrens vorgelegt. Dies ist mein erstes und einziges Promotionsverfahren. Kiel, den 01.10.2014 Pham Thi Mien iii Scientific contribution Sampling of soft corals in the Baltic Sea was done by Gregor Steffen (GEOMAR). Sampling of soft corals in the North Sea was done by SCUBA diving team of AWI staff at Helgoland. All bioassays of crude extracts and pure compounds were done by Arlette Wenzel-Storjohann (Marine Microbiology, GEOMAR). NMR measurement was performed at the Otto-Diels Institute of Organic Chemistry (Christian-Albrechts University of Kiel) and NMR data analysis was done by Bin Wu (GEOMAR). All other experiments were done by Pham Thi Mien under supervision of Johannes F. Imhoff. iv Table of Contents Summary ......................................................................................................... 1 Introduction ............................................................................................................ 5 1. Soft coral Alcyonium digitatum (Linnaeus, 1758) ......................................... 5 2. Bacteria and coral host ................................................................................... 7 2.1 Where we found the bacteria in coral hosts? ........................................... 7 2.2 Coral associated bacteria ......................................................................... 8 3. Biotechnological potential of soft corals and coral associated bacteria ....... 11 4. Thesis outline ................................................................................................. 14 Material and Methods ................................................................................... 15 1. Sampling and cultivation processes ............................................................... 15 2. DNA extraction and phylogenetic classification ........................................... 16 3. Extraction procedure for all isolates .............................................................. 18 4. Chemical and antimicrobial screening ........................................................... 18 4.1 Chemical screening .................................................................................. 18 4.2 Antimicrobial activities screening ........................................................... 19 5. Cultivation and extraction procedures for four selected strains .................... 20 6. Compound purification of four selected strains ............................................. 20 6.1 Preparative HPLC with the VWR International LaPrep system ............. 20 6.2 Preparative HPLC with the Merck Hitachi Elite LaChrom system 1 ...... 21 6.3 Preparative HPLC with the Merck Hitachi Elite LaChrom system 2 ...... 21 7. Bioassays of purified compounds .................................................................. 21 8. NMR analyses ................................................................................................ 22 Results ............................................................................................................. 23 1. Phylogenetic analysis and identification of the isolates ................................ 23 2. Antimicrobial activities of all isolates ........................................................... 27 3. Chemical substances produced by soft coral-associated bacteria .................. 35 3.1 Lysinibacillus sp. C-2-29 .............................................................................. 37 3.1.1 Origin, cultivation and identification ................................................... 37 3.1.2 Cultivation and extraction of the strain C-2-29 .................................... 37 3.1.3 Compound purification ......................................................................... 38 v 3.1.4 Metabolic profile .................................................................................. 38 3.1.5 Bioactivities of the isolated compounds ............................................... 42 3.1.6 Discussion ............................................................................................. 42 3.2 Bacillus sp. H-6 ............................................................................................. 44 3.2.1 Origin, cultivation and identification .................................................... 44 3.2.2 Cultivation and extraction of the strain H-6 ......................................... 44 3.2.3 Compound purification ......................................................................... 44 3.2.4 Metabolic profile .................................................................................. 45 3.2.5 Bioactivities of the isolated compounds ............................................... 48 3.2.6 Discussion ............................................................................................. 48 3.3 Micrococcus sp. A-2-28 ................................................................................ 52 3.3.1 Origin, cultivation and identification ................................................... 52 3.3.2 Cultivation and extraction of the strain A-2-28 .................................... 52 3.3.3 Compound purification ......................................................................... 52 3.3.4 Metabolic profile .................................................................................. 53 3.3.5 Bioactivities of the isolated compounds ............................................... 56 3.3.6 Discussion ............................................................................................. 56 3.4 Bacillus sp. C-1-10 ........................................................................................ 59 3.4.1 Origin, cultivation and identification ................................................... 59 3.4.2 Cultivation and extraction of the strain C-1-10 .................................... 59 3.4.3 Compound purification ......................................................................... 59 3.4.4 Metabolic profile .................................................................................. 60 3.4.5 Bioactivities of the isolated compounds ............................................... 66 3.4.6 Discussion ............................................................................................. 66 Discussion ........................................................................................................ 75 1. Phylogenetic classification of coral associated bacteria .......................... 75 2. Coral hosts and their associated bacteria ................................................. 78 3. Antimicrobial activity of isolates ............................................................. 80 4. Biotechnological potential of soft coral associated isolates .................... 83 Conclusions ............................................................................................................. 86 Acknowledgment ...................................................................................................... 87 List of Abbreviations ................................................................................................. 88 References ............................................................................................................... 91 vi List of Figures Figure 1.1 The annual cycle of A. digitatum ............................................................. 6 Figure 1.2 Microorganisms in coral host ................................................................... 7 Figure 1.3 Distribution in drug classes of marine bioactive compounds .................. 11 Figure 3.1 Antimicrobial activity of isolates using two different media ................... 27 Figure 3.2 UV spectrums of C-2-29 in GYM medium (100 ml volume) .................. 39 Figure 3.3 UV spectrums of C-2-29 in BM medium (100 ml volume) ..................... 40 Figure 3.4 UV spectrums of C-2-29 in 1 L cultures in GYM medium ..................... 40 Figure 3.5 Structure of the compound 3 ................................................................... 41 Figure 3.6 Structure of anisomycin (I) and deacetylanisomycin ............................... 43 Figure 3.7 Chemical profiles of H-6 in 100 ml cultures ............................................ 47 Figure 3.8 Chemical profiles of H-6 in 1 L cultures in BM medium ....................... 47 Figure 3.9 Structure of amicoumacin A from Bacillus sp.. ....................................... 48 Figure 3.10 Structure of bacilosarcin B from
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