Title Antimicrobial activities and diversity of sponge derived microbes Author(s) Flemer, Burkhardt Publication date 2013 Original citation Flemer, B. 2013. Antimicrobial activities and diversity of sponge derived microbes. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2013, Burkhardt Flemer http://creativecommons.org/licenses/by-nc-nd/3.0/ Embargo information No embargo required Item downloaded http://hdl.handle.net/10468/1192 from Downloaded on 2017-02-12T12:58:08Z University College Cork Coláiste na hOllscoile Corcaigh Antimicrobial activities and diversity of sponge derived microbes A thesis presented to the National University of Ireland for the degree of Doctor of Philosophy Burkhardt Flemer, Dipl. Ing. (FH) National University of Ireland, Cork Department of Microbiology January 2013 Head of Department: Professor Gerald F. Fitzgerald Supervisors: Professor Alan Dobson, Professor Fergal O’Gara This thesis is dedicated to my family, my inspiration DECLARATION 4 ABSTRACT 5 CHAPTER 1 8 INTRODUCTION BODY PLAN OF MARINE SPONGES 9 Demosponges 9 MICROBIAL SPONGE ASSOCIATES 10 HIGHTHROUGHPUT SEQUENCING AS A TOOL TO ESTIMATE SPONGE- 16 ASSOCIATED MICROBIOTA Studies employing pyrosequencing for the analysis of sponge-associated 17 microbiota ESTABLISHMENT AND MAINTENANCE OF SPONGE MICROBE 21 INTERACTIONS SYMBIONT FUNCTION 22 Carbon metabolism 23 Nitrogen metabolism 23 Sulphur metabolism 25 SPONGE ASSOCIATED FUNGI 26 DEEP SEA SPONGES 28 SECONDARY METABOLITES FROM MARINE SPONGES AND SPONGE 29 DERIVED MICROBES Potential of sponge-ass. MO for the production of bioactive metabolites 30 Bioactive natural products from marine sponges 32 POLYKETIDE SYNTHASES (PKS) AND NON RIBOSOMAL PEPTIDE 33 SYNTHASES (NRPS) NRPSs 33 PKSs 34 AIMS AND OVERVIEW OF THE PRESENTED STUDY 36 REFERENCES 36 CHAPTER 2 54 DIVERSITY AND ANTIMICROBIAL ACTIVITIES OF MICROBES FROM TWO IRISH MARINE SPONGES, SUBERITES CARNOSUS AND LEUCOSOLENIA SP. ABSTRACT 55 INTRODUCTION 56 MATERIALS AND METHODS 58 Sponge sampling 58 Isolation of bacteria 58 Deferred antagonism assay 58 Well diffusion assay 59 PCR anplificaton of 16S-rRNA genes 59 Sequencing and phylogenetic analysis of 16S-rRNA gene products 60 RESULTS 61 Isolation of bacteria 61 Antimicrobial activity - deferred antagonism assay 63 Antimicrobial activity - well diffusion assay 68 1 DISCUSSION 68 REFERENCES 73 SUPPLEMENTARY FIGURES 78 CHAPTER 3 83 DIVERSITY AND ANTIMICROBIAL ACTIVITIES OF FUNGAL ISOLATES OBTAINED FROM 12 MARINE SPONGES SAMPLED IN LOUGH HYNE, IRELAND ABSTRACT 84 INTRODUCTION 85 MATERIALS AND METHODS 86 Sponge sampling 86 Isolation of fungi 86 Deferred antagonism assay 87 Well diffusion assay 87 Preparation and testing of antibiotic discs 88 Isolation of genomic DNA from fungal isolates and PCR anplificaton of 18S- 89 rRNA genes Sequencing and phylogenetic analysis of 18S-rRNA gene products 89 Analysis of secondary metabolite genes 90 RESULTS 92 Isolation of fungi 92 Antimicrobial activity - deferred antagonism assay 92 Antimicrobial activity - well diffusion assay and antibiotic discs 97 Secondary metabolite genes in the genome of W9F6 97 DISCUSSION 102 Diversity 102 Bioactivity 104 Secondary metabolite genes 104 CONCLUSION 106 REFERENCES 107 CHAPTER 4 112 ANTIMICROBIAL ACTIVITY AND CHEMICAL CHARAC- TERIZATION OF A SECONDARY METABOLITE FROM A SPONGE DERIVED MICROMONOSPORA SP. ABSTRACT 113 INTRODUCTION 114 MATERIALS AND METHODS 115 Sponge sampling 115 Isolation of bacteria 115 Deferred antagonism assay 115 Well diffusion assay 116 Fermentation and preparation of CFS for the extraction of antimicrobial 116 compounds Extraction of antimicrobial compounds 117 Liquid-liquid extraction 118 Extraction of microbial pellet 118 Resazurin assay 118 2 Thin layer and preparative scale chromatography 119 Analytical procedures 119 RESULTS AND DISCUSSION 120 Scale up 126 Purification of bioactive compounds 129 LC-MS and NMR analysis 129 LC-MS and NMR of fractions 5,6,8a, 8b and 9 134 CONCLUSION 135 REFERENCES 140 CHAPTER 5 142 DISTINCT PROKARYOTIC DIVERSITY AND SPATIAL DISTRIBUTION OF MICROBES ASSOCIATED WITH DEEP SEA SPONGES REVEALED BY PYROSEQUENCING ABSTRACT 143 INTRODUCTION 144 MATERIALS AND METHODS 146 Sponge sampling 146 Metagenomic DNA extraction from seawater and sponge samples 146 PCR amplicon library preparation for pyrosequencing 147 Pyrosequencing data analysis 147 Preparation of Phylogenetic trees 148 RESULTS 149 Sponge sampling 149 Initial analysis 149 Bacterial vs. archaeal diversity 152 Archaeal diversity 152 Bacterial diversity 154 Classification into lower taxonomic levels 155 DISCUSSION 163 Bacterial community 163 Archaeal community 166 Analysis of shared OTUs 167 Spatial distribution of sponge-associated microbes in S. normani 168 Deep water sponges’ microbiota vs. microbiota of shallow water sponges 169 CONCLUSION 171 REFERENCES 172 SUPPLEMENTARY TABLES 178 CHAPTER 6 189 GENERAL DISCUSSION GENERAL DISCUSSION 190 REFERENCES 199 ACKNOWLEDGEMENTS 204 3 I herewith declare that the thesis is my own work and has not been submitted for another degree, either at University College Cork or elsewhere. Burkhardt Flemer _____________________________ 4 ABSTRACT Marine sponges are the single best marine source for novel secondary metabolites with bioactivity and play host to a microbiota with a remarkable diversity. In this study, marine sponges collected in Irish waters were analysed for their associated microbiota. Bacteria and fungi were isolated from several sponge species and analysed for their diversity and antimicrobial activity and deep water sponges were analysed for their associated bacteria and Archaea employing 454 pyrosequencing. In order to evaluate the diversity and antimicrobial activity of bacteria from the marine sponges Suberites carnosus (class Demospongiae ) and Leucosolenia sp. (class Calcaraea ) collected at Lough Hyne, Ireland, two hundred and thirty seven bacteria were isolated. Isolates from the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were obtained. Isolates of the genus Pseudovibrio were dominant among the bacteria from S. carnosus whereas Pseudoalteromonas and Vibrio were the dominant genera isolated from Leucosolenia sp. Approximately 50% of the isolates from S. carnosus displayed antibacterial activity and ~15% of the isolates from Leucosolenia sp. demonstrated activity against the test fungal strains. The antibacterial activity observed was mostly from Pseudovibrio and Spongiobacter isolates, while the majority of the antifungal activity was observed from the Pseudoalteromonas, Bacillus and Vibrio isolates. Overall, both sponges possess a diverse range of bioactive and potentially novel bacteria. Differences observed from the sponge-derived groups of isolates in terms of bioactivity suggests that S. carnosus isolates may be a better source of antibacterial compounds, while Leucosolenia sp. isolates appear to be a better source of antifungal compounds. Both sponge species have been evaluated for the first time for their associated microbiota. 12 marine sponge samples from the same site, among them the S. carnosus and Leucosolenia sp. sample, were analysed for their associated culturable fungi and 71 fungal isolates were obtained. Based on their 18S-rRNA gene sequence the isolates were found to be very diverse with 22 OTUs determined at a sequence identity of 98.5% and different phylotypes present in some of the OTUs. Together with genera 5 or orders typically isolated from marine sponges such as Hypocreales, Pleosporales and Eurotiales some isolates were representatives of taxa seldomly found in marine sponges. All isolates were assessed for their antimicrobial activity against 2 Gram postitive and 1 Gram negative bacterium in the deferred antagonism assay and more than 60% of the isolates tested positive in at least one assay against at least one of the test strains. Isolates with clear and/ or broad range activity were also tested in the well diffusion assay and the disc diffusion assay against the same bacterial as well as 5 fungal test strains (4 yeasts and 1 mycelium forming fungi). Isolates grouping into three OTUs of the orders Hypocreales, Pleosporales and Eurotiales showed especially strong and broad range activity. One of the isolates, which was closely related to Fusarium oxysporum and showed activity against bacteria and fungi, was investigated for its secondary metabolite genes (NRPS and PKS). At least 5 different NRPS genes were identified in its genome based on partial gene sequences obtained from a clone library. The sequence identity to published NRPS sequences of one gene was as low as 50 % highlighting the likelihood that this isolate may be capable of producing potentially novel secondary metabolites. A Micromonospora sp. was isolated from a Haliclona simulans sample collected in Irish waters. The isolate inhibited the growth of Gram positive bacterial test strains in three different antimicrobial assays. Several production media were subsequently employed to optimize the production of the antimicrobial compound(s) and a scale up culture with the best medium was extracted for its antimicrobial compounds. The purification of a yellow-orange compound with strong bioactivity against B. subtilis, S. aureus and P. aeruginosa was facilitated by bio-assay guided chromatography and the compound was tried to be characterized utilising LC-MS and NMR. By LC-MS two compounds of bioactivity were detected in the extracts, one with an m/z=617.21 (positive mode) and one with an m/z=619.21. Another compound with