Title Exploiting the diverse microbial ecology of marine sponges Author(s) Jackson, Stephen A. Publication date 2013 Original citation Jackson, S. A. 2013. Exploiting the diverse microbial ecology of marine sponges. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2013, Stephen A. Jackson http://creativecommons.org/licenses/by-nc-nd/3.0/ Embargo information Restricted to everyone for three years Item downloaded http://hdl.handle.net/10468/1037 from Downloaded on 2017-02-12T13:45:47Z Exploiting the Diverse Microbial Ecology of Marine Sponges A Thesis presented to the National University of Ireland for the Degree of Doctor of Philosophy Stephen Anthony Jackson, BSc. Department of Microbiology National University of Ireland Cork Head of Department: Dr Gerald Fitzgerald Research Supervisors: Prof. Alan Dobson & Dr John Morrissey 2013 1 For Daniel, My inspiration 2 Index 1 Abstract 7 Chapter 1: Literature Review 10 1.1 Marine sponges 11 1.1.1 Sponge anatomy and physiology 12 1.1.1.1 Sponge skeletons 12 1.1.1.2 Sponge cell types 13 1.1.1.3 Sponge physiology 14 1.2 Sponge associated microorganisms 15 1.2.1 Sponge associated bacteria 15 1.2.1.1 Culture dependent analyses 15 1.2.1.2 Culture independent analyses 16 1.2.1.2.1 Microscopy 16 1.2.1.2.2 16S rRNA clone libraries 18 1.2.1.2.3 Pyrosequencing 20 1.2.2 Sponge associated archaea 22 1.2.3 Sponge associated eukaryota 22 1.2.3.1 Sponge associated fungi 22 1.2.3.2 Sponge associated diatoms 23 1.2.3.3 Sponge associated dinoflagellates 23 1.2.3.4 Other sponge associated eukaryota 24 1.2.4 Sponge-specific microorganisms 24 1.3 Symbiotic functions of sponge associated microbes 27 1.3.1 Methods to elucidate sponge symbiont functions 27 1.3.2 Discrimination between food microbes and symbiotic microbes 28 1.3.3 Nutrient cycling in sponges 29 1.3.3.1 Carbon cycling 29 1.3.3.2 Nitrogen cycling 30 1.3.3.2.1 Nitrogen fixation 30 1.3.3.2.2 Nitrification 31 1.3.3.2.3 Denitrification 32 1.3.3.3 Sulphur cycling 33 1 1.3.4 Other putative symbiosis factors 36 1.4 Pharmacological potential of marine sponges 37 1.5 Exploiting the pharmacological potential of marine sponges 42 1.6 Metagenomic strategies for the discovery and production of novel industrial and pharmacological products 43 1.6.1 Problems associated with large insert metagenomic clone libraries 45 1.7 Summary 46 1.8 References 47 2.0 Chapter 2: Diverse and distinct sponge-specific bacterial communities in sponges from a single geographical location in Irish waters and antimicrobial activities of sponge isolates 103 2.1 Abstract 104 2.2 Introduction 105 2.3 Materials & Methods 107 2.3.1 Sponge sampling 107 2.3.2 Culture isolation 108 2.3.2.1 General isolation 108 2.3.2.2 Targeted isolation 108 2.3.3 Phylogenetic analysis of cultured isolates 110 2.3.4 Antimicrobial assays 111 2.3.5 Metagenomic DNA extraction from sponges 111 2.3.6 Metagenomic DNA extraction from seawater 111 2.3.7 PCR amplicon library preparation for pyrosequencing 112 2.3.8 Pyrosequencing data analysis 113 2.4 Results 114 2.4.1 Culture isolation 114 2.4.2 Antimicrobial assay 116 2.4.3 Pyrosequencing 119 2.5 Discussion 125 2.5.1 Isolated bacteria 125 2.5.2 Antimicrobial activities 126 2.5.3 Pyrosequencing 127 2 2.5.4 Community analysis 128 2.5.5 Sponge-specific phylotypes 130 2.5.6 Linking taxonomy to function 134 2.6 Conclusion 135 2.7 Acknowledgements 136 2.8 References 137 3.0 Chapter 3: Archaea dominate the microbial communities in the marine sponge Inflatella pellicula in the deep sea as revealed by pyrosequencing 147 3.1 Abstract 148 3.2 Introduction 148 3.3 Materials & Methods 150 3.3.1 Sampling 150 3.3.2 Metagenomic DNA extraction from sponges 151 3.3.3 Metagenomic DNA extraction from seawater 151 3.3.4 PCR amplicon library preparation for pyrosequencing 151 3.3.5 Pyrosequencing data analysis 152 3.4 Results 153 3.4.1 Sequencing 153 3.4.2 Sequence classification 154 3.4.3 Relative abundances of archaea and bacteria 154 3.4.4 Sponge and seawater from 780 m 155 3.4.5 Sponges and seawater from 2900 m 160 3.4.6 Phylogeny of clustered sponge sequences 162 3.5 Discussion 165 3.5.1 Context of this study 165 3.5.2 Archaeal relative abundance and diversity 166 3.5.3 Bacterial diversity 167 3.5.4 Functional capabilities of sponge symbionts 169 3.6 Conclusions 170 3.7 Acknowledgements 171 3.8 References 172 3 4.0 Chapter 4: ‘Mining’ the metagenomes of marine sponges 179 4.1 Abstract 180 4.2 Introduction 181 4.3 Materials & Methods 183 4.3.1 Sponge sampling 183 4.3.2 Metagenomic DNA extraction from sponges 183 4.3.3 Polymerase chain reaction 184 4.3.3.1 Polyketide synthase PCR 184 4.3.3.2 Non-ribosomal peptide synthetase PCR 184 4.3.3.3 Laccase PCR 184 4.3.4 Cloning and sequencing of PCR amplicons 185 4.3.4.1 Cloning PCR products 185 4.3.4.2 M13 PCR 185 4.3.4.3 Sequencing and analysis of cloned PCR products 186 4.3.5 Large insert metagenomic clone library construction 186 4.3.5.1 Insert preparation – DNA fractionation 186 4.3.5.2 Insert preparation 187 4.3.5.3 Vector preparation 187 4.3.5.4 Ligation, phage packaging & transfection 188 4.3.6 Clone library functional screening 189 4.3.6.1 Antimicrobial activity screening 189 4.3.6.2 Laccase activity screening 190 4.3.6.3 Lipase activity screening 190 4.3.7 Fosmid analysis 191 4.4 Results 192 4.4.1 Polyketide synthase genes 192 4.4.2 Non-ribosomal synthetase genes 194 4.4.3 Laccase genes 196 4.4.4 Functional screening of clone library 198 4.4.5 Fosmid end-sequencing 201 4.5 Discussion 202 4.5.1 PKS and NRPS 202 4.5.2 Laccase genes and clone library screening 203 4 4.6 Acknowledgements 206 4.7 References 207 5.0 Chapter 5: Maribacter spongiicola sp. nov., and Maribacter vaceletii sp. nov., isolated from marine sponges and emended description of the genus Maribacter 216 5.1 Abstract 217 5.2 Introduction 217 5.3 Materials & Methods 218 5.3.1 Sampling & culture isolation 218 5.3.2 Phylogenetic analysis 219 5.3.3 Phenotypic & biochemical characterisation 220 5.4 Results 222 5.4.1 Phylogenetic analysis 222 5.4.2 Biochemical characterisation 224 5.4.3 Phenotypic characterisation 226 5.5 Description of novel Maribacter spp. sponge isolates and emended description of the genus Maribacter 228 5.5.1 Emended description of the genus Maribacter Nedashkovskaya et al ., 2004 emend. Nedashkovskaya et al ., 2010 228 5.5.2 Description of Maribacter spongiicola sp. nov. 228 5.5.3 Description of Maribacter vaceletii sp. nov. 230 5.6 Acknowledgements 231 5.7 References 232 6.0 General discussion 239 6.1 References 250 7.0 Appendix 259 Supplementary Table S2.1 260 Supplementary Table S3.1 274 8.0 Acknowledgements 288 5 Declaration: I, the undersigned, hereby declare that the work herein is my own and that it has not been submitted for any other degree, either at University College Cork, or elsewhere. _____________________ Stephen Jackson 6 Abstract 7 Marine sponges (phylum Porifera ) are the oldest extant metazoan animals on earth today and they host large populations of symbiotic microbes: Bacteria , Archaea and unicellular Eukaryota . Those microbes play various ecological functions which are essential to the health of the host. Their functions include carbon, nitrogen and sulfur cycling as well as defence of the host through the production of bioactive secondary metabolites which protect against infection and predation. The diversity of sponge- associated microbes is remarkable with thousands of OTUs reported from individual sponge species. Amongst those populations are sponge-specific microbes which may be specific to sponges or specific to sponge species. Sponges are a source of a vast array of chemical entities with many bioactive properties of interest to industry and pharmacology. While marine natural product discovery concerns many animal phyla, Porifera account for the largest proportion of novel compounds. Evidence suggests that many of these compounds of interest are the products of symbiotic microbes. Descriptions of sponge-associated microbial community structures has been greatly advanced by the development of next-generation sequencing technologies while the discovery and exploitation of sponge derived biocatalysts and bioactive compounds has increased due to developments in sequence-based and function-based metagenomics. Here we use pyrosequencing to describe the bacterial communities associated with two shallow, temperate water sponges namely Raspailia ramosa and Stelligera stuposa from Irish coastal waters and to describe the bacterial and archaeal communities from three individuals of a single sponge species ( Inflatella pellicula ) from two different depths in cold, deep waters in the Atlantic Ocean in Irish waters, including at a depth of 2900 m, a depth far greater than that of any previous sequence-based sponge-microbe investigation. We identified diverse microbial communities in all sponges and the presence of sponge-specific taxa recruiting to previously described sponge-specific clusters and also to novel sponge-specific clusters. We also identified archaeal communities which dominated sponge-microbe communities. We demonstrate that sponge-associated microbial communities differ from ambient seawater communities indicating host selection processes.