AN INVESTIGATION INTO THE BACTERIAL DIVERSITY ASSOCIATED WITH SOUTH AFRICAN LATRUNCULID SPONGES THAT PRODUCE BIOACTIVE SECONDARY METABOLITES A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY RHODES UNIVERSITY TARA AISLING WALMSLEY FEBRUARY 2013 „All are but parts of one stupendous whole, whose body Nature is, and God the soul‟ Tara A. Walmsley PhD Thesis i TABLE OF CONTENTS List of Figures ........................................................................................................ vi List of Tables ...................................................................................................... xiii List of Abbreviations ............................................................................................. xiv Acknowledgements .............................................................................................. xvi Abstract .................................................................................................... xviii Chapter One ........................................................................................................ 1 Literature Review ................................................................................................... 1 1.1 Sponge taxonomy ......................................................................................... 3 1.2 Sponges as the dominant source of biologically active marine natural products .............................................................................................................. 4 1.2.1 Psammaplin A ........................................................................................ 5 1.2.2 Spermatinamine ..................................................................................... 6 1.2.3 Crambescidin 800 .................................................................................. 7 1.2.4 Candidaspongiolide ................................................................................ 8 1.2.5 Bengamides ........................................................................................... 9 1.2.6 Halichondrin B ...................................................................................... 10 1.2.7 Pyrroloiminoquinones ........................................................................... 12 1.3 Diversity of sponge-associated microorganisms ......................................... 17 1.3.1 Sponge-associated microbial communities – a product of habitat or maternal instinct? .......................................................................................... 19 1.3.2 Sponge-bacteria symbiosis .................................................................. 20 1.4 The biotechnological potential of sponge associated microorganisms ....... 23 1.4.1 Theonella swinhoei ............................................................................... 25 1.4.2 Manzamine A ....................................................................................... 27 Table of Contents 1.4.3 Genomic approaches for the production of secondary metabolites associated with sponge symbionts ................................................................ 29 1.5 The sponge genus Tsitsikamma, a source of biologically active pyrroloiminoquinone metabolites and novel bacterial communities .................. 31 1.6 Hypothesis .................................................................................................. 39 1.7. Research Objectives .................................................................................. 39 1.8 References ................................................................................................. 40 Chapter Two ...................................................................................................... 53 Sponge Identification and Chemical Characterisation .......................................... 53 2.1 Introduction ................................................................................................. 53 2.2 Methods and Materials ................................................................................ 55 2.2.1 Sponge collection ................................................................................. 55 2.2.2 Morphological identification of sponge specimens ............................... 56 2.2.3 Total genomic DNA extraction from sponge tissue............................... 56 2.2.4 General PCR amplification protocol ..................................................... 57 2.2.5 Molecular identification and phylogenetic analysis of sponge species . 57 2.2.6 Isolation of the major secondary metabolite from a batch collection of T. favus.............................................................................................................. 58 2.2.7 NMR stack plot comparison of a crude extract of T. favus specimens Sp01, Sp02 and Sp03 ................................................................................... 60 2.2.8 NMR stack plot comparison and mass spectrometry analysis of a crude extract of T. scurra and T. favus .................................................................... 60 2.3 Results ........................................................................................................ 61 2.3.1 Sponge taxonomy ................................................................................ 61 2.3.2 Molecular Identification of sponges using the COX1 and 28S rRNA gene fragments ...................................................................................................... 62 Tara A. Walmsley PhD Thesis iii 2.3.3 Isolation and characterisation of the major secondary metabolite in Tsitsikamma favus ........................................................................................ 65 2.3.4 NMR and LCMS analysis of the secondary metabolite profile of T. favus and T. scurra specimens ............................................................................... 67 2.4 Discussion .................................................................................................. 70 2.5 References ................................................................................................. 73 Chapter Three ..................................................................................................... 75 Diversity and population structure of Tsitsikamma favus- associated bacteria ..... 75 3.1 Introduction ................................................................................................. 75 3.2 Materials and Methods ................................................................................ 76 3.2.1 Extraction of total genomic DNA .......................................................... 76 3.2.2 Primers used in the 16S rRNA molecular analysis ............................... 76 3.2.3 DGGE analysis of bacterial 16S rRNA gene amplicons ....................... 77 3.2.4 Preparation and sequencing of 16S rRNA clonal libraries derived from T. favus ......................................................................................................... 77 3.2.5 Phylogenetic analysis of the 16S rRNA clonal libraries of T. favus associated bacteria ....................................................................................... 78 3.2.6 454 pyrosequencing analysis of the T. favus-associated 16S rRNA amplicons ...................................................................................................... 78 3.3 Results ........................................................................................................ 80 3.3.1 DGGE analysis of sponge-associated bacterial diversity ..................... 80 3.3.2 16S rRNA clone library analysis of sponge-associated bacterial communities .................................................................................................. 82 3.3.3 Direct comparison of T. favus DGGE profiles with the dominant 16S rRNA gene clones ......................................................................................... 86 3.3.4 454 pyrosequencing analysis of the bacterial community associated with T. favus ......................................................................................................... 88 3.4 Discussion .................................................................................................. 92 Table of Contents 3.5 References ................................................................................................. 95 Chapter Four ...................................................................................................... 99 Comparing the Bacterial Diversity associated with Algoa Bay Latrunculid Sponges ...................................................................................................... 99 4.1 Introduction ................................................................................................. 99 4.2 Methods and Materials .............................................................................. 100 4.2.1 Isolation of total genomic DNA followed by DGGE and NGS analysis of sponge-associated 16S rRNA amplicons .................................................... 100 4.3 Results ...................................................................................................... 102 4.3.1 Comparison of the bacterial communities associated with Algoa Bay Tsitsikamma and Latrunculia sponges using DGGE profiling...................... 102 4.3.2 454 pyrosequencing analysis of the bacterial communities associated with Algoa Bay Latrunculid sponges ..........................................................