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Biodiversity of Actinomycetes Associated with Caribbean Sponges of Puerto Rico, and Their Metabolic Profiles

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Biodiversity of Actinomycetes Associated with Caribbean Sponges of Puerto Rico, and Their Metabolic Profiles BIODIVERSITY OF ACTINOMYCETES ASSOCIATED WITH CARIBBEAN SPONGES OF PUERTO RICO, AND THEIR METABOLIC PROFILES Jan Vicente Raczkowski A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Center for Marine Science University of North Carolina Wilmington 2010 Approved by Advisory Committee Wilson Freshwater Bongkeun Song Pamela Seaton Jeffrey L. Wright Chair Accepted by Dean, Graduate School TABLE OF CONTENTS ABSTRACT .........................................................................................................................v ACKNOWLEDGEMENTS .............................................................................................. vii LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ........................................................................................................... xi INTRODUCTION ...............................................................................................................1 Sponges and their associated micro biota ................................................................1 Actinomycetes: Distribution in terrestrial and marine environments ......................5 Actinomycetes in the marine environment and their association with marine sponges ..............................................................................................8 Actinomycetes are a diverse resource for the production of natural products .......12 Proposed study and objectives ..............................................................................17 Hypothesis..............................................................................................................19 METHODS ........................................................................................................................20 Site description and sponge collection ...................................................................20 DNA isolation and PCR from actinomycete isolates .............................................24 Sequencing and phylogenetic analysis...................................................................25 Chemical analysis of actinomycetes ......................................................................26 General experimental procedure for isolating frigocyclinone derivatives and dehydroquinones ....................................................................................................27 Purification of frigocyclinones from Streptomyces sp. M7-15 ..............................28 Purification of dihydroquinones from Streptomyces sp. M18-3 ............................29 Comparison of the chemical profile of Micromonospora aquatica isolated from sponges and marine sediments of Puerto Rico ..............................................30 iii Antibiotic and antifungal assay ..............................................................................30 Anticancer bioassay with SJCRH30 (rhabdomyosarcoma) ...................................31 RESULTS ...........................................................................................................................33 Distribution and phylogenetic analysis of actinomycetes associated with sediments and sponges of Puerto Rico...................................................................33 Isolation and structure elucidation of frigocyclinone derivatives ..........................52 Isolation of dihydroquinones from Streptomyces sp. M18_3 ................................78 Antibiotic and anticancer bioactivity of frigocyclinone and chloro-dihydroquinone Derivatives .......................................................................86 Metabolic Profile Analysis for Micromonospora aquatica Associated with Sediments and sponges of Puerto Rico ..................................................................90 DISCUSSION ....................................................................................................................95 Diversity of culturable actinomycetes in sponges and sediments of Puerto Rico.............................................................................................................95 Discovery of frigocyclinone derivatives in Streptomyces sp. M17_15 ...............100 Discovery of dihydroquinone derivatives from Streptomyces sp. M18_3 ...........104 Chemical profiles between Micromonospora aquatica strains associated to sponges and sediments of Puerto Rico ............................................................106 CONCLUSION ................................................................................................................109 LITERATURE CITED ....................................................................................................111 APPENDIX ......................................................................................................................118 iv ABSTRACT Marine actinomycetes provide a flourishing source of structurally unique and bioactive secondary metabolites that account for 75% of our antibiotics. Almost all of these products have come from land-based organisms and now attention is turning towards marine-derived species as a source of new structures and bioactivity. Reports to date indicate this group of bacteria are adaptable to a variety of environmental conditions, and can be isolated from multiple marine habitats with immense species diversity. Numerous genera of marine actinomycetes have been isolated from marine sediments and some strains have been isolated from several sponge species. Even so, the discovery of actinomycetes in Caribbean sponges remains underexplored. In this study, ten different species of Caribbean sponges and sediments were collected from four different locations in the coastal waters off Puerto Rico to examine diversity and bioactive compound production of marine actinomycetes in Caribbean sponges. A total of 180 actinomycetes, were isolated and identified based on 16S rRNA gene analysis. Phylogenetic analysis revealed the presence of at least 15 new phylotypes belonging to the genera Micromonospora, Verruscosispora, Streptomyces, Salinospora, Solwaraspora and Rhodococcus. Accordingly, 72 of the isolates had a100% identical sequences with Micromonospora aquatica. Despite having identical 16S rRNA sequences, when fermented, the majority of Micromonospora isolates from sponges produced a large diversity of antibiotic secondary metabolites in comparison to those that were isolated from the sediments. Sponges also showed a higher diversity of culturable actinomycetes than sediments. In addition, 17 antibiotic compounds have been purified and characterized from two Streptomyces spp. isolates, of which nine are new derivatives of frigocyclinone, a compound previously isolated from a Streptomyces griseus found in Antarctica (Bruntner et al., 2005). v ACKNOWLEDGEMENTS I would first like to thank my advisor Dr. Jeffrey Wright for making me realize how valuable one cubic centimeter of sponge can be to the field of microbiology and natural product chemistry. I would like to thank him for giving me the opportunity to design a multidisciplinary project that would satisfy both an ecological, and a biotechnological perspective with sponge associated bacteria. I would also like to extend my sincere gratitude to Mrs. Allison Stewart for teaching me everything that needs to be known about the biology, chemistry, and culturing techniques of actinomycetes. I also thank Dr. Ryan Van Wagoner for his intellectual and constructive guidance during the compound purification and characterization of the 17 metabolites isolated in this study. Thanks to the past and present Wright Lab members (Dr. Yanhui Meng, Dr. Bharat Bashyal, Dr. Rudy Ravindra, Mrs. Eve Wright, and Justin Isaacs) who have given me academic and moral support throughout this project. I am also greatly indebted for having guidance and support from my committee member Dr. Bongkeun Song, who taught me all the molecular techniques involved in this project. I also thank his lab members who welcomed me into their lab as one of their own. In particular, I extend my deep appreciation to Matthew Hirsch and Jennifer Bagwell for dedicating endless hours of their time to PCR troubleshooting, construction of phylogenetic trees, and statistical analyses of my data. I would like to thank my other committee members Dr. Wilson Freshwater, for assistance in constructing logical phylogenetic trees with my sequence library, and Dr. Pamela Seaton, for providing guidance in the structure elucidation of unknown compounds. A special thanks goes to Ms. Elizabeth Elliott for running the bioassay against human rhabdomyosarcoma SJCRH30 cells and Dr. Hugh Crews for calculating the median effective vi concentrations of these compounds. I also acknowledge Dr. Ron Sizemore for the use of lab space and instruments for the molecular aspects of this project. I am also indebted to Mr. Alex Méndez who made the travel arrangements and was responsible for all of the logistics involved in the very precarious voyage to Mona Island. I also would like thank my father, Dr. Vance P. Vicente who has introduced me to the marine sciences and has inspired me to overcome all of my challenges thus far. During the course of this project I was supported by Dr Wright‟s Carl B. Brown Trust Fund, and the state of North Carolina that provided funding to MARBIONC, a marine biotechnology initiative at UNCW. Additional support
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