UNIVERSITY of CALIFORNIA, SAN DIEGO Phylogenetic and Chemical
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UNIVERSITY OF CALIFORNIA, SAN DIEGO Phylogenetic and Chemical Diversity of Marine-Derived Actinomycetes from Southern California Sediments A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Oceanography by Alejandra Prieto-Davó Committee in charge: William Fenical, Chair Lihini Aluwihare Douglas H. Bartlett Paul R. Jensen Brian Palenik Christopher Wills 2008 The Dissertation of Alejandra Prieto-Davó is approved, and it is acceptable in quality and form for publication on microfilm: ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ ____________________________________ Chair University of California, San Diego 2008 iii DEDICATION To my father, who I have admired throughout my life and who has always been an example of strength and perseverance. ***** To my mother, who has taught me that a woman’s place is not in the kitchen and who has always supported all my crazy dreams. ***** To my husband Luis Alfonso, for showing me that love has no borders. Te amo. iv TABLE OF CONTENTS Signature Page………………………………………………………………….…..iii Dedication……………………………………………………………………….….iv Table of Contents……………………………………………………………………v List of Figures ……………………………………………………………………..vii List of Tables………………………………………………………………………..ix Acknowledgements……………………………………………………...……….….x Vita………………………………………………………………………...…....…xiv Abstract………………………………………………...…………...………….…..xv Chapter 1: Introduction: Marine Microbiology And Microbial Ecology...…..……..1 The Beginnings of Marine Microbiology ………………………………….1 Ocean processes and marine bacteria…………………………………….…2 The 16S rRNA gene: A new era for microbiology …………………………4 Marine Sediment Microbiology……………………………………………..8 Actinomycetes………………………………...……………………………11 Genomics and Metagenomics: An Exciting Future Has Already Dawned ………………………………………………….……….12 Chapter 2: Actinomycete Diversity In Marine Sediments ……..………….…...…15 Abstract……………...…………………………………………………….15 Introduction………....……………………………………………………..16 Materials and Methods…………………………………………………….18 v Results…………..…………………………………………………….……22 Discussion …………………………………….…………………………...30 Acknowledgements………………………………………………………...36 Chapter 3: Molecular Identification Of Streptomycetaceae and Micromonosporaceae In Offshore San Diego Sediments …………………………47 Introduction ………....……………………………………………………..47 Methods ………………………………………………...………………….50 Results …………..…………………………………………………….…...54 Discussion…………………………………….…………………………… 63 Acknowledgements………………………………………………………. ..71 Chapter 4: Screening Of Marine Derived Actinomycetes For The Production Of Biologically Active Compounds …………...…………………………………...90 Introduction…………………………………………………………………90 Methods……………………………………………………………………..94 Results………………………………………………………………………97 Discussion………………………………………………………………. ..104 Conclusions……………………………………………………….……….106 Acknowledgements………………………………………………………..107 Supplemental Information 1-2 References………………………………………………...…………...…………..113 vi LIST OF FIGURES CHAPTER 2 Figure 2.1 Number of seawater dependent, non-seawater dependent, and variable OTUs at consensus groups ranging from 97-100% for combined near-shore and offshore data sets.……………………….…...................................41 Figure 2.2a Neighbor-joining distance trees detailing the phylogenetic relationships among representatives of the OTUs (based on 98% sequence identity, 1378 bp) cultured from near-shore marine sediments........................……43 Figure 2.2b Neighbor-joining distance trees detailing the phylogenetic relationships among representatives of the OTUs (based on 98% sequence identity, 1378 bp) cultured from offshore marine sediments...........................……44 Figure 2.3a Observed number of OTUs (accumulation curve,) and calculated diversity estimates for near-shore actinomycete community..............................….45 Figure 2.3b Observed number of OTUs (accumulation curve,) and calculated diversity estimates for offshore actinomycete community...................................…45 Figure 2.4a Rank abundance curves for OTU diversity at 98% sequence identity from near-shore sediments.............. ......................………….……………46 Figure 2.4b Rank abundance curves for OTU diversity at 98% sequence identity from offshore sediments.......................................………….……….….…46 CHAPTER 3 Figure 3.1 Diversity estimators calculated for 26 Streptomycetaceae clone sequences ………………………………………………………………………..…72 Figure 3.2 Neighbor-joining phylogenetic tree of total Streptomyces diversity observed combining cultivation dependent and independent methods .…………………………………………………………………………..73 vii Figure 3.3 Streptomyces diversity estimated to be present on San Diego, CA marine sediments....................……………………………......…..76 Figure 3.4 Neighbor-joining phylogenetic tree (536 bp) of total Micromonosporaceae diversity observed combining cultivation dependent and independent methods ………………………..................................77 Figure 3.5 Diversity of Micromonosporaceae estimated to be present on San Diego, CA marine sediments..........…………..…………..…………..…...79 Figure 3.6 Neighbor-joining tree (560 bp) of cultured and cloned 16S rRNA gene sequences belonging to the Family Streptomycetaceae from five offshore samples and 16 offshore samples, respectively.........................81 Figure 3.7 Neighbor-joining phylogenetic tree of Streptomyces clones obtained from near-shore and offshore sediment samples...................................…84 Figure 3.8 Neighbor-joining phylogenetic tree (560 bp) of Streptomyces clones from different sections of a core sample.................................................…..87 CHAPTER 4 Figure 4.1 Neighbor-joining phylogenetic tree of the nearly complete (1354 bp) 16S rRNA gene from strains belonging to MAR2, MAR4 and CNQ-418........................................................................……………..……...108 Figure 4.2 Marinomycins A-C...........................................................................…110 Figure 4.3 Napyradiomycins from CNQ-525....................................................….111 Figure 4.4 Marinopyrroles A(1) and B(2).........................................................….112 viii LIST OF TABLES CHAPTER 2 Table 2.1 Marine sediment sampling sites, ocean depth, distance from shore, number of actinomycetes isolated, and the effects of seawater for growth.............................................................................…………...37 Table 2.2 Number of Operational Taxonomic Units (OTUs) recovered from near-shore (NS) and offshore (OS) marine sediments...................................38 Table 2.3 Generic affiliation and abundance of actinomycete strains and OTUs recovered from near-shore and offshore samples..................................39 Table 2.4 Actinomycete richness and diversity estimates based on 16S rRNA gene sequence data for San Diego samples ..........................................40 CHAPTER 3 Table 3.1 Overall comparison of Streptomycetaceae diversity observed with cultivation independent and cultivation dependent methods...........75 Table 3.2 Overall comparison of Micromonosporaceae diversity observed with cultivation independent and cultivation dependent methods...........80 Table 3.3a Comparison between Streptomyces clones from the top 5 cm of five offshore samples and cultivated Streptomyces strains from the top 5 cm of 16 offshore samples....................................................83 Table 3.3b Sample-specific comparison between Streptomyces clones and cultivated strains from five offshore samples....................................................83 Table 3.4 Comparison between Streptomyces OTUs from a near-shore and an offshore sediment sample. ......................................................................….86 Table 3.5 Comparison between Streptomyces OTUs from different sections of a core sample......................................................................................…89 ix ACKNOWLEDGEMENTS It would take another whole thesis to thank everyone that has made it possible for me to be where I am today, nevertheless there are a few people who were fundamental for accomplishing this great task. I must start by thanking my parents, who have always been incredibly supportive no matter how far I decide to go to follow my dreams. Secondly, I have to thank my husband Luis Alfonso, who since the day we met has been excited about my work and has encouraged me to keep going even in the most difficult times. Gracias Luis por todo este tiempo que me has apoyado y por todos los esfuerzos que has hecho por estar junto a mi, eres en verdad mi complemento, te amo. I would like to thank my sister Lolina and my brother Jorge, who have come to visit me so many times even though we live so far away from each other, you guys are the best, thank you for keeping me hanging in there and for making it easy to still be a family, I love you both. To the rest of my family, I will be eternally grateful for all your support. To my aunts: Gloria, Adriana and Anabella, thank you for so many family dinners and nights spent over, you have definitely made me feel at home. You are the best aunts anyone could ask for. I would also like to thank my new family, thank you Lulys and señora Lourdes, you have made me part of your family and you have always been there for me and Luis. Thank you Richi, Juan, Ricardo and señor Ricardo for being so much fun and so supportive of my work.