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UC San Diego UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title Chemical Ecology of the Marine Actinomycete Genus Salinispora Permalink https://escholarship.org/uc/item/46j900gw Author Patin, Nastassia Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Chemical Ecology of the Marine Actinomycete Genus Salinispora A dissertation submitted in partial satisfaction of the requirements for the Degree Doctor of Philosophy in Marine Biology by Nastassia Virginia Patin Committee in charge: Professor Paul R. Jensen, Chair Professor Eric Allen Professor Farooq Azam Professor Doug Bartlett Dr. Bianca Brahamsha Professor Pieter Dorrestein 2016 Copyright Nastassia Virginia Patin, 2016 All Rights Reserved ii The dissertation of Nastassia Virginia Patin is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ Chair University of California, San Diego 2016 iii DEDICATION For my mother iv EPIGRAPH The more I handled things and learned their names and uses, the more joyous and confident grew my sense of kinship with the rest of the world. --Helen Keller v TABLE OF CONTENTS SIGNATURE PAGE.…..………………..………….....…………….…………..........……………………….iii DEDICATION ....................................................................................................... iv EPIGRAPH ........................................................................................................ v TABLE OF CONTENTS ........................................................................................ vi LIST OF ABBREVIATIONS .................................................................................. ix LIST OF FIGURES ................................................................................................ x LIST OF TABLES ................................................................................................ xiii ACKNOWLEDGMENTS ..................................................................................... xiv VITA ..................................................................................................... xvi ABSTRACT OF THE DISSERTATION .............................................................. xviii CHAPTER 1 Introduction ..................................................................................... 1 1.1 Chemical ecology ............................................................................... 2 1.2 Microbial secondary metabolites ........................................................ 4 1.2.1 Siderophores ............................................................................. 5 1.2.2 Quorum sensing molecules ...................................................... 5 1.2.3 Antibiotics .................................................................................. 6 1.3 Actinomycetes .................................................................................... 8 1.4 The genus Salinispora ..................................................................... 10 1.5 Overview of the dissertation ............................................................. 10 1.6 References ....................................................................................... 12 CHAPTER 2 Competitive strategies differentiate two closely related species of marine Actinobacteria ............................................................................... 19 2.1 Abstract ............................................................................................ 20 2.2 Introduction ...................................................................................... 21 2.3 Methods ........................................................................................... 24 2.3.1 Sediment collection and processing ....................................... 24 2.3.2 Strain isolation ........................................................................ 25 2.3.3 DNA extraction, PCR, and 16S rRNA gene sequencing ........ 25 2.3.4 Direct challenge assays .......................................................... 27 2.3.5 Interference vs. exploitation competition assays .................... 28 2.3.6 Growth rates ........................................................................... 28 2.3.7 Chemical extraction and disc diffusion assays ....................... 29 2.3.8 LC-MS/MS and MALDI-TOF imaging mass spectrometry ...... 30 2.3.9 Statistical analyses ................................................................. 31 2.3.10 Genome sequencing and analysis ......................................... 32 2.4 Results ............................................................................................. 33 2.4.1 Strain isolation and identification ............................................ 33 2.4.2 Direct challenge assays .......................................................... 43 vi 2.4.3 Temporal variability ................................................................. 49 2.4.4 Interference vs. exploitation competition ................................ 52 2.4.5 Growth rates ........................................................................... 55 2.4.6 Bioinformatic analyses ............................................................ 57 2.4.7 Chemical analyses .................................................................. 59 2.5 Discussion ........................................................................................ 61 2.6 Acknowledgments ............................................................................ 67 2.7 References ....................................................................................... 68 CHAPTER 3 The role of inter-species interactions in Salinispora tropica secondary metabolite production .............................................................. 76 3.1 Abstract ............................................................................................ 77 3.2 Introduction ...................................................................................... 77 3.3 Methods ........................................................................................... 80 3.3.1 Strain selection and initial co-culture experiments .................. 80 3.3.2 Organic extracts and sensitivity testing .................................. 81 3.3.3 Time course experiments........................................................ 82 3.3.4 Vibrio sp. interaction ............................................................... 83 3.3.5 HPLC analysis ........................................................................ 85 3.3.6 LC-MS analysis and MS/MS molecular networking ................ 86 3.4 Results ............................................................................................. 88 3.4.1 Co-culture survey: inhibition and sensitivity ............................ 88 3.4.2 Co-culture survey: up regulation ............................................. 88 3.4.3 Time course experiment ......................................................... 93 3.4.4 MS/MS networking of co-culture and time course samples .... 97 3.4.5 Vibrio sp. co-culture .............................................................. 101 3.5 Discussion ...................................................................................... 105 3.6 Acknowledgments .......................................................................... 110 3.7 References ..................................................................................... 111 CHAPTER 4 Effects of actinomycete secondary metabolites on sediment microbial communities ............................................................................ 117 4.1 Abstract .......................................................................................... 118 4.2 Introduction .................................................................................... 119 4.3 Methods ......................................................................................... 122 4.3.1 Sediment collection ............................................................... 122 4.3.2 Sediment processing and DNA extraction ............................ 123 4.3.3 Library preparation and sequencing ..................................... 123 4.3.4 Sequence data processing ................................................... 126 4.3.5 Mesocosm experimental setup ............................................. 126 vii 4.3.6 Mesocosm sample processing and data analysis ................ 127 4.3.7 Bacterial sensitivity assays ................................................... 129 4.3.8 MS/MS molecular networking ............................................... 130 4.4 Results ........................................................................................... 131 4.4.1 Sediment communities ......................................................... 131 4.4.2 Mesocosm diversity .............................................................. 133 4.4.3 Mesocosm time course ......................................................... 135 4.4.4 Effects of extracts ................................................................
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