UNIVERSITY of CALIFORNIA, SAN DIEGO Molecular Diversity And
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UNIVERSITY OF CALIFORNIA, SAN DIEGO Molecular diversity and adaptations of microorganisms from the deep ocean A dissertation submitted in partial satisfaction of the Requirements for the degree Doctor of Philosophy in Marine Biology by Emiley Ansley Eloe Committee in charge: Professor Douglas H. Bartlett, Chair Professor Eric E. Allen Professor Lihini I. Aluwihare Professor Farooq Azam Professor Kit Pogliano 2011 Copyright Emiley Ansley Eloe, 2011 All rights reserved. ii The Dissertation of Emiley Ansley Eloe is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ Chair University of California, San Diego 2011 iii DEDICATION For my family – Mama, Dad, Tommy And for my Doug With all my love, always. iv EPIGRAPH The reports of biologists are the measure, not of the science, but of the men themselves. John Steinbeck The Log from the Sea of Cortez v TABLE OF CONTENTS Signature Page…………………………………………………………………. iii Dedication………..……………………………………………………………. iv Epigraph……….………………………………………………………………. v Table of Contents……………………………………………………………… vi List of Abbreviations…………………………………………………..……… vii List of Figures………………………………………………………………… viii List of Tables……………………………………………………………….… xi Acknowledgements………………………………………………………..…. xii Vita…………………………………………………………………………… xv Abstract of the Dissertation.…………………………………………….…… xvi Chapter I Into the deep: An Introduction………………………………. 1 Chapter II Compositional differences in particle-associated and free-living microbial assemblages from an extreme deep-ocean environment…………………………………….. 15 Chapter III Going deeper: Metagenome of a hadopelagic microbial community…………………………………...…… 39 Chapter IV The elusive art of microbial cultivation……………………… 94 Chapter V Isolation and characterization of the first psychropiezophilic Alphaproteobacterium………………….. 124 Chapter VI The deep-sea bacterium Photobacterium profundum SS9 utilizes separate flagellar systems for swimming and swarming under high-pressure conditions…………………… 160 Chapter VII Deep perspectives: Concluding remarks…………………….. 172 vi LIST OF ABBREVIATIONS AABW Antarctic bottom water AT atmospheric pressure BLAST basic local alignment search tool Cm chloramphenicol COG cluster of orthologous groups DOC dissolved organic carbon DNA deoxyribonucleic acid EGS estimated genome size FACS Fluorescence-activated cell sorting HP high pressure HSP high-scoring pair HV high viscosity KEGG Kyoto Encyclopedia of Genes and Genomes Kn kanamycin MDA Multiple Displacement Amplification MPa Megapascal (0.101 MPa = 1 atmosphere) NADW North Atlantic deep water ORF open reading frame OTU operational taxonomic unit PCR polymerase chain reaction POM particulate organic matter Rif rifampicin RNA ribonucleic acid rRNA ribosomal RNA RT-PCR reverse transcription PCR tRNA transfer RNA vii LIST OF FIGURES Chapter II Figure 1 Rarefaction analysis for bacterial and archaeal ribosomal small-subunit rRNA libraries………………………………… 18 Figure 2 Heatmap of classified bacterial orders from the free-living and particle-associated libraries……………………………… 19 Figure 3 UPGMA hierarchical clustering based on (A) phylum-level and (B) OTU-level groupings………………………………… 22 Figure S1 Coverage plots for the bacterial and archaeal libraries……… 26 Figure S2 Bacterial phylogenetic trees depicting the relationship of the particle-associated and free-living sequences from the Puerto Rico Trench……………………………………… 27 Figure S3 Archaeal phylogenetic tree depicting the relationship of the particle-associated and free-living sequences from the Puerto Rico Trench……………………………………… 31 Figure S4 Eukaryal phylogenetic trees depicting the relationship of (A) Opisthokonta and (B) non-Opisthokonta sequences from the Puerto Rico Trench……………………. 32 Figure S5 Heatmap of relative abundances of bacterial phyla for deep ocean datasets……………………………………... 34 Chapter III Figure 1 Phylogenetic distribution of partial SSU ribosomal genes…. 77 Figure 2 Phylogenetic distribution of PRT protein sequences based on APIS classifications……………………………..... 78 Figure 3 Cluster analysis of (A) COG categories and (B) KEGG pathways within each metagenome………………………… 79 Figure 4 Metagenomic profile comparisons of COG families for the PRT and Sargasso Sea metagenomes……………….. 80 Figure 5 Transporter family distribution……………………………… 81 viii Figure 6 Fragment recruitment for the PRT metagenome compared to the GS00d metagenome………………………. 82 Figure S1 Metagenomic profile comparison of COG families for the PRT and HOT4000 metagenomes…….…………….. 83 Figure S2 Abundance of the functional OG category Signal Transduction (T)……………………….…………….. 84 Figure S3 Abundance of the functional OG category Transcription (K)……………………….…….…………….. 85 Figure S4 Abundance of the functional OG category Inorganic ion transport and metabolism (P)………………….…………….. 86 Figure S5 Relative abundance of COG categories and distribution within phylum-level groupings based on APIS…….……….. 87 Chapter IV Figure 1 Phylogenetic novelty: The case for ‘Candidate’ divisions…. 119 Figure 2 The ‘cultivation landscape’…….…………………………… 120 Chapter V Figure 1 Growth of PRT in natural seawater medium……………….. 151 Figure 2 Morphological features of PRT1…………………………… 152 Figure 3 Phylogenetic placement of PRT1 within the Roseobacter clade…………………………………………… 153 Figure 4 Interpolated contour diagram of the temperature-pressure dependence of the exponential growth rate constant (k)……. 154 Figure S1 Phylogenetic placement of the PRT1 g5 GTA protein……... 158 Chapter VI Figure 1 Polar (A) and lateral (B) flagellar gene clusters in P. profundum SS9……………………………………….. 162 Figure 2 Growth-based 0.3% agar bulb assay to qualitatively assess motility for P. profundum strain 3TCK and SS9…………… 164 ix Figure 3 Growth-based agar bulb assay to qualitatively assess motility at high hydrostatic pressure and high viscosity……. 164 Figure 4 Flagella visualized with NanoOrange staining for P. profundum SS9 and deletion mutants……………………. 165 Figure 5 Flagellin B increases under conditions of high pressure and increased viscosity……………………………………… 166 Figure 6 Swimming velocity as a function of increasing hydrostatic pressure for three strains……………………………………. 166 x LIST OF TABLES Chapter II Table 1 Chemical and biological constituents of unfiltered surface (1 m) and hadal (6,000 m) seawater………………… 17 Table 2 Similarity-based OTUs and species richness estimates……... 18 Table 3 Eukaryal classifications for partial 18S ribosomal gene sequences……………………………………………… 20 Chapter III Table 1 General features of the metagenomic dataset from the Puerto Rico Trench……………………………………… 73 Table 2 Functional annotations for non-redundant proteins for the PRT metagenome and comparison metagenomes………. 74 Table 3 Single cell sequencing, assembly, and annotation statistics… 75 Table 4 Comparison of single cell phylogeny to closest sequenced isolate genome…………………………………… 76 Table S1 Detailed assembly and putative contaminant statistics for single-cell genomes……………………………………… 88 Chapter IV Table 1 Single-cell technologies implemented for cultivation……….. 121 Chapter V Table 1 Growth properties of piezotolerant and piezophilic isolates… 155 Chapter VI Table 1 Bacterial strains and plasmids used in this study…………… 163 Table S1 Primer sequences used in this study………………………… 169 Table S2 Swimming velocity as a function of increasing hydrostatic pressure………………………………………… 170 xi ACKNOWLEDGEMENTS I am indebted to countless individuals with whom I have shared this adventure over the past few years. I have been exceedingly fortunate to work alongside superb scientists as well as dear friends. I would first like to sincerely thank Doug Bartlett for giving me the independence to develop as a critical thinker, the guidance and financial security when I needed support, and a true sense of what it means to be a scientist. His uncanny optimism and dedication have helped to inspire and inform my research. I would also like to sincerely thank my committee members, Eric Allen, Lihini Aluwihare, Farooq Azam, and Kit Pogliano. They have all welcomed me into their labs and provided invaluable insight in the construction of this body of research. It was an honor to learn from such a phenomenal group of scientists. Thanks to past and present members of the illustrious Bartlett lab, particularly Federico Lauro, Taylor Stratton, Ian Kerman, Lauren Franco, and Angeliki Marietou. I would like to thank Kevin Hardy for constructing and allowing me to use his ‘pick-up truck’ to the deep sea. I would also like to thank Roger Chastain, the guardian of all pressure equipment. Many thanks as well to past and present members of the Azam and Allen labs for technical assistance, helpful advice, and riveting discussions. I am grateful for the many collaborators I have had the pleasure to work with, particularly Shannon Williamson, Rudi Vogel, Shibu Yooseph, and Roger Lasken. Instrumental in these collaborations were Mark Novotny, Mary-Jane Lombardo, Joyclyn Yee-Greenbaum, Lisa Zeigler Allen, Maria Kim, and Jennifer Gutierrez. xii I would like to particularly thank my dear friends Francesca Malfatti, Christine Shulse, and Nellie Shaul for being both my scientific and personal advocates. It is with all my heart that I