AN ABSTRACT OF THE DISSERTATION OF H. James Tripp for the degree of Doctor of Philosophy in Molecular and Cellular Biology presented on June 7, 2007. Title: Genomic-Assisted Determination of the Natural Nutrient Requirements of the Cosmopolitan Marine Bacterium ‘Candidatus Pelagibacter ubique’ Abstract approved: Stephen J. Giovannoni To identify factors limiting ‘Candidatus Pelagibacter ubique’ maximum cell density and growth rate in pure culture on seawater, the genome sequence of ‘Cand. P. ubique’ was analyzed, culturing experiments with organic and inorganic nutrient additions were made, and radiotracer uptake experiments were performed. The genome was sequenced, custom data mining tools were developed, and all major biosynthetic and energy pathways were reconstructed from a genome annotation. Analysis of the genomic data suggested ‘Cand. P. ubique’ might be limited by the availability of reduced sulfur, since genes cysDNCHIJ, used for assimilatory sulfate reduction, were missing. When reduced sulfur in the form of 3-dimethylsulfoniopropionate (DMSP), methionine, or cysteine was added to filtered, autoclaved seawater containing all other nutrients in excess, maximum cell yield increased an order of magnitude. But, increasing DMSP additions from 50 nM to 10 μM did not lead to higher cell densities at a normal growth rate; instead, cultures increased in cell density from 3.03 ± 106 cells ml-1 to 2.1 ± 107 cells ml-1, at a growth rate of 0.110 d-1 instead of 0.664 d-1. An additional hypothesis was that, due to the inefficiency with which threonine degradation to glycine compensates for missing serBC genes, and the presence of a glycine-activated riboswitch on malate synthase, the growth rate using DMSP as a sole sulfur source would be improved by addition of glycine betaine because glycine can be used as a methyl acceptor to regenerate methyl carriers needed to demethylate DMSP. When glycine betaine was added to filtered, autoclaved seawater containing DMSP and all other nutrients in excess, growth rate increased. ©Copyright by H. James Tripp June 7, 2007 All Rights Reserved Genomic-Assisted Determination of the Natural Nutrient Requirements of the Cosmopolitan Marine Bacterium ‘Candidatus Pelagibacter ubique’ by H. James Tripp A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented June 7, 2007 Commencement June 2008 Doctor of Philosophy dissertation of H. James Tripp presented June 7, 2007. APPROVED: Major Professor, representing Molecular and Cellular Biology Director of the Molecular and Cellular Biology Program Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. H. James Tripp, Author ACKNOWLEDGEMENTS The ambitious scope and scientific relevance of this project are due to the vision of my Principal Investigator and Major Professor, Stephen Giovannoni, who graciously afforded me the opportunity in mid-life to launch a second career in functional genomics. The help and interest of all co-authors is gratefully acknowledged. The author also acknowledges helpful discussions on microbial physiology, oceanography, and microbial ecology with all past and present members of the Giovannoni lab, particularly Ulrich Stingl, Alexander Treusch, Olivia Mason, and Hyun-Myung Oh. Corroborating proteomic, genomic, and metagenomic data was shared by lab members Sarah Sowell and Daniel Smith. Training in SAR11 cultivation techniques, radiotracer assays, and microbiological molecular techniques came from Jang-Cheon Cho and Kevin Vergin. Joshua Kitner worked tirelessly on the final stages of culturing experiments confirming bioinformatic predictions. Scott Givan provided copious bioinformatic support including software training, design, development, maintenance, installation and enhancement. Ron Kiene provided 35S-DMSP and John Dacey provided unlabelled DMSP. The author gratefully acknowledges funding from Diversa Corporation and the Gordon and Betty Moore Foundation. I must also thank my wife Shelley for the sacrifices that came from consenting to this major realignment of my career and for her faithful and unflagging support for the effort it took to complete it. CONTRIBUTION OF AUTHORS Dr. Stephen J. Giovannoni conceptualized this project and was consulted throughout the course of all the work done. Dr. Michael S. Schwalbach reviewed manuscripts and experimental designs. Joshua B. Kitner assisted in data collection for radiotracer uptake studies and cell cultivation and counting. Larry J. Wilhelm provided bioinformatics support. Daniel Smith provided nucleotide sequences for analysis. Dr. John Dacey provided unlabeled DMSP and data on DMSP concentrations in the ocean. TABLE OF CONTENTS Page 1. INTRODUCTION ....................................................................................................1 1.1 Difficulties and Special Issues Studying SAR11 Metabolism..........................6 1.2 Pre-genomic Nutrient Studies...........................................................................8 1.3 Genome Sequencing and Metagenomics........................................................10 1.4 Metabolic Reconstructions..............................................................................12 2. METABOLIC RECONSTRUCTIONS FOR MAJOR BIOSYNTHETIC AND ENERGY PATHWAYS IN ‘CANDIDATUS PELAGIBACTER UBIQUE’ strain HTCC1062......................................................................................................................15 2.1 Abstract...........................................................................................................16 2.2 Introduction.....................................................................................................16 2.3 Nutrient Requirements of ‘Cand. P. ubique’ ..................................................19 2.4 Central Metabolism.........................................................................................20 2.4.1 Glycolysis/Gluconeogenesis.......................................................................21 2.4.2 Pentose Phosphate Cycle ............................................................................22 2.4.3 Energy Generation......................................................................................23 2.5 Major Biosynthetic Pathways .........................................................................25 2.5.1 Anaplerotic Sequences................................................................................26 2.5.2 Nucleotide Biosynthesis..............................................................................28 2.5.3 Amino Acid Biosynthesis ...........................................................................28 2.5.4 Fatty Acid Biosynthesis..............................................................................36 2.5.5 Cell Wall Biosynthesis................................................................................37 2.5.6 D-amino acid Isomerization........................................................................37 2.5.7 Aminosugar Metabolism.............................................................................38 2.5.8 Peptidoglycan Biosynthesis........................................................................38 2.5.9 Heme Biosynthesis......................................................................................39 2.5.10 Ubiquinone Biosynthesis............................................................................40 2.5.11 Vitamin biosynthesis...................................................................................40 2.6 Type II Secretion Type IV Pili........................................................................45 2.6.1 Horizontal Gene Transfer and Cosmopolitan Distribution of pil Genes ....45 2.6.2 Uptake Signal Sequence .............................................................................46 2.7 Conclusions.....................................................................................................48 TABLE OF CONTENTS (continued) Page 3. PHYTOPLANKTON LYSATES SUPPLY COMPOUNDS THAT ARE NEEDED BY ‘CANDIDATUS PELAGIBACTER UBIQUE TO ATTAIN HIGHER CELL DENSITIES IN SEAWATER .............................................................................82 3.1 Abstract...........................................................................................................83 3.2 Introduction.....................................................................................................83 3.3 Materials and Methods....................................................................................84 3.3.1 Preparation of Phytoplankton Lysate..........................................................84 3.3.2 Fractionation procedures.............................................................................85 3.4 Results and Discussion...................................................................................86 3.5 Figures.............................................................................................................88 4. SAR11 MARINE BACTERIA REQUIRE EXOGENOUS REDUCED SULPHUR FOR GROWTH...........................................................................................91 4.1 Abstract...........................................................................................................92 4.2 Introduction.....................................................................................................92