AN ABSTRACT OF THE THESIS OF Marcelino T. Suzuki for the degree of Doctor of Philosophy in Oceanography presented on October 14th 1997. Title: The Effect of Protistan Bacterivory on Bacterioplankton Community Structure. Redacted for privacy Abstract approved: Barry F. Sherr A series of experiments were performed to test the hypothesis that bacterivorous protists selectively feed on specific genotypes of marine bacterioplankton, affecting bacterioplankton community structure. A study comparing the bacterioplankton community diversity estimated by a SSU rDNA gene (SSU rDNA) clone library and a collection of cultivated bacteria from the same water sample suggests that bacterioplankton is dominated by organisms that do not grow easily in enrichmentcultures and are underrepresented in culture collections. Therefore, a new method, length heterogeneity analysis by PCR (LH-PCR) was developed to assess the community structure of in situ bacterioplankton communities. In LH-PCR, a region of the SSU rDNA which exhibits length variations among different phylogenetic groups isamplified from environmental DNA by PCR. Fragments originating from different organisms are discriminated by their length and quantified by the fluorescence emission of a labeled primer. Since the method is based on PCR, a study was performed to evaluate the introduction of bias by the reaction.Using pairwise mixtures of rDNAs I described reannealing bias, a new source of PCR bias by PCR. This bias is caused byself-inhibitionofPCR ampliconsthatattainelevated concentrations after several reaction cycles;thus templates with higher concentration in original gene mixtures tend to be underrepresented in products. This bias can be minimized by limitingfinal PCR product concentrations.I applied LH-PCR to assess changes in bacterioplankton communities in four protist exclusion experiments. Changes in the genotypic composition of the bacterial community of water samples with protists removed by filtration was followed and compared to the changes in community structure of control water samples.There were differences between filtered water samples and controls incubated for 24 to 48 hours. In the absence of bacterivores some SSU rDNAs that were insignificant in the original water samples dominated the bacterioplankton SSU rDNA pool after48hours. Protistsappearedtobecapableofcontrolling bacterioplankton taxonomic diversity under manipulated conditions.This supports the hypothesis that aquatic bacterioplankton communities are dominated by relatively inactive cells that are less susceptible to grazing by bacterivorous protists. The Effect of Protistan Bacterivory on Bacterioplankton Community Structure Marcelino 1. Suzuki A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed September14th,1997 Commencement June 1998 Doctor of Philosophy thesis of Marcelin.o T. Suzuki presented on October 14th1997 APPROVED: Redacted for privacy Co-Major Professot, representing Oceanography Redacted for privacy Co-Major Professor, representing Oceanography Redacted for privacy Dean of the College of Oceanic and Atmospheric Sciences Redacted for privacy Dean of Gradfjte School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted.. for privacy Marcelino I; ACKNOWLEDGMENTS First of all I would like to thank my major professors Drs. Barry and Evelyn Sherr for all the enthusiasm, patience and support in my seven years in Corvallis, I will always consider them as my American step-parents.I would also like to thank my co-major professor, Dr. Stephen Giovannoni, for all his support to this dissertation, and for introducing me to the molecular approach in microbial ecology, which is a major part of my dissertation. Thanks as well for my past and present committee members, p Dr. Pat Wheeler, Dr. Kate Field, Dr. Anne-Michelle Wood and Dr. Rebecca Donatelle. Many thanks go as well to all the people in the College of Oceanic and Atmospheric Sciences and Department of Microbiology, Irma Delson, Robin Hzlobeczy, Kelly Gibbs, Marylin Wallace and Donna Obert who saved my life so many times. I would also like to thank Dr. Charlie Miller, for his excellent lectures and discussions. Thanks to Dr. Dick Morita for such enjoyableandenlighteningdiscussions,andsuggestionsforthe Introduction on this thesis. Thanks as well to my lab. mates, in special to Nanci Adair for teaching me the basics molecular techniques, and my co- workers Mike Rappé and Zara Haimberger, it was sure lots of fun to work with you. I would like to also thanks all the people that made my stay in Corvallis so much more enjoyable. Thanks to my friends, most of whom are miles away from Corvallis for such good times:Luca Tallini, Axel Fabritius, Vasilis Zervakis, Ulrike Hahn and Iris Bechinger, just to name a few.Last, but not least, many thanks to my parents,for without their support for all those years, this thesis would have never have been possible. CONTRIBUTION OF AUTHORS Dr. Stephen Giovannoni was involved in the design, analysis and writing of the first three manuscripts. Dr. Michael Rappé was involved in the construction of the clone libraries, DNA sequence analysis and writing of two manuscripts. Zara W. Haimberger helped in the screening and mantainance of the culture collection, and in the DNA sequencing. Harry Winfield, Nanci Adair, JUrgen Ströbel helped in the isolation and screening of the culture collection. TABLE OF CONTENTS Page Introduction 1 Bacterioplankton Community Structure 1 Controls of Bacterioplankton Biomass 5 Controls of Bacterioplankton Production 8 Bacterioplankton Diversity 10 Molecular Methods for the Study of Bacterioplankton Taxonomic Community Structure 13 Environmental Clone Libraries of Small Subunit Ribosomal RNA genes 13 Oligonucleotide Hybridization 14 PCR-Based Methods 15 Controls of Bacterioplankton Diversity 18 Chapter 1. Bacterial Diversity Among SSU rDNA Gene Clones and Cellular Isolates from the Same Seawater Sample 21 Abstract Introduction 23 Material and Methods 25 Water samples 25 Isolation of cellular clones 25 Cell counts 27 SSU rRNA gene clone library 27 RFLP patterns: cellular clones 29 RFLP patterns: gene clones 30 TABLE OF CONTENTS (Continued) Page Sequencing: cellular clones 30 Sequencing: gene clones 31 Phylogenetic analyses 31 Accession numbers 32 Results 32 Discussion 44 Acknowledgments 47 References 47 Chapter 2. Bias Caused by Template Annealing in the Amplification of Mixtures of 16S rRNA Genes by PCR 52 Abstract 53 Introduction 54 Material and methods 57 Templates 57 PCR reaction conditions 59 PCR primers 59 Detection of PCR products 60 Kinetic models 61 Effect of the number of cycles 61 Results 61 519F11406R-HEX primer pair 62 27F-FAM/338R primer pair 63 Kinetic models 65 TABLE OF CONTENTS (Continued) Page Calculating the values of f and k 67 Discusssion 71 Acknowledgments 76 References 77 Chapter 3. Estimating Coastal Picoplankton Community Structure by LH- PCR 80 Abstract 81 Introduction 82 Material and Methods 84 Water samples 84 LH-PCR Clone library and Culture Collection 86 Length heterogeneity analysis of published sequences 87 Bias by PCR 87 Results 88 Predicted Length Heterogeneity of the 5' regions of 16S rRNAs 88 Analyses of Coastal Bacterioplankton Diversity 100 Kinetic bias in gene frequencies 105 LH-PCR 105 Discussion 107 Acknowledgments 110 References 110 TABLE OF CONTENTS (Continued) Page Chapter 4. The Effect of Protistan Bacterivory on Bacterioplankton Community Structure 113 Abstract 114 Introduction 115 Material and methods 117 Water samples 117 Protist Exclusion Experiment 1 118 Protist Exclusion Experiment 2 118 Protist Exclusion Experiment 3 and 4 119 Cell Counts 119 LH-PCR 120 Results 121 Cell Counts 121 LH-PCR 122 Discusssion 135 Acknowledgments 139 Literature cited 139 Conclusions Bias by PCR 144 LH-PCR 146 Bacterioplankton diversity 147 Controls of Bacterioplankton Community Structure 150 Bibliography 154 TABLE OF CONTENTS (Continued) Page Appendix 191 List of bacterial strains isolated from seawater and their SSU rDNA sequences. 192 WWW addresses of online databases searched 193 Culture collections-databases 193 Gene sequence databases 193 Abbreviations 193 LiST OF FIGURES Figure Page 1.1 HaellIRFLPpatterns common to cellular isolates and environmental geneclone SSU rDNAs ...................................................................................... 35 1.2. Taxonomic grouping of Oregon coast cellular clones A) and gene clones B), obtained by a comparison of partial (5' end) SSU rDNA sequences to sequences available through the RDP using the program SIMILARITY_RANK......................................................................................... 37 1.3. Phylogenetic tree generated by the neighbor-joining method from a mask of Ca. 200 nucleotide positions, showing the relationships between Oregon coast cellular clones (R2A) and gene clones (OCS) related to the gammaProteobacteria ........................................................................................ 41 1.4. Phylogenetic tree generated by the neighbor-joining method from a mask of Ca. 200 nucleotide positions, showing the phylogenetic relationships among Oregon coast cellular and gene clones within the alpha subdivision of the Proteobacteria.........................................................42 2.1. Locations of PCR primers and cleavage sites of the