University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2012 Diversity and Activity of Roseobacters and Roseophage Charles Ryan Budinoff [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Bacteriology Commons, Biodiversity Commons, Bioinformatics Commons, Ecology and Evolutionary Biology Commons, Environmental Microbiology and Microbial Ecology Commons, and the Marine Biology Commons Recommended Citation Budinoff, Charles Ryan, "Diversity and Activity of Roseobacters and Roseophage. " PhD diss., University of Tennessee, 2012. https://trace.tennessee.edu/utk_graddiss/1276 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Charles Ryan Budinoff entitled "Diversity and Activity of Roseobacters and Roseophage." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Microbiology. Alison Buchan, Major Professor We have read this dissertation and recommend its acceptance: Steven W. Wilhelm, Erik Zinser, Mark Radosevich Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Diversity and Activity of Roseobacters and Roseophage A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Charles Ryan Budinoff May 2012 Copyright © 2011 by Charles Ryan Budinoff All rights reserved. ii DEDICATION I dedicate my dissertation to my maternal grandmother, for her unwavering love and support. iii ACKNOWLEDGEMENTS Dr. Alison Buchan, your mentorship was essential for the completion of this dissertation and of my doctoral degree. I have learned many things from you over the past four years. Your expertise in bacterial diversity and your knowledge of molecular biology were of great value. But the most important lesson I learned was how to be a good mentor and leader. Your extraordinary interpersonal skills and astounding work ethic allow you to effectively lead a large university laboratory. In my opinion, these are two of the most important traits of a supervisor. Through leading by example, you undoubtedly earn the respect of fellow faculty and staff, but also of every student in the department. I am honored to have been in your lab and know that the examples you have set will guide me for the rest of my life. Dr. Gary LeCleir, your friendship over the past decade cannot be summed in a few sentences. I will never be able to repay you for the help, advice, and laughter you gave to me, nor will I ever forget it. You are a person who never says ‘no’ to his friends. No matter how difficult the favor, I know you will be there with a big heart and a big smile. I hope to pay this forward by providing such guidance and friendship to others whenever I can. Kellina Morris, thanks for being by my side this past year. You listened to all my griping, all my lapses in confidence, and never let me think about giving up. Knowing that my graduation would bring us one step closer to each other was the extra motivation I needed. Your support has allowed us to close our first chapter and will be the springboard for our future together. Mary Hadden, thanks for keeping the lab in tip-top shape. I could not of had so many successful experiments without your hard work and devotion. iv ABSTRACT Bacteria of the Roseobacter lineage are dominant bacterioplankton in coastal systems and contribute significantly to secondary production in oceanic environments. Generalities of Roseobacter ecology, diversity, and distributions are known, but the intraspecific differences between species and their dynamics over short temporal periods is not well understood. Bacteriophage that infect Roseobacters (‘roseophage’) have the potential to shunt secondary production into the dissolved carbon pool and through the process of infection alter Roseobacter physiology. Despite their significance, little effort was made prior to the onset of this study to characterize roseophage. Using culture dependent and independent approaches, I describe the diversity and activity of Roseobacters and roseophage from two distinct coastal environments. Chapter 2 describes the development of an alternative method to enumerate viruses using epifluorescence microscopy that not only reduces sample processing costs, but also the total volume of sample required. A novel species of the Roseobacter lineage (Marivita roseacus) is proposed in Chapter 3. M. roseacus is unique in its needle-like morphology, forming long, relatively inflexible chains of cells. The Marivita genus is characterized by a distinct ecology, being closely associated with algae, resistant to grazing, and present in numerous marine and saline environments. Chapter 4 details the use of deep-amplicon sequencing (16S rDNA) to describe bacterial succession patterns during a mesocosm algal bloom, revealing the temporal dynamics of ~100 distinct phylotypes. A multivariate analysis showed that temporal portioning amongst the bacterial community was occurring at both high and low taxonomic levels. Chapter 5 details the isolation and genomic characterization of roseophage and describes their ecology using publically available metagenomic databases collected from throughout the world. Four distinct phage were isolated and sequenced including an N4-like strain, a novel Siphoviridae, and two temperate Podoviridae. The two temperate phage were practically identical at the nucleotide level, except for a 3000 bp putative replication module, which showed no homology between the two. Overall, this dissertation suggests that ecological partitioning within the Roseobacter lineage is occurring at and arguably below traditional species level taxonomic classifications and microdiversity amongst closely related marine bacteria is likely the norm rather than the exception. v TABLE OF CONTENTS Chapter 1 - Introduction ...................................................................................................... 1! References ....................................................................................................................... 5! Chapter 2 - A protocol for enumeration of aquatic viruses by epifluorescence microscopy using Anodisc™ 13 membranes ......................................................................................... 6! Abstract ........................................................................................................................... 7! Background ................................................................................................................. 7! Results ......................................................................................................................... 7! Conclusions ................................................................................................................. 8! Background ..................................................................................................................... 8! Results and Discussion ................................................................................................... 9! Construction of custom filter holders for 13 mm Anodisc membranes ...................... 9! Enumeration of VLP using 13mm Anodisc membranes .......................................... 10! Comparison of VLP counts using Anodisc membranes and evaluation of staining methods ..................................................................................................................... 11! Analysis of Nuclepore membranes ........................................................................... 12! Conclusions ................................................................................................................... 13! Methods......................................................................................................................... 13! Sample collection and preparation ............................................................................ 13! Enumeration of viruses using 25 mm Anodisc membranes ...................................... 13! Enumeration of viruses using Nuclepore membranes .............................................. 14! SEM imaging of Nuclepore membranes ................................................................... 15! Statistical comparison of virus counts from the Anodisc membranes ...................... 15! Acknowledgements ....................................................................................................... 15! References ..................................................................................................................... 16! Appendix ....................................................................................................................... 18! Tables ........................................................................................................................ 18! vi Chapter 3 - Marivita roseacus sp. nov., of the family Rhodobacteraceae, isolated from a temperate estuary