Abundance, Diversity, and Distribution of Aerobic
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ABUNDANCE, DIVERSITY, AND DISTRIBUTION OF AEROBIC ANOXYGENIC PHOTOTROPHIC BACTERIA IN THE DELAWARE ESTUARY by Lisa A. Waidner A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Marine Studies Spring 2007 Copyright 2007 Author All Rights Reserved UMI Number: 3267188 UMI Microform 3267188 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ABUNDANCE, DIVERSITY, AND DISTRIBUTION OF AEROBIC ANOXYGENIC PHOTOTROPHIC BACTERIA IN THE DELAWARE ESTUARY by Lisa A. Waidner Approved: _____________________________________________________ Nancy M. Targett, Ph.D. Dean of the College of Marine and Earth Studies Approved: _____________________________________________________ Carolyn A. Thoroughgood, Ph.D. Vice Provost for Research and Graduate Studies I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: _____________________________________________________ David L. Kirchman, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: _____________________________________________________ Thomas E. Hanson, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: _____________________________________________________ K. Eric Wommack, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: _____________________________________________________ Marcelino T. Suzuki, Ph.D. Member of dissertation committee ACKNOWLEDGEMENTS I would like to thank Louis Waidner and Phyllis Waidner for all their support in my education and life, from kindergarten all the way through the Ph.D. I couldn’t have done any of this work without them. I love you, Mom and Dad. To Dan Stentz, I extend my gratitude for an unending supply of understanding and patience in the last decade. Additionally, I would like to thank all my fellow Kirchman lab students, including Dawn Castle, Katie Preen, Rex Malmstrom, Paul Jones, Tiffany Straza, and Glenn Christman. A special “thank you” goes to Hila Elifantz. She was always available to listen to my presentations, critique my graphs, and remind me of the proper use of statistical tests. Most importantly, she is a great friend; I’ll never forget her. Vanessa Michelou is also a very special labmate, who helped me with sampling on multiple cruises, but also she offered her friendship. She always kept her home open to me whenever I needed to stay overnight in Lewes. LiYing Yu was always willing to share her expertise in molecular biology, and gave me her time even when she was busy with her own work. She and her husband Ming Xu have been kind friends, frequently inviting me to their house for wonderful feasts. To Matt Cottrell, I would like to offer thanks for all the advice and support for counting AAP bacteria, but also for his great sense of humor, even when the roof was leaking, and all the equipment in the lab became water-logged. Barb Campbell was always willing to engage in science discussions that were both illuminating as well as entertaining. She has been my mentor iv at CMES for the last six years, and I hold the utmost respect for her opinions and ideas. She and her husband, Jim Williams, have been very generous with friendship and hospitality at their home, and it has been a joy to watch Jason grow and mature. Kathy Coyne has been a great resource for qPCR advice; and professionally and personally, she has been a good friend. Of course, none of the sampling would have been possible without the professionalism and hard work of Bill Byam and the crew of the R/V’s Cape Henlopen and Hugh R. Sharp. I would like to extend a huge thanks to my committee members, who each provided important contributions to help formulate the ideas that went into this dissertation. To Eric Wommack, I am thankful for his expertise in microbial ecology, but also for letting me tag along on his MOVE cruises; they were a lot of fun. To Marcelino Suzuki, who is one of the world’s experts in quantifying microbes using qPCR, I thank for all the phone conversations, which were always been thought-provoking and fruitful. And to Tom Hanson, I’m especially grateful for his insights into anoxygenic phototrophy, but also for giving me some great ideas for my metagenomics and qPCR analyses. And last, but of course not least, I cannot thank Dave Kirchman enough. He has been very generous with his time, sharing his extensive knowledge of aquatic bacteria, writing styles, and data analysis. He took me under his wing and showed me the ropes on my first research cruise on the Henlopen. Dave and Ana have opened their home for some fantastic parties throughout the years, making the Kirchman lab group feel like family. v TABLE OF CONTENTS LIST OF TABLES………………………………………………………………….. viii LIST OF FIGURES………………………………………………………………… x ABSTRACT…………………………………………………………………………. xiii 1 INTRODUCTION…………………………………………………………………… 1 References…………………………………………………………………. 9 2 AEROBIC ANOXYGENIC PHOTOTROPHIC BACTERIA ATTACHED TO PARTICLES IN TURBID WATERS OF THE DELAWARE AND CHESAPEAKE ESTUARIES……………………………………………... 17 Abstract……………………………………………………………………… 17 Introduction…………………………………………………………………. 18 Materials and Methods……………………………………………………. 20 Sampling and environmental parameters………………………. 20 Microscopic detection of bChl a –containing cells……………… 21 Quantitative PCR of pufM…………………………………………. 22 Supplementary materials………………………………………….. 24 Results………………………………………………………………………. 25 Direct enumeration of bChl a-containing bacteria……………… 25 Particle-attached and free-living AAP bacteria…………………. 27 Abundance estimates by qPCR and microscopy………………. 30 Discussion………………………………………………………………….. 32 Acknowledgements………………………………………………………… 38 References…………………………………………………………………. 39 3 AEROBIC ANOXYGENIC PHOTOSYNTHESIS GENES AND OPERONS IN UNCULTURED BACTERIA IN THE DELAWARE RIVER…………. 54 Abstract……………………………………………………………………… 54 Introduction…………………………………………………………………. 55 Materials and Methods……………………………………………………. 57 Screening the fosmid library for photosynthesis operons……... 57 Sequencing and sequence analyses…………………………….. 58 Gene order of puf operon…………………………………………. 59 Accession numbers………………………………………………... 60 Supplementary materials………………………………………….. 60 Results and Discussion…………………………………………………… 61 Beta-proteobacterial fosmid clone……………………………….. 61 Alpha-3-proteobacterial fosmid clone……………………………. 63 Freshwater cluster of AAP beta-proteobacteria………………… 65 vi Gene order of the puf operon…………………………………….. 67 Conclusions………………………………………………………………… 69 Acknowledgements………………………………………………………… 70 References…………………………………………………………………. 71 4 DIVERSITY, DISTRIBUTION AND EXPRESSION OF DISTINCT ECOTYPES OF THE AEROBIC ANOXYGENIC PHOTOTROPH GENE, PUFM………………………………………………………………. 87 Abstract……………………………………………………………………… 87 Introduction…………………………………………………………………. 88 Materials and Methods……………………………………………………. 92 Sampling and environmental parameters……………………….. 92 pufM clone libraries………………………………………………… 93 Quantitative PCR of pufM amplicons……………………………. 96 Supplementary materials………………………………………….. 98 Results………………………………………………………………………. 99 Diversity and composition of Delaware estuary pufM………….. 99 pufM libraries……………………………………………………….. 102 pufM ecotypes……………………………………………………… 104 Particle-attached and free-living pufM ecotypes……………….. 106 Discussion………………………………………………………………….. 109 Acknowledgements………………………………………………………… 116 References…………………………………………………………………. 116 5 Conclusions………………………………………………………………………… 135 References…………………………………………………………………. 144 6 Appendices…………………………………………………………………………. 151 Appendix A………………………………………………………………….. 151 Appendix B………………………………………………………………….. 164 Appendix C………………………………………………………………..... 180 vii LIST OF TABLES Table 2.1 Average (± SE) prokaryote and AAP bacterial abundances in the Delaware estuary……………………………………………………………. 44 Table 2.2 Correlation of total and AAP bacterial abundances in whole water in the Delaware estuary……………………………………………………….. 45 Table 2.3 Comparison of two methods for separating free-living and particle- attached prokaryotes and AAP bacteria………………………………….. 46 Table 2.4 Average percentages (± SE) of cells associated with particles in the Delaware estuary……………………………………………………………. 47 Table 3.1 Analysis of open reading frames (ORFs) of beta-proteobacterial clone DelRiverFos06H03…………………….................................................... 77 Table 3.2 Analysis of open reading frames (ORFs) of alpha-3-proteobacterial clone DelRiverFos13D03…………………….......................................... 78 Table 3.3 Gene orders in puf operons of cultured and uncultured proteobacteria……………………………………………………................ 79 Table 4.1 Primers used in general pufM cloning and ecotype-specific