Old Dominion University ODU Digital Commons OES Theses and Dissertations Ocean & Earth Sciences Spring 2019 Coupling Metaproteomics with Taxonomy to Determine Responses of Bacterioplankton to Organic Perturbations in the Western Arctic Ocean Molly P. Mikan Old Dominion University, [email protected] Follow this and additional works at: https://digitalcommons.odu.edu/oeas_etds Part of the Biogeochemistry Commons, Biology Commons, Microbiology Commons, and the Oceanography Commons Recommended Citation Mikan, Molly P.. "Coupling Metaproteomics with Taxonomy to Determine Responses of Bacterioplankton to Organic Perturbations in the Western Arctic Ocean" (2019). Doctor of Philosophy (PhD), Dissertation, Ocean & Earth Sciences, Old Dominion University, DOI: 10.25777/p8gg-tr58 https://digitalcommons.odu.edu/oeas_etds/85 This Dissertation is brought to you for free and open access by the Ocean & Earth Sciences at ODU Digital Commons. It has been accepted for inclusion in OES Theses and Dissertations by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. COUPLING METAPROTEOMICS WITH TAXONOMY TO DETERMINE RESPONSES OF BACTERIOPLANKTON TO ORGANIC PERTURBATIONS IN THE WESTERN ARCTIC OCEAN by Molly P. Mikan B.S. December 2004, Colorado State University M.S. December 2012, North Carolina State University A Dissertation Submitted to the Faculty of Old Dominion University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY OCEANOGRAPHY OLD DOMINION UNIVERSITY May 2019 Approved by: H. Rodger Harvey (Director) Brook L. Nunn (Member) P. Dreux Chappell (Member) David Gauthier (Member) ABSTRACT COUPLING METAPROTEOMICS WITH TAXONOMY TO DETERMINE RESPONSES OF BACTERIOPLANKTON TO ORGANIC PERTURBATIONS IN THE WESTERN ARCTIC OCEAN Molly P. Mikan Old Dominion University, 2019 Director: Dr. H. Rodger Harvey Understanding how the functionality of marine microbial communities change over time and space, and which taxonomic groups dominate distinct metabolic pathways, are essential to understanding the ecology of these microbiomes and the factors contributing to their regulation of elemental cycles in the oceans. The primary goal of this dissertation was to investigate the community metabolic and taxonomic responses and the degradation potential of two compositionally distinct marine microbiomes within the shallow shelf ecosystem of the Chukchi Sea after rapid fluctuations in algal organic matter availability. Novel bioinformatics tools were collaboratively developed and used together with community proteomics (metaproteomics) to characterize and quantify changes in bacterial community functioning and taxonomic composition over time. 16S rRNA sequencing was employed to confirm bacterial taxonomic dynamics. These approaches were linked to particulate analyses for lipids and amino acids in order to track temporal changes in organic substrate composition. Results obtained using these improved methodological standards and the multidisciplinary approach demonstrated that organic perturbations within these systems stimulated changes to microbial taxonomic composition and functionality. The removal of organic particles seen within the control initiated a divergence between the two microbiomes while substrate abundance, as algal inputs, led to a convergence in community function. Despite the functional and taxonomic overlap seen as dominant features characterizing the responses to rapid influxes of algal organic matter, unique metabolic traits differentiated the major bacterial groups of each microbiome. This was most apparent in the recycling of nitrogen and carbon as well as substrate acquisition, suggesting that conditions which select for certain bacterial groups in the western Arctic Ocean may impact local chemical gradients. The large dataset of information obtained from this dissertation provides insight into the timing and characterization of Arctic bacterial community responses to environmental perturbations and in turn how they influence changes in substrate composition through selective degradation of labile lipid classes. In addition, this work demonstrates the applicability of trait-based methodologies to inform on how environmental conditions may drive niche formation within complex microbial communities. iv Copyright, 2019, by Molly P. Mikan, All Rights Reserved. v This dissertation is dedicated to my husband, daughter and son, and to my parents. This finished body of work would never have been completed without their continuous love, support and patience. vi ACKNOWLEDGMENTS I express sincere appreciation for everyone that contributed to the design, analysis and completion of this dissertation. In particular, I am especially grateful for my academic advisor, H. Rodger Harvey, whose sustained support and kindness guided me throughout the many segmented stages of this dissertation. Collaboration with a group of talented scientists from the University of Washington, Genome Sciences Department (Emma Timmins-Schiffman, Michael Riffle, Damon H. May, William S. Noble and Brook L. Nunn) was essential for the development of bioinformatics tools vital to my research. This group demonstrated that a gathering of the minds and fearless approach to tackle inherent limitations within a scientific discipline can result in powerful outcomes; through this collaboration, we raised the standards for applying metaproteomic techniques to complex and uncultured natural bacterial communities. Additionally, Ian Salter assisted with experimental design, collection of samples and long hours of filtering seawater while at sea aboard the USCGC Healy, all while providing a dose of good humor. Lab work completed for this research was possible because of the expertise of so many different people, including Brook Nunn (metaproteomics and tandem mass spectrometry), Ian Salter (16S rRNA sequencing), Fred Dobbs (microbial techniques) and many members of the Harvey lab group (amino acids and lipids analysis). Last, but not least, a thank you to my academic committee members (H. Rodger Harvey, P. Dreux Chappell, Dave Gauthier and Brook L. Nunn), who provided important feedback that improved this final product. vii NOMENCLATURE Functional traits: “Morphological, physiological, phenological, or behavioral features measured on organisms that can ultimately be linked to their performance.” Violle et al. [1] Functional-trait ecology: An ecosystem-based approach recognizing that “the health of an ecosystem may depend not only on the number of species present, but also on the diversity of their traits.” Cernansky [2] Functional redundancy: “The ability of one microbial taxon to carry out a process at the same rate as another under the same environmental conditions.” Allison and Martiny [3] Strict functional redundancy: “The coexistence of organisms that share the exact same set of functions and that can readily replace each other.” Galand et al. [4] Metabolic plasticity: “The potential [of bacterioplankton populations] to achieve similar ecosystem process rates [in response to environmental disturbances].” Lindh et al. [5] Community resilience: “The rate at which microbial composition returns to its original composition after being disturbed.” Allison and Martiny [3] Community resistance: “The degree to which microbial composition remains unchanged in the face of a disturbance.” Allison and Martiny [3] Replacement effect: “Replacement of OTUs, leading to changes in community composition and functioning.” Lindh et al. [5] Priming effect: “Short-term changes in the turnover rate of [soil] organic matter induced by the addition of carbon and/or nutrients [to soil].” Bird et al. [6] Insurance hypothesis: “Any long-term effects of biodiversity that contribute to maintain or enhance ecosystem functioning in the face of environmental fluctuations.” Yachi and Loreau [7] viii TABLE OF CONTENTS Page LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ...................................................................................................................... xii Chapter 1. INTRODUCTION AND OVERVIEW ...................................................................................... 1 2. METAPROTEOMICS REVEAL THAT RAPID PERTURBATIONS IN ORGANIC MATTER INPUTS STIMULATE FUNCTIONAL RESTRUCTURING IN ARCTIC OCEAN MICROBIOMES ............................................................................................................................ 6 2.1 Introduction ........................................................................................................................... 6 2.2 Methods ................................................................................................................................. 8 2.2.1 Seawater sample collection ............................................................................................ 8 2.2.2 Shipboard incubation set-up with organic amendments ............................................... 11 2.2.3 16S rRNA: DNA extraction, library construction, and sequencing ............................. 11 2.2.4 16S rRNA: Bioinformatic and Statistical Analyses ..................................................... 14 2.2.5 Metagenomics: sample preparation and data analysis .................................................. 14 2.2.6 Metaproteomics: sample preparation and data analysis ............................................... 15 2.2.7 Peptide-based