Graduate Thesis Or Dissertation

 

Single-Cell Genomics of Uncultivated Marine Bacteria Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/s7526g485

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  • Historically, the difficulty of obtaining pure cultures of abundant marine microbial plankton has an obstacle to reconstructing the underlying mechanisms of biogeochemistry in the ocean. While a number of dominant marine species from the ocean surface have been cultured, the dominant microbial plankton of the dark ocean proved far more difficult to tame. Genomic analyses of single cells emerged as a powerful means to expand knowledge of the diverse biochemical potential of these communities. Chapter 1 reviews the timeline of events in this field and summarizes current research with single-cell genomics and metagenomics within the framework of marine microbial ecology. The defining step in single-cell genomics approaches to environmental studies is the physical isolation of wild-type cells from heterogeneous microbial populations. In Chapter two I detail the construction and application of new instrumentation for optical trapping in conjunction with microfluidic devices (optofluidics) that allows for the selection of individual cells for genome amplification and sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy approaches to cell visualization.Fluorescence-activated cell sorting (FACS) approaches to single-cell genomics have reached full development and have been applied effectively to explore microbial diversity in the deep. In Chapter 3 I explore single amplified genomes obtained with FACS approaches, from several single-amplified genomes (SAGs) of the SAR202 clade, which has been shown to be ubiquitously abundant in the meso- and bathypelagic waters of the open ocean. Prior to this study the metabolism and geochemical role of the SAR202 clade was unknown, but their high abundance suggested they played an important role in nutrient cycling in the dark ocean. Due to their distinctive vertical profile, early accounts of the SAR202 clade speculated that they might be major mediators of recalcitrant organic carbon sequestration and turnover in the deep ocean, contributing to the "microbial carbon pump" through the conversion of labile carbon forms to more heterogeneous and refractory forms that could remain in the deep sea for thousands of years. I discovered that SAR202 encodes several families of oxidative enzymes and hypothesize that they are involved in the cycling of a major class of refractory deep-water marine dissolved organic matter (DOM), known as carboxyl-rich alicyclic matter, or CRAM. In Chapter 4 I revisit the optofluidic approach and describe its use to isolate single-amplified genomes (SAGS) from the marine environment. Several of these SAGs were shown to be representatives of groups of microbial plankton that are abundant in the ocean but not represented by genome sequences. In this chapter we evaluate the performance of this technique for single-cell genomics and outline the encoded metabolic features of three relatively-unstudied groups of marine microbes isolated using this technique. In Chapter 5, I outline potential areas of improvement for the optofluidic technology described in this thesis and discuss where the future of single-cell genomics technology.
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  • 2017-08-16 to 2018-06-11

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