Graduate Thesis Or Dissertation
 

Development and application of genomic DNA based methods for the identification and characterization of marine bacterioplankton

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  • A series of experiments was performed to determine the feasibility of genomic DNA-based methods for ecological and physiological studies of dominant bacteria from complex natural systems. Two approaches, bacterial chromosomal painting (BCP) and environmental genomics (EG), were evaluated for the potential to identify and enumerate specific bacterial groups in situ and to reconstruct the complete genome of uncultivated bacteria. Methods which are commonly used to assess microbial ecology questions are subject to several biases, including enrichment and growth rate effects. BCP is a new method adapted from eukaryotic cell biology designed to avoid these biases. In BCP, genomic DNA is fluorescently labeled and used for in situ hybridization to allow detection of single cells related to the probe DNA at the genus level and below. Initial studies with cultured isolates showed that BCP could differentiate between closely related bacterial groups of various types. BCP was used to determine the spatio-temporal variability in abundance of a dominant culturable bacterioplankton species from the western Sargasso Sea. The resulting data were used in conjunction with physiological studies in the laboratory as well as physical and chemical data to form hypotheses explaining this variability. Environmental genomics has been used to obtain information about metabolically relevant genes from uncultured prokaryotes. One of the goals of this approach is the reconstruction of the complete genome of uncultivated bacteria. To estimate the number of clones required to obtain the complete genome of a single bacterioplankton with EG, I used pulsed field gel electrophoresis to estimate the genome size of Pseudoalteromonas haloplanktis, a common marine isolate. During this analysis, I determined that the genome of this organism is composed of two large genetic elements. Although multiple large genetic elements are thought to be relatively rare in bacteria, this result provides a caveat for research which attempts to reconstruct the complete genome of an uncultured microorganism from EG clone libraries. These two methods, BCP and EG, were shown to be useful for ecological and physiological studies of cultured bacteria. Further, ongoing studies will attempt to merge these techniques for examining distributions and physiology of uncultured bacteria in natural systems.
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file details LanoilBrianD1999.pdf 2017-08-01 Pubblico Scaricare