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Thesis: Sequencing, Pipeline Development, and Select Comparative Analysis of 64 High-Quality Draft Genomes of Extremophilic Bacteria Isolated from Communities in Carboxylate Platform Fermentations.
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Microbial extremophiles have the potential for a wide variety of biotechnological and industrial applications and yet extremophiles are underrepresented in whole genome sequencing efforts to date. The generation of whole genome sequences allows for gene calling, function prediction, and creation of evolutionary models and adds to the richness of extant knowledge of the bacterial world. The sequencing of extremophiles is thus of high value. Previous efforts collected 501 soil samples from 77 thermal and saline sites across the United States and Puerto Rico and used these in an effort to optimize the microbial communities in a carboxylate biofuel platform. The 34 best performing inocula were used to isolate 1866 strains using a variety of media in a low-oxygen and high-temperature environment. A diverse subset of this isolate library was screened for traits of industrial relevance. In this project I created a model to choose a characteristic subset of these isolates while maintaining the phylogenetic, phenotypic, and geographic diversity of the isolate library. Using this subset I created a pipeline to sequence, assemble, annotate, and disseminate high quality draft-genomes of these microbes. In this work I created high-quality draft genome sequences of 64 isolates from 22 sites across the United States and Puerto Rico. I inferred phylogeny of a subset (N=48) of these isolates using multilocus sequence analysis of four housekeeping genes and discovered three potentially novel genera. Using the Joint Genome Institutes Integrated Microbial Genomes system I was able to annotate and make functional assertions about these isolates. These isolates display a diverse range of carbohydrate utilization that is directly related to their phylogeny, and many isolates show industrially relevant carbohydrate utilization pathways such as cellulose, arabinose, and xylose. Many of the isolates sequenced also show a pathway for degradation of furfural, an inhibitory compound that causes issues in second-generation biofuel platforms. The furfural degradation pathway is shown to be rare among extant sequenced prokaryotes. The Opu operon was found in many of these isolates, which when complete transports the compatible solute glycine betaine into the cell. This pathway has been implicated in osmoregulation, thermotolerance, and cold-show protection. Finally, four isolates were found to have a group II intron interrupting the housekeeping gene recA, which codes for a protein related to DNA repair. The insertion of a group II intron into a housekeeping gene is extremely rare and has potential implications for our existing knowledge about the role of group II introns. This work creates 64 high-quality draft genome sequences and annotations as well as select analyses, clearly demonstrating the potential of these resources for future applications.
multilocus sequence analysis
Caraway, Emma Britain (2016). Thesis: Sequencing, Pipeline Development, and Select Comparative Analysis of 64 High-Quality Draft Genomes of Extremophilic Bacteria Isolated from Communities in Carboxylate Platform Fermentations.. Master's thesis, Texas A & M University. Available electronically from