| Summary: | Gas clathrates are composed of a latticework of water molecules that trap guest gas molecules and form at high pressure and low temperatures. Methane clathrates along continental margins and in permafrost store thousands of gigatons of carbon in sediments and serve as a habitat for a unique deep subsurface biosphere. Microbes in gas clathrate-bearing sediments may influence clathrate stability and thereby play a role in the release of greenhouse gases to overlying sediments. Gas clathrates also pose a clogging hazard in natural gas pipelines, which has led to a search for environmentally friendly gas clathrate inhibitors. Antifreeze proteins (AFPs) were found to bind to gas clathrates and suppress clathrate growth. This dissertation evaluated whether proteins from methane clathrate-bearing sediment microbes influence clathrate growth and morphology. Bacterial protein sequences with sequence similarity to alpha helical Type I AFPs in cold-water fish were identified in metagenomes from gas clathrate-bearing sediment from Hydrate Ridge, offshore Oregon, and Shimokita Peninsula, offshore Japan. In the search for AFP-like sequences, two Type I AFP amino acid motifs were used as search queries of Hydrate Ridge metagenomes. Homology modeling software and antifreeze prediction software were used to predict antifreeze properties of the resulting sequences. Homologous sequences with predicted antifreeze properties from sediments in Hydrate Ridge were also detected in Shimokita Peninsula sediments. These Clathrate-Binding Protein (CBP) genes (cbpA) and upstream genes (cbpB,C,D) are likely from Dehalococcoidia bacteria, of the phylum Chloroflexi, which are known to occur in methane clathrates. To date, CBPs are unique to gas clathrates. Recombinantly expressed CbpAs were first tested on tetrahydrofuran (THF) clathrate, a structure II clathrate that is stable at atmospheric pressures and 4˚C. In the absence of CbpAs, large (~1 cm diameter) single THF clathrate crystals formed. In the presence of Type I AFPs, CbpA2, and ...
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