Deep biosphere microbial protein interactions with clathrates

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Johnson, Abigail Marie
Glass, Jennifer B.
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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 CbpA3, small branching THF clathrate crystals formed. In the presence of CbpA5 and CbpA6, THF clathrates formed a few flat, interconnected sheets with hexagonal growth parallel to the [1 1 1] crystal face. Like Type I AFPs, CbpA, were concentrated in the clathrate crystal compared to the controls and Green Fluorescent Protein labeled CbpAs were observed to bind to THF clathrate. These data show that CbpAs bind to and alter the morphology of THF clathrate and the different morphologies likely represent two distinct binding modes. I then tested the effect of CbpAs on growth of methane clathrate using a high-pressure cell. Methane clathrate shells were synthesized on water droplets at 5 MPa in the presence or absence of CbpAs. Gas consumption was determined after depressurization. Significantly less methane clathrate formed in the presence of CbpAs and the commercial gas clathrate inhibitor, polyvinylpyrrolidone, relative to controls. Type I AFP did not significantly alter methane clathrate formation compared to controls at the high driving force used in this study. Dome-shaped shells formed in treatments with suppressed clathrate growth, whereas treatments with more clathrate formed cratered shells. Overall, I found that CbpAs alter methane clathrate morphology and inhibit methane clathrate formation similarly to the commercial inhibitor, polyvinylpyrrolidone. Discovery of clathrate-binding proteins in gas clathrate-bearing sediments is unprecedented and has many implications: climate change, natural gas pipeline flow assurance and transport, microbial survival strategies, and searching for life on other planetary bodies. CBPs produced by microbes living in clathrate-bearing sediments may be useful as an ecofriendly clathrate inhibitor in natural gas pipelines. CBPs may influence clathrate stability in situ, with important climate impacts. CBPs should be considered in astrobiology as a potential microbial habitat when searching for life on other planetary bodies.
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