Ice-associating proteins (IAPs) are proteins that interact directly with ice crystals, either by offering a site for nucleation, i.e. ice nucleating proteins (INPs), or by binding to nascent crystals to prevent addition of more water molecules, i.e. antifreeze proteins (AFPs). AFPs have been found to inhibit the formation of clathrate-hydrates, ice-like crystalline solids composed of water-encaged guest molecules. Study of AFP-hydrate interaction is leading to a greater understanding of AFP adsorption and of the mechanism behind the “memory effect” in hydrates, wherein previously frozen crystals reform more quickly after a brief melt. AFP is currently the only known memory inhibitor. Such a low-dosage hydrate inhibitor (LDHI) is of great interest to the oil and gas industry, as hydrate formation and reformation in the field is a huge problem. Bacterial AFPs, though largely uncharacterized, may be the best candidates for large-scale production of hydrate inhibitors, given the difficulties in obtaining AFP from other sources. The popular kinetic inhibitors (KIs) polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap) were used for points of comparison in experiments exploring the hydrate-inhibition activity of several ice-associating bacteria and proteins. The addition of the soil microbe, Chryseobacterium, increased the average lag-time to tetrahydrofuran (THF) hydrate formation by 14-fold, comparable to PVP or PVCap. Samples containing Pseudomonas putida, a bacterium having both ice-nucleation protein (INP) and AFP activity, had lag-times double that of the control. Solutions with P. putida and Chryseobacterium sometimes formed hydrate slurries of stunted crystal nuclei instead of solid crystals. No inhibition of memory or nucleation was noted in bacterial assays, however bacteria with INP activity was linked to unusually rapid memory reformation. Quartz crystal microbalance experiments with dissipation (QCM-D) showed that a tight adsorption to SiO2 and resistance to rinsing are correlated with a molecule’s inhibition of hydrate formation and reformation. These results support a heterogeneous nucleation model of the memory effect, and point to the affinity of AFP for heterogeneous nucleating particles as an important component of memory inhibition. / Thesis (Master, Biology) -- Queen's University, 2008-05-30 15:20:38.749

An ice nucleating protein (INP) with 66% amino acid sequence identity to the better-known INP of Pseudomonas syringae has been described in an environmental isolate of P. borealis and designated InaPb. Despite the fact that INPs are classified as ice-binding proteins, InaPb showed little affinity for pre-formed ice and showed incorporation rates similar to Ina- strains. Additionally, it appeared to lack in the ability to shape ice or limit its growth. However, it was an effective ice nucleator. Using the coding sequence for InaPb and a green fluorescent protein tag (GFP), an InaPb-GFP fusion protein construct was inserted into a broad host expression vector in order to visualize the expression and localization of the protein in E. coli and an Ina- strain of P.syringae. The InaPb-GFP protein appears to localize at the poles of E. coli, but the nucleation temperature for these cells was only marginally above -9°C, which indicated poor nucleation activity. When expressed in Ina- P. syringae, the proteins showed clustering throughout the cell and an increased ability to nucleate ice following cold conditioning. The ability to nucleate ice was further increased by the removal of the GFP tag resulting in an average nucleation temperature more consistent with that seen in the native host P. borealis. Since inaPb transcript levels did not appear to change after cold conditioning, the clustering seen using fluorescence microscopy was likely the result of increased aggregation of protein in the membrane. Most INP- producing bacteria are associated with plant disease, but experiments with P. borealis suggested that the Ina+ phenotype was not indicative of pathogenicity in this strain. It is hoped that my contribution to the functional characterization of this INP will lead to a better understanding of these special proteins and their importance to the handful of bacteria that exhibit this activity. / Thesis (Master, Biology) -- Queen's University, 2012-05-15 09:55:52.506

Certain organisms, including some bugs (both insects and microbes) are able to survive low temperatures by the production of either ice nucleating proteins (INPs) or antifreeze proteins (AFPs). INPs direct crystal growth by inducing rapid ice formation whereas AFPs adsorb to ice embryos and decrease the temperature at which the ice grows. We have also shown that certain AFPs can inhibit the crystallization of clathrate hydrates and eliminate more rapid recrystallization or “memory effect”. Here we examine several bacterial species with iceassociating properties for their effect on tetrahydrofuran (THF) hydrate crystallization. The bacteria Chryseobacterium sp. C14, which shares the ice recrystallization inhibition ability of AFPs, increased induction time to THF hydrate crystallization in isothermal experiments. In an effort to understand the association between AFPs and THF hydrate we have produced bacterially-expressed AFPs as probes for hydrate binding. Although the structure of hydrates is clearly distinct from ice, the apparent potential for these products to perturb clathrate hydrate growth compels us to explore new techniques to uncover “green inhibitors” for hydrate binding.