Deletion, Mutation, and Biochemical Analyses of the Ice Nucleation Protein from Pseudomonas borealis
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Authors
Forbes, Jordan
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thesis
Language
eng
Keyword
Bacterial ice nucleation , Ice nucleation protein , Structure-function relationship , Gram-negative bacteria , Membrane-bound protein , Structural models , Antifreeze protein
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Abstract
Outer membrane-associated ice nucleation proteins (INPs) in certain Gram-negative bacteria can cause frost damage to plants by nucleating ice in supercooled water. The INP central domain consists of ~55-80 tandem sixteen-residue repeats that are flanked by short N- and C-terminal domains. Structural models suggest that INP repeats resemble a much larger version of some beta-solenoid antifreeze proteins. Whereas antifreeze proteins organize ice-like water molecules to facilitate ice adsorption, the larger INPs likely order sufficient water molecules to initiate ice nucleation. When Pseudomonas borealis INP (PbINP) was expressed in Escherichia coli, it triggered ice nucleation at high sub-zero temperatures within 2 °C of P. borealis activity. Deletions within the central 65-repeat region lowered ice nucleation temperatures in droplet freezing assays, with sharp decreases below 24 remaining repeats and with 13-15 repeats required for INP activity, including some closest to the C-terminal domain. Nanoliter-sized droplets containing a few E. coli showed uniform freezing at a temperature comparable to freezing by thousands of cells; similar dilutions with P. borealis revealed that 0.5-1% of cells are more active. Mutation of subsets of putative water-organizing and dimerization residues caused decreases in ice nucleation activity that were larger than when the corresponding segment was deleted. This importance of central domain continuity was supported by two different mRuby2 insertions, which also demonstrated functional protein insertion within the repetitive domain. These data show the correlation between central domain length and INP activity and support the hypothesis that repetitive water-ordering motifs in INPs are necessary for potent ice nucleation.
N-terminal domain truncations of PbINP caused only 1-2 °C decreases in freezing temperatures, as seen with deletions on a P. syringae INP. Through truncation of the first 18 residues, INPs are retained in the cytoplasm; a construct of cytoplasmic PbINP with 18 repeats and a polyhistidine tag was produced in E. coli and purified. It appeared to form a heterogeneous aggregate that induced freezing near -11 °C. Heterologous PbINP in E. coli showed strong reactivity with anti-INP, yet weak reactivity was observed with P. borealis lysate. These miscellaneous results serve as points of departure for future experiments.
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ProQuest PhD and Master's Theses International Dissemination Agreement
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Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.