Team:KULeuven/Inp

Ice-nucleating Protein (Inp)

Did you know that water does not always freeze at 0°C and it can remain super cool till -40°C if free of ice nucleating species? Moreover, water can freeze around 0°C but there will almost be no crystallization. Ice flakes are a good example of crystallized ice and are formed at temperatures around -28°C, high in the sky. In order for water to crystallize into ice, water molecules have to be structurally ordered around a particle. Ice crystallization around 0°C can also occur with the help of nature’s Ice Nucleating Proteins (INPs).

There has been an interest in the study of bacterial ice nucleation since the early 1980’s. Different types of Gram-negative bacteria possess the ability to catalyze ice formation in temperatures ranging from -2 to -12°C [1, 2]. Most of the ice nucleating active (INA) bacteria are known to be found in plants [3] and animals [4, 5]. Other microorganisms with several genres of fungi [6] and lichens [7] are also reported to have the ability to induce formation. INA bacteria could induce frost formation in crops leading to big losses for agriculture production [1].

Certain species of Pseudomonas were found living on the surface of many crops. Further studies provided the evidence of a positive correlation between the presence of Pseudomonas syringae [Figure 1] and freezing of the crops. This is due to the fact that P. syringae’s outer cell membrane is coated with ice nucleating proteins and thus induces ice nucleation. It has been shown that the presence of P. Syringae lowers the freezing temperature on the surface of plant tissues [Figure 2]. It can be seen on the graph that corn leaves sprayed with P. Syringae freeze around -3°C, whereas, when sterile water was used, the leaves only freeze around -11°C. These results led researchers to conclude that bacteria on the surfaces of leaves play a major role in plant freezing.

Figure 1. P. syringae shown using SEM, Source [8].		Figure 2. Ice nucleation activity of corn leaf disks from plants with and without leaf surface populations of Pseudomonas syringae. Plants were sprayed with suspensions of 2 x 108 cells/ml. Source: Lindow et al., 1982.

Graether and Jia (2001) designed a structural model for the INP of P. Syringae, based on a comparison of two insect AFP structures [8]. After analyzing the INP sequence, they found a 16-residue that repeated itself sixty times and thus they proposed a 16-reside loop for P. Syringae [Figure 3].

Figure 3. Cross section of modeled INP and a β-helical protein. All figures show a wire frame representation of one loop. (A) Cross section of the modeled INP after 100 steps of energy minimization. (B) Cross section of the modeled INP after 3000 steps of energy minimization. (C) Cross section of UDP-acetylglucosamine acyltransferase, residues 36–53 [8].

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References

[1] Lindow et al., Bacterial Ice Nucleation: A Factor in Frost Injury to Plants, Plant Physiol. (1982) 70, 1084-1089
[2] Hirano et al., Ice Nucleation Temperature of Individual Leaves in Relation to Population Sizes of Ice Nucleation Active Bacteria and Frost Injury,Plant Physiol. (1985) 77, 259-265
[3] Lindow et al., Distribution of ice nucleation-active bacteria on plants in nature, Applied and Environmental Microbiology, DEC. 1978, p. 831-838
[4] Worland and Block, Ice-Nucleating Bacteria from the Guts of Two Sub-Antarctic Beetles, Hydromedion sparsutum and Perimylops antarcticus (Perimylopidae), Cryobiology 38, 60 – 67 (1999)
[5] J. S. Bale et al., Effects of summer frost exposures on the cold tolerance strategy of a sub-Antarctic beetle, Journal of Insect Physiology 47 (2001) 1161–1167
[6] C. Richard et al., Ice nucleation activity identified in some phytopathogenic Fusarium species, Phytoprotection, vol. 77, n° 2, 1996, p. 83-92.
[7] Kieft et al., Biological Ice Nucleation Activity in Lichen Mycobionts and Photobionts, The Lichenologist (1989), 21: 355-362
[8] Graether, S.P., and Z. Jia, Modeling Pseudomonas syringae ice-nucleation protein as a B-helical protein, Biophysical Journal 80, 2001: 1169-1173.