Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

K, van Zee; Da, Baertlein; Se, Lindow; Panopoulas, N; Th, Chen

doi:10.1007/bf00017816

Cited by 5 publications

(3 citation statements)

References 18 publications

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“…4; Table 5) (76), which may be due to improper protein folding or differences in posttranslational modification, especially since Xu et al (76) reported that ice nucleation activity decreased when the AfpA was deglycosylated experimentally. Another possibility is that highly active INPs and AFPs may be produced only at cold temperatures, as observed when inaZ was expressed in plants (65).…”

Section: Discussionmentioning

confidence: 99%

Cloning and Expression of afpA , a Gene Encoding an Antifreeze Protein from the Arctic Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2

Muryoi

Sato²,

Kaneko³

et al. 2004

J Bacteriol

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The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.

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Section: Discussionmentioning

confidence: 99%

Cloning and Expression of afpA , a Gene Encoding an Antifreeze Protein from the Arctic Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2

Muryoi

Sato²,

Kaneko³

et al. 2004

J Bacteriol

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“…Previous SFG studies have shown that P. syringae and INPs anchored to the surface of hollow Escherichia coli cell envelopes can order water at the bacterial surface 31 – 33 , and that water interaction is enhanced close to the water melting point 31 , 34 . However, the molecular basis for water ordering and the mechanism behind the activation at low temperatures remain unclear.…”

Section: Introductionmentioning

confidence: 99%

Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water

et al. 2021

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Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Using specialized ice-nucleating proteins (INPs), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds, which may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INP-induced freezing have remained largely elusive. We investigate the structural basis for the interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae. Using vibrational sum-frequency generation (SFG) and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces. In this configuration, interaction between INPs and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with molecular-dynamics simulations and spectral calculations show that InaZ reorients at lower temperatures. This reorientation can enhance water interactions, and thereby the effectiveness of ice nucleation.

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“…29,30 It has also become evident that the ability of P. syringae InaZ to control water orientation is enhanced when cooled to temperatures close to the water melting point. 29,31 However, since bacterial cell surfaces contain a variety of many different proteins and other biomolecules, these studies could not track the molecular origin of this activation at the protein level. Our current picture of the interaction of INPros with water is still, to a large extent, based on theoretical models and molecular dynamics (MD) simulations 3,32,33 , with contradicting conclusions for protein folding and the role of side chain motifs.…”

Section: Introductionmentioning

confidence: 99%

The Ice Nucleating Protein InaZ is Activated by Low Temperature

Roeters¹,

Golbek²,

Bregnhøj³

et al. 2020

Preprint

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Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Utilizing specialized ice-nucleating proteins (INPros), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds and may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INProinduced freezing have remained largely elusive. In the present study, we investigated the folding and the structural basis for interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae strain R10.79. Using vibrational sum-frequency generation and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces.In this configuration, hydrogen bonding between INPros and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with spectral calculations and molecular-dynamics simulations shows that the INPro reorients at lower temperatures. We suggest that the reorientation can enhance order-inducing water interactions and, thereby, the effectiveness of ice nucleation by InaZ.

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Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

Cited by 5 publications

References 18 publications

Cloning and Expression of afpA , a Gene Encoding an Antifreeze Protein from the Arctic Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2

Cloning and Expression of afpA , a Gene Encoding an Antifreeze Protein from the Arctic Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2

Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water

The Ice Nucleating Protein InaZ is Activated by Low Temperature

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