Isolation and characterization of an antifreeze protein with ice nucleation activity from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2

1998 ◽  
Vol 44 (1) ◽  
pp. 64-73 ◽  
Author(s):  
Hao Xu ◽  
Marilyn Griffith ◽  
Cheryl L Patten ◽  
Bernard R Glick
Keyword(s):  
Plant Growth ◽  
Pseudomonas Putida ◽  
Antifreeze Protein ◽  
Ice Nucleation ◽  
Isolation And Characterization ◽  
Apparent Homogeneity ◽  
Plant Growth Promoting ◽  
Nucleation Activity ◽  
Growth Promoting ◽  
Ice Nucleation Activity

An antifreeze protein secreted to the growth medium by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was purified to apparent homogeneity. The purified protein has a molecular mass of 164 ± 15 kDa and an isoelectric point of 5.3, contains both carbohydrate and lipid moieties, and is relatively rich in glycine and alanine. The properties of the purified antifreeze protein are similar to the properties previously reported for bacterial ice-nucleation proteins. In fact, the purified antifreeze protein also displays a low level of ice-nucleation activity. Removal of approximately 92 kDa of carbohydrate from the 164-kDa antifreeze glycoprotein did not noticeably alter the antifreeze activity of the molecule, although it did diminish the ice-nucleation activity. This is the first report of an antifreeze protein that also is active as an ice-nucleation protein.Key words: antifreeze protein, plant growth promoting rhizobacteria, freezing tolerance, ice-nucleation protein.

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

2004 ◽  
Vol 186 (17) ◽  
pp. 5661-5671 ◽  
Author(s):  
Naomi Muryoi ◽  
Mika Sato ◽  
Shoji Kaneko ◽  
Hidehisa Kawahara ◽  
Hitoshi Obata ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plant Growth ◽  
Pseudomonas Putida ◽  
Ice Nucleation ◽  
The Arctic ◽  
Gene Encoding ◽  
Plant Growth Promoting ◽  
Nucleation Activity ◽  
Growth Promoting ◽  
Ice Nucleation Activity

ABSTRACT 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.

Low temperature growth, freezing survival, and production of antifreeze protein by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2

1995 ◽  
Vol 41 (9) ◽  
pp. 776-784 ◽  
Author(s):  
Xiuying Sun ◽  
Marilyn Griffith ◽  
J. J. Pasternak ◽  
Bernard R. Glick
Keyword(s):  
Plant Growth ◽  
Pseudomonas Putida ◽  
Growth Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Plant Growth Promoting Rhizobacteria ◽  
Antifreeze Protein ◽  
High Arctic ◽  
Major Protein ◽  
Plant Growth Promoting ◽  
Growth Promoting

The plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was originally isolated from the rhizosphere of plants growing in the Canadian High Arctic. Here we report that this bacterium was able to grow and promote root elongation of both spring and winter canola at 5 °C, a temperature at which only a relatively small number of bacteria are able to proliferate and function. In addition, the bacterium survived exposure to freezing temperatures, i.e., −20 and −50 °C. In an effort to determine the mechanistic basis for this behaviour, it was discovered that following growth at 5 °C, P. putida GR12-2 synthesized and secreted to the growth medium a protein with antifreeze activity. Analysis of the spent growth medium, following concentration by ultrafiltration, by SDS-polyacrylamide gel electrophoresis revealed the presence of one major protein with a molecular mass of approximately 32–34 kDa and a number of minor proteins. However, at this point it is not known which of these proteins contains the antifreeze activity.Key words: plant growth promoting rhizobacteria, PGPR, bacterial fertilizer, soil bacteria, antifreeze protein.

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