Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2

Naomi Muryoi, Mika Sato, Shoji Kaneko, Hidehisa Kawahara, Hitoshi Obata, Mahmoud W F Yaish, Marilyn Griffith, Bernard R. Glick

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)5661-5671
Number of pages11
JournalJournal of Bacteriology
Volume186
Issue number17
DOIs
Publication statusPublished - Sep 2004

Fingerprint

Antifreeze Proteins
Pseudomonas putida
Ice
Organism Cloning
Growth
Genes
Proteins
Amino Acid Sequence
Polymerase Chain Reaction
Hemolysin Proteins
Protein Sorting Signals
Gram-Negative Bacteria
Recombinant Proteins
Cell Wall
Open Reading Frames
Sequence Analysis
Carbohydrates
Escherichia coli
Calcium
Lipids

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2. / Muryoi, Naomi; Sato, Mika; Kaneko, Shoji; Kawahara, Hidehisa; Obata, Hitoshi; Yaish, Mahmoud W F; Griffith, Marilyn; Glick, Bernard R.

In: Journal of Bacteriology, Vol. 186, No. 17, 09.2004, p. 5661-5671.

Research output: Contribution to journalArticle

Muryoi, Naomi ; Sato, Mika ; Kaneko, Shoji ; Kawahara, Hidehisa ; Obata, Hitoshi ; Yaish, Mahmoud W F ; Griffith, Marilyn ; Glick, Bernard R. / Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2. In: Journal of Bacteriology. 2004 ; Vol. 186, No. 17. pp. 5661-5671.
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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.",
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AU - Sato, Mika

AU - Kaneko, Shoji

AU - Kawahara, Hidehisa

AU - Obata, Hitoshi

AU - Yaish, Mahmoud W F

AU - Griffith, Marilyn

AU - Glick, Bernard R.

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