Stabilized mutants of quorum quenching lactonase and use thereof in treatment of pathogens
Mutated phosphotriesterase-like lactonases or functional fragments can be used in methods for treating or preventing infection of a bacterium in a host, such as a plant or a part, organ or a plant propagation material. The methods include applying the mutated phosphotriesterase-like lactonases or the wild-type enzyme to the host Cells expressing the mutated phosphotriesterase-like lactonases can also be produced using nucleic acid molecules and vectors encoding the mutated phosphotriesterase-like lactonases or functional fragments.
1 . A mutated phosphotriesterase-like lactonase having at least 90% identity with the wild-type putative parathion hydrolase from M. tuberclorosis of SEQ ID NO: 1, in which an amino acid residue corresponding to position 59 or 172 of SEQ ID NO: 1 is substituted, wherein a glycine residue corresponding to G59 is substituted by an amino acid residue selected from valine, alanine, leucine, and isoleucine, or a histidine residue corresponding to H172 is substituted by an amino acid residue selected from tyrosine, phenylalanine and tryptophan, and said mutated phosphotriesterase-like lactonase has substantially identical TIM-barrel fold to a wild-type phosphotriesterase-like lactonase and preserves catalytic residues in its active site.
2 . The mutated phosphotriesterase-like lactonase of claim 1 , wherein a glycine residue corresponding to G59 of SEQ ID NO: 1 is substituted by valine; or a histidine residue corresponding to H172 of SEQ ID NO: 1 is substituted by tyrosine.
3 . The mutated phosphotriesterase-like lactonase of claim 2 comprising or essentially consisting of the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
4 . The mutated phosphotriesterase-like lactonase of claim 1 , wherein said mutated phosphotriesterase-like lactonase has an increased thermostability in comparison with thermostability of a non-mutated wild-type phosphotriesterase-like lactonase or substantially similar or higher lactonase catalytic activity provided with N-(3-oxo-hexanoyl)-homoserine lactone as a substrate in comparison with said non-mutated wild-type phosphotriesterase-like lactonase.
5 . The mutated phosphotriesterase-like lactonase of claim 4 , wherein said increased thermostability expressed as T 50 is about 50° C. to about 80° C.
6 . The mutated phosphotriesterase-like lactonase of claim 4 , having an extended shelf-life as compared with said non-mutated wild-type phosphotriesterase-like lactonase.
7 . The mutated phosphotriesterase-like lactonase of claim 1 , wherein a glycine residue corresponding to G59 of SEQ ID NO: 1 is substituted by valine or a histidine residue corresponding to H172 of SEQ ID NO: 1 is substituted by tyrosine; and said mutated phosphotriesterase-like lactonase has an increased thermostability in comparison with thermostability of a non-mutated wild-type phosphotriesterase-like lactonase or substantially similar or higher lactonase catalytic activity provided with N-(3-oxo-hexanoyl)-homoserine lactone as a substrate in comparison with said non-mutated wild-type phosphotriesterase-like lactonase.
8 . The mutated phosphotriesterase-like lactonase of claim 7 , comprising or essentially consisting of the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3; said increased thermostability expressed as T 50 is about 55° C. to about 80° C., or said mutated phosphotriesterase-like lactonase has an extended shelf-life as compared with said non-mutated wild-type phosphotriesterase-like lactonase.
9 . The mutated phosphotriesterase-like lactonase of claim 1 , comprising the PPH of SEQ ID NO: 1 in which the glycine residue at position 59 is substituted by an amino acid residue selected from valine, alanine, leucine, and isoleucine, or the histidine residue at position 172 is substituted by an amino acid residue selected from tyrosine, phenylalanine and tryptophan.
10 . A composition comprising the mutated phosphotriesterase-like lactonase of claim 1 , optionally further comprising a copper salt.
11 . The composition of claim 10 , wherein said copper salt is CuSO 4 .
12 . An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a mutated phosphotriesterase-like lactonase of claim 1 .
13 . The nucleic acid molecule of claim 12 comprising a nucleic acid sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14 or SEQ ID NO: 15.
14 . An expression vector comprising the nucleic acid molecule of claim 12 operatively linked to a promoter.
15 . A cell comprising the isolated nucleic acid molecule of claim 12 or the expression vector of claim 14 .
16 . The cell of claim 15 , selected from a bacterial, fungal, mammal or plant cell.
17 . The cell of claim 16 , wherein said cell is E. coli.
18 . A plant or a part, organ or a plant propagation material thereof, at least partly covered or coated with a mutated phosphotriesterase-like lactonase of claim 1 .
19 . A plant or a part, organ or a plant propagation material thereof, at least partly covered or coated with a composition of claim 10 .
20 . A method for treating or preventing infection of a bacterium in a plant or a part, organ or a plant propagation material thereof, said plant being infected by or susceptible to a bacterium secreting a lactone selected from N-(3-hydroxybutanoyl)-L-homoserine lactone (C4-HSL), N-(3-oxo-hexanoyl)-homoserine lactone (C6-oxo-HSL), N-[(3S)-tetrahydro-2-oxo-3-furanyl]octanamide (C8-oxo-HSL), and N-[(3S)-tetrahydro-furanyl]decanamide (C10-HSL),
said method comprising applying on said plant or said part, organ or plant propagation material thereof, the wild-type putative parathion hydrolase from M. tuberclorosis of SEQ ID NO: 1, the mutated phosphotriesterase-like lactonase of claim 1 , or the composition of claim 10 .
21 . The method of claim 20 , wherein said bacterium is selected from the group consisting of Erwinia amylovora, Pectobacterium carotovorum, Pseudomonas syringae, Pseudomonas corrugata, Burkholderia vietnamiensis, Burkholderia cepacia, Burkholderia thailandensis and Pseudomonas aeruginosa , including any pathovars.
22 . The method of claim 21 , wherein said bacterium is Erwinia amylovora, Pectobacterium carotovorum , or Pseudomonas syringae.
23 . The method of claim 22 , wherein said Pseudomonas syringae is Pseudomonas tomato (formerly known as Pseudomonas syringae pv. tomato ).
24 . The method of claim 20 , wherein said putative parathion hydrolase from M. tuberclorosis , said mutated phosphotriesterase-like lactonase, or said composition, and a separate composition comprising a copper salt, are separately applied to said plant, part, organ or plant propagation material of said plant.
25 . The method of claim 20 , comprising applying the mutated phosphotriesterase-like lactonase wherein a glycine residue corresponding to G59 of SEQ ID NO: 1 is substituted by valine, or the phosphotriesterase-like lactonase wherein a histidine residue corresponding to H172 of SEQ ID NO: 1 is substituted by tyrosine.
26 . The method of claim 24 , wherein said copper salt is CuSO 4 .
27 . The method of claim 25 , wherein said mutated phosphotriesterase-like lactonase wherein a glycine residue corresponding to G59 of SEQ ID NO: 1 is substituted by valine comprises or essentially consists of the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 11; or the phosphotriesterase-like lactonase wherein a histidine residue corresponding to H172 of SEQ ID NO: 1 is substituted by tyrosine comprises or essentially consists of the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 12.