Methods and strain

Abstract

The present invention relates to a method for transforming a strain of the Lactococcus genus through natural competence. The present invention further relates to strains obtained or obtainable by said method. The present invention also relates to a method for identifying a strain of the Lactococcus genus which is transformable through natural competence.

1 . A method for transforming a strain of the  Lactococcus  genus with an exogenous DNA polynucleotide comprising the steps of:
 (a) providing a strain of the  Lactococcus  genus, wherein said strain is transformable through natural competence;   (b) modulating the production of a ComX protein in said strain;   (c) contacting said strain of step (b) with an exogenous DNA polynucleotide in a medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide into the genome of said strain; and   (d) selecting a strain which has integrated the exogenous DNA polynucleotide into its genome.   
     
     
         2 . A method according to  claim 1 , wherein the step of modulating the production of a ComX protein is performed by expressing a comX gene in said strain or increasing the expression of a comX gene in said strain. 
     
     
         3 . A method according to  claim 2 , wherein said comX gene is an exogenous comX gene. 
     
     
         4 . A method according to  claim 3 , wherein said exogenous comX gene is transferred into said strain by conjugation, transduction, or transformation. 
     
     
         5 . A method according to  claim 2 , wherein said comX gene is the endogenous comX gene of said strain. 
     
     
         6 . A method according to  claim 5 , wherein the method comprises carrying out step (b) and then carrying out step (c) or comprises carrying out step (b) and step (c) simultaneously. 
     
     
         7 . A method according to  claim 1 , wherein said ComX protein has:
 the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22;   an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22; or   an amino acid sequence having at least 90% similarity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.   
     
     
         8 . A method according to  claim 1 , wherein said comX gene has:
 the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21; or   a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21.   
     
     
         9 . A method according to  claim 1 , wherein said medium of step (c) is a chemically defined medium. 
     
     
         10 . A method according to  claim 1 , wherein prior to step (c) said strain is incubated in a pre-culture medium. 
     
     
         11 . A method according to  claim 1 , wherein said strain is incubated with the exogenous DNA polynucleotide for around 4-8 hours at around 30° C. and said medium of step (c) is supplemented with an osmo-stablizer. 
     
     
         12 . A method according to  claim 1 , wherein said strain of the  Lactococcus  genus of step (a) is a strain of the  Lactococcus raffinolactis  species or a strain of the  Lactococcus lactis  species. 
     
     
         13 . A method according to  claim 1 , wherein said exogenous DNA polynucleotide used in step (c) is obtained from a strain of the same species as the strain provided in step (a). 
     
     
         14 . A strain of the  Lactococcus  genus obtained by the method of  claim 1 . 
     
     
         15 . A method for identifying a strain of the  Lactococcus  genus which is transformable through natural competence comprising the steps of:
 (a) providing a strain of the  Lactococcus  genus;   (b) transforming said strain with a plasmid expressing a comX gene having at least 90% identity to the endogenous comX gene of said strain;   (c) contacting said strain obtained in step (b) with an exogenous DNA polynucleotide encoding a marker gene in a medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide into the genome of said strain; and   (d) determining the rate of recombination events;   wherein a rate of at least 1×10 −6  transformants per μg of DNA is indicative of a strain which is transformable through natural competence.   
     
     
         16 . A method according  claim 1 , wherein said strain of step (a) is identified using a method for identifying a strain of the  Lactococcus  genus which is transformable through natural competence comprising:
 (a) providing a strain of the  Lactococcus  genus;   (b) transforming said strain with a plasmid expressing a comX gene having at least 90% identity to the endogenous comX gene of said strain;   (c) contacting said strain obtained in step (b) with an exogenous DNA polynucleotide encoding a marker gene in a medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide into the genome of said strain; and   (d) determining the rate of recombination events;   wherein a rate of at least 1×10 −6  transformants per μg of DNA is indicative of a strain which is transformable through natural competence.   
     
     
         17 . A method according to  claim 1 , wherein said strain of step (a) is identified using assay A, which is performed as follows:
 i) providing a strain of the  Lactococcus  genus;   ii) transforming the strain with a plasmid expressing a comX gene having at least 90% identity to the endogenous comX gene of the strain;   iii) pre-culturing the transformed strain overnight in a complex medium supplemented with glucose;   iv) diluting about 1.5 mL of the pre-culture in 8.5 mL of fresh medium;   v) after 2 hr of further growth at 30° C., washing the cells twice with distilled water and adjusting the OD 600  to 0.05 in a chemically defined medium comprising 5 μg mL −1  erythromycin and an osmo-stabilizer;   vi) adding 5 μg of exogenous DNA polynucleotide bearing an antibiotic resistance gene to 300 al of the culture medium;   vii) incubating the resulting culture for 6 hr at 30° C.;   viii) plating the cells onto agar plates comprising the complex medium supplemented with glucose and antibiotic corresponding to the antibiotic resistance gene of the exogenous DNA polynucleotide and incubating for 48 hr;   ix) counting the colony forming units and determining the transformation rate, wherein:
 the transformation rate equals the number of antibiotic-resistance colony forming units per mL divided by the total number of viable colony forming units per mL, and 
 a transformation rate of at least 1×10 −6  transformants per μg of DNA is indicative of a strain that is transformable through natural competence. 
   
     
     
         18 . A method according to  claim 1 , wherein said ComX protein has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22. 
     
     
         19 . A method according to  claim 1 , wherein said comX gene has the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21. 
     
     
         20 . A method according to  claim 11 , wherein the osmo-stabilizer is glycerol or mannitol.