Competence stimulating peptide
View Patent ↗The present invention concerns a new competence stimulating peptide identified in Firmicutes , in particular Streptococcus , and more preferably S. thermophilus and methods of producing transformation competent Firmicutes , in particular Streptococcus , and more preferably S. thermophilus bacteria.
1. A method for producing a mutant bacterium of the phylum Firmicutes which comprises the steps of:
(a) producing transformation competent bacteria of the phylum Firmicutes comprising contacting said bacteria with an effective amount of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (LKTLKIFVLFSLLIAILPYFAGCL), a derivative thereof having a percentage of identity of at least 90% with the amino acid sequence SEQ ID NO: 1; or a fragment of SEQ ID NO: 1, wherein said derivative or fragment is capable of stimulating competence in Streptococcus ; and
(b) contacting said transformation competent bacteria with homologous DNA comprising a mutation under conditions allowing transformation by homologous recombination of said bacteria with said homologous DNA comprising said mutation to produce said mutant bacterium.
2. The method of claim 1 wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 12.
3. The method of claim 2 wherein said contacting of step (a) comprises culturing said bacteria in a peptide-free medium and adding the effective amount of the polypeptide to the peptide-free medium.
4. The method of claim 1 wherein the bacteria are of the genus Streptococcus.
5. The method of claim 1 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or of a derivative thereof having a percentage of identity of at least 90% with the amino acid sequence of SEQ ID NO: 1.
6. The method of claim 1 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1.
7. The method of claim 1 , wherein the length of the polypeptide is less than 100 amino acids.
8. The method of claim 1 , wherein the polypeptide further comprises an amino acid sequence corresponding to a signal peptide.
9. The method of claim 1 , wherein said step of producing transformation competent bacteria is performed in a culture medium comprising an effective amount of said polypeptide.
10. The method of claim 1 , wherein the bacteria are of the genus Streptococcus.
11. The method of claim 1 , wherein the bacteria are of the species Streptococcus thermophilus.
12. The method of claim 1 , wherein said contacting of step (a) comprised expression in said bacteria of a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (LKTLKIFVLFSLLIAILPYFAGCL) a derivative thereof having a percentage of identity of at least 90% with the amino acid sequence SEQ ID NO: 1; or a fragment of SEQ ID NO: 1, wherein said derivative or fragment is capable of stimulating competence in Streptococcus and wherein the nucleotide sequence encoding the polypeptide is operably linked to a gene expression sequence functional in said bacterium.
13. The method of claim 12 , wherein the gene expression sequence comprises a constitutive promoter or an inducible promoter.
14. The method of claim 12 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or of a derivative thereof having a percentage of identity of at least 90% with the amino acid sequence of SEQ ID NO: 1.
15. The method of claim 12 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1.
16. The method of claim 12 , wherein the nucleotide further encodes a signal peptide.
17. The method of claim 1 , wherein said contacting of step (a) comprises culturing said bacteria in a peptide-free medium and adding the effective amount of the polypeptide to the peptide-free medium.
18. The method of claim 1 , wherein the contacting of step (a) comprises the steps of (i) culturing said bacteria in a peptide-free medium to an OD600 between 1.5 and 2.5 and (ii) diluting the culture of step (i) to an OD600 between 0.01 and 0.1 prior to adding the effective amount of the polypeptide to the growth medium.