Monocins and methods of use
The disclosure relates to the identification, cloning, and expression of a genetic locus within a Listeria monocytogenes genome that encodes a phage tail-like bacteriocin (PTLB), termed a monocin. Also provided are non-natural monocins, which have been engineered to have altered bactericidal specificity. Nucleic acid molecules encoding natural or non-natural monocins, vector constructs containing such nucleic acids operably linked to a heterologous promoter, producer cells containing such vectors, the encoded monocins, as well as methods of making and using such monocins are described.
1. An isolated nucleic acid molecule encoding an F-type bacteriocin, wherein the nucleic acid molecule comprises a first polynucleotide sequence that encodes the following structural proteins: a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; and a structural protein at least 90% identical to SEQ ID NO: 16,
wherein the nucleic acid molecule further comprises a heterologous second polynucleotide sequence encoding a heterologous receptor binding protein (RBP), and wherein the RBP comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29,
and wherein the F-type bacteriocin has bactericidal activity against at least one strain of Listeria monocytogenes.
2. A vector comprising the nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is operably linked to an inducible promoter.
3. The vector of claim 2 , wherein the promoter is a small molecule inducible promoter.
4. An isolated nucleic acid molecule encoding an F-type bacteriocin of claim 1 , wherein the nucleic acid molecule further comprises a heterologous promoter inducible by a small molecule, wherein the first polynucleotide sequence is operably linked to the heterologous promoter.
5. The nucleic acid molecule of claim 4 , wherein said nucleic acid molecule further comprises a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 5, and wherein the promoter is placed at 11, 14, 17, 20, or 23 nucleotides upstream of the nucleic acid sequence encoding SEQ ID NO: 5.
6. A vector comprising the nucleic acid molecule of claim 5 .
7. An isolated F-type bacteriocin producer cell comprising the vector of claim 2 .
8. The isolated nucleic acid molecule of claim 1 , wherein the RBP comprises the amino acid sequence of SEQ ID NO: 29.
9. An isolated F-type bacteriocin producer cell comprising a first foreign polynucleotide sequence that encodes the following structural proteins: a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; and a structural protein at least 90% identical to SEQ ID NO: 16, and
wherein said producer cell further comprises a second foreign polynucleotide sequence encoding a heterologous receptor binding protein (RBP), and wherein the RBP comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29,
and wherein the F-type bacteriocin has bactericidal activity against at least one strain of Listeria monocytogenes.
10. The isolated producer cell of claim 9 , wherein the first and second foreign polynucleotide sequences are contained within separate nucleic acid molecules.
11. The isolated producer cell of claim 9 , wherein the first and second foreign polynucleotides are contained within one nucleic acid molecule.
12. The isolated producer cell of claim 9 , wherein said first and second foreign polynucleotide sequences are operably linked to a heterologous promoter inducible by a small molecule.
13. A method of producing an F-type bacteriocin, comprising exposing the F-type bacteriocin producer cell of claim 12 to an inducing agent in a concentration effective to induce expression of the F-type bacteriocin via the inducible promoter, thereby producing the F-type bacteriocin.
14. The isolated producer cell of claim 9 , wherein the RBP comprises the amino acid sequence of SEQ ID NO: 29.
15. An F-type bacteriocin, wherein said F-type bacteriocin is encoded by a nucleic acid molecule comprising a first polynucleotide sequence and a second polynucleotide sequence,
wherein said first polynucleotide sequence encodes the following structural proteins: a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; and a structural protein at least 90% identical to SEQ ID NO: 16, and
wherein said second polynucleotide sequence encodes a heterologous receptor binding protein (RBP), and wherein the RBP comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29,
and wherein the F-type bacteriocin has bactericidal specificity against at least one strain of Listeria monocytogenes.
16. A method of killing a Listeria monocytogenes, comprising contacting the L. monocytogenes with an effective amount of the F-type bacteriocin of claim 15 , whereby the F-type bacteriocin binds and kills the L. monocytogenes.
17. The method of claim 16 , wherein the contacting is with a surface contaminated with L. monocytogenes.
18. The method of claim 17 , wherein the contacting and killing are at 4°-10° C.
19. The F-type bacteriocin of claim 15 , wherein the RBP comprises the amino acid sequence of SEQ ID NO: 29.
20. An isolated nucleic acid molecule encoding an F-type bacteriocin, wherein the nucleic acid molecule comprises a polynucleotide sequence that encodes the following proteins:
a structural protein at least 90% identical to SEQ ID NO: 7; a structural protein at least 90% identical to SEQ ID NO: 8; a structural protein at least 90% identical to SEQ ID NO: 9; a structural protein at least 90% identical to SEQ ID NO: 10; a structural protein at least 90% identical to SEQ ID NO: 11; a structural protein at least 90% identical to SEQ ID NO: 12; a structural protein at least 90% identical to SEQ ID NO: 13; a structural protein at least 90% identical to SEQ ID NO: 14; a structural protein at least 90% identical to SEQ ID NO: 15; a structural protein at least 90% identical to SEQ ID NO: 16; and a protein at least 90% identical to SEQ ID NO: 17,
wherein said polynucleotide sequence is operably linked to a heterologous inducible promoter.
21. A vector comprising the nucleic acid molecule of claim 20 .
22. The nucleic acid molecule of claim 20 , wherein the promoter is a small molecule inducible promoter.
23. The nucleic acid molecule of claim 20 , wherein said nucleic acid molecule further comprises a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 5, and wherein the promoter is placed at 11, 14, 17, 20, or 23 nucleotides upstream of the nucleic acid sequence encoding SEQ ID NO: 5.
24. A vector comprising the nucleic acid molecule of claim 23 .
25. An isolated F-type bacteriocin producer cell comprising the nucleic acid molecule of claim 20 .
26. An isolated F-type bacteriocin producer cell comprising the nucleic acid molecule of claim 22 .
27. A method of producing an F-type bacteriocin, comprising exposing the F-type bacteriocin producer cell of claim 25 to an inducing agent in a concentration effective to induce expression of the F-type bacteriocin via the inducible promoter, thereby producing the F-type bacteriocin.