US 10,767,156 B2Granted

Polynucleotides encoding BREX system polypeptides and methods of using same

AssigneeYeda Research and Development Co. Ltd.

InventorsRotem Sorek, Hila Sberro, Tamara Goldfarb

Life of this patent

Estimating the term…

Loading patent…

Abstract

Isolated polynucleotides encoding a BREX system are provided. Accordingly there is provided an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ and at least one of pglX, pglXI, brxP, brxHI, brxHII, brxL, brxD, brxA, brxB, brxF, and brxE, with the proviso that said BREX system does not comprise pglW, and wherein said BREX system confers phage resistance to a bacteria recombinantly expressing same; Also provided is an isolated polynucleotide encoding a BREX system comprising a nucleic acid sequence encoding the BREX system comprising brxC/pglY, pglZ, pglX, pglW and at least one of brxD and brxHI, and wherein said BREX system confers phage resistance to a bacteria recombinantly expressing same. Also provided are compositions and methods for conferring phage resistance to bacteria or for conferring bacterial susceptibility to phages.

Claims9

Dependent claims

An expression vector comprising a nucleic acid sequence encoding a functionally active type 1 Bacteriophage Exclusion (BREX) system comprising a pglX polypeptide comprising an adenine-specific methylase domain (pfam13659, COG1002/COG0286), a brxC/pglY polypeptide comprising an ATP binding domain (pfam10923), a pglZ polypeptide comprising an alkaline phosphatase domain (pfam08665), a brxL polypeptide comprising a Lon-like protease domain (COG4930), a brxA polypeptide comprising a DUF1819 domain (pfam08849), and a brxB polypeptide comprising a DUF1788 domain (pfam08747);

wherein the amino acid sequences of each of said type 1 BREX system polypeptides, are set forth as follows:
(a) the brxA polypeptide amino acid sequence is selected from the group consisting of SEQ ID NOs:
4953-5064, 5102-5367, 5399-5414, and 6225;
(b) the brxB polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
5570-5686, 5698-5939, 5944-5947, 6206, and 6227;
(c) the brxC/pglY polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
615, 617-735, 818-1110, 1170-1175, and 6229;
(d) a pglX polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
2766-2916, 2954-3251, 3276-3280, 6182, 6190, 6192, 6194, 6196, 6198, and 6231;
(e) a pglZ polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
1716-1834, 1905-2192, 2248, 2250, 6204, and 6233;
and
(f) a brxL polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
4028-4298, 4300-4402, 6165, and 6235;
said expression vector further comprising a heterologous cis-acting regulatory element for directing expression of said nucleic acid sequence encoding the type 1 BREX system, wherein said type 1 BREX system confers phage resistance to a bacterium recombinantly expressing same, wherein said bacterium does not endogenously express a functional type 1 BREX system, and wherein said bacterium is a species selected from the group consisting of a Lactococcus species, a Streptococcus species, a Lactobacillus species, a Leuconostoc species, an Oenococcus species, a Pediococcus species, a Bifidobacterium species, and a Propionibacterium species.

12 more elements

An isolated, genetically modified bacterium, wherein said bacterium does not express a functional type 1 Bacteriophage Exclusion (BREX) system endogenously, modified to express a functionally active type 1 BREX system that confers phage resistance to said bacterium, said type 1 BREX system comprising a pglX polypeptide comprising an adenine-specific methylase domain (pfam13659, COG1002/COG0286), a brxC/pglY polypeptide comprising an ATP binding domain (pfam10923), a pglZ polypeptide comprising an alkaline phosphatase domain (pfam08665), a brxL polypeptide comprising a Lon-like protease domain (COG4930), a brxA polypeptide comprising a DUF1819 domain (pfam08849), and a brxB polypeptide comprising a DUF1788 domain (pfam08747);

wherein the amino acid sequences of each of said type 1 BREX system polypeptides, are set forth as follows:
(a) the brxA polypeptide amino acid sequence is selected from the group consisting of SEQ ID NOs:
4953-5064, 5102-5367, 5399-5414, and 6225;
(b) the brxB polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
5570-5686, 5698-5939, 5944-5947, 6206, and 6227;
(c) the brxC/pglY polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
615, 617-735, 818-1110, 1170-1175, and 6229;
(d) a pglX polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
2766-2916, 2954-3251, 3276-3280, 6182, 6190, 6192, 6194, 6196, 6198, and 6231;
(e) a pglZ polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
1716-1834, 1905-2192, 2248, 2250, 6204, and 6233;
and
(f) a brxL polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
4028-4298, 4300-4402, 6165, and 6235;
and wherein said bacterium is a species selected from the group consisting of a Lactococcus species, a Streptococcus species, a Lactobacillus species, a Leuconostoc species, an Oenococcus species, a Pediococcus species, a Bifidobacterium species, and a Propionibacterium species.

12 more elements

A method of protecting a bacterium from phage attack, wherein said bacterium does not express a functional type 1 Bacteriophage Exclusion (BREX) system endogenously, the method comprising:

(1) introducing into the bacterium an expression vector comprising a nucleic acid sequence encoding a functionally active type 1 Bacteriophage Exclusion (BREX) system comprising a pglX polypeptide comprising an adenine-specific methylase domain (pfam13659, COG1002/COG0286), a brxC/pglY polypeptide comprising an ATP binding domain (pfam10923), a pglZ polypeptide comprising an alkaline phosphatase domain (pfam08665), a brxL polypeptide comprising a Lon-like protease domain (COG4930), a brxA polypeptide comprising a DUF1819 domain (pfam08849), and a brxB polypeptide comprising a DUF1788 domain (pfam08747);
wherein the amino acid sequences of each of said type 1 BREX system polypeptides, are set forth as follows:
(a) the brxA polypeptide amino acid sequence is selected from the group consisting of SEQ ID NOs:
4953-5064, 5102-5367, 5399-5414, and 6225;
(b) the brxB polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
5570-5686, 5698-5939, 5944-5947, 6206, and 6227;
(c) the brxC/pglY polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
615, 617-735, 818-1110, 1170-1175, and 6229;
(d) a pglX polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
2766-2916, 2954-3251, 3276-3280, 6182, 6190, 6192, 6194, 6196, 6198, and 6231;
(e) a pglZ polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
1716-1834, 1905-2192, 2248 2250, and 6204, and 6233;
and
(f) a brxL polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
4028-4298, 4300-4402, 6165, and 6235;
and
(2) expressing in the bacterium said functional type 1 BREX system from said expression vector;
wherein upon phage attack, said functional type 1 BREX system confers phage resistance to the bacterium and thereby protects the bacterium from phage attack;
wherein said expression vector comprises a heterologous cis-acting regulatory element for directing expression of said nucleic acid sequence;
and wherein said bacterium is a species selected from the group consisting of a Lactococcus species, a Streptococcus species, a Lactobacillus species, a Leuconostoc species, an Oenococcus species, a Pediococcus species, a Bifidobacterium species, and a Propionibacterium species.

17 more elements

An isolated bacterium comprising a nucleic acid sequence encoding a functionally active type 1 Bacteriophage Exclusion (BREX) system, said type 1 BREX system expressed from a plasmid or a transposon, wherein said isolated bacterium does not endogenously express said type 1 BREX system, wherein said functionally active type 1 BREX system confers phage resistance to the bacterium recombinantly expressing same, and wherein said type 1 BREX system comprises a pglX polypeptide comprising an adenine-specific methylase domain (pfam13659, COG1002/COG0286), a brxC/pglY polypeptide comprising an ATP binding domain (pfam10923), a pglZ polypeptide comprising an alkaline phosphatase domain (pfam08665), a brxL polypeptide comprising a Lon-like protease domain (COG4930), a brxA polypeptide comprising a DUF1819 domain (pfam08849), and a brxB polypeptide comprising a DUF1788 domain (pfam08747);

wherein the amino acid sequences of each of said type 1 BREX system polypeptides, are set forth as follows:
(a) the brxA polypeptide amino acid sequence is selected from the group consisting of SEQ ID NOs:
4953-5064, 5102-5367, 5399-5414, and 6225;
(b) the brxB polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
5570-5686, 5698-5939, 5944-5947, 6206, and 6227;
(c) the brxC/pglY polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
615, 617-735, 818-1110, 1170-1175, and 6229;
(d) a pglX polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
2766-2916, 2954-3251, 3276-3280, 6182, 6190, 6192, 6194, 6196, 6198, and 6231;
(e) a pglZ polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
1716-1834, 1905-2192, 2248 2250, and 6204, and 6233;
and
(f) a brxL polypeptide amino acid sequence selected from the group consisting of SEQ ID NO:
4028-4298, 4300-4402, 6165, and 6235;
wherein said bacterium is a species selected from the group consisting of a Lactococcus species, a Streptococcus species, a Lactobacillus species, a Leuconostoc species, an Oenococcus species, a Pediococcus species, a Bifidobacterium species, and a Propionibacterium species.

12 more elements

Dependencies are inferred from the claim text.

Facts

Application: 15/030,876Filed 14 Oct 2014 · us provisional application
Priority: 24 Oct 2013
Published: US 2016/0281053 A129 Sep 2016
Granted: 8 Sep 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification24 codes

C12N 1/20
Microorganisms; Compositions thereof ; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of…:…
A23C 9/1234
Using only microorganisms of the genus lactobacteriaceae; Yoghurt: characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus,…
C07K 14/195
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from bacteria
C07K 14/245
From Enterobacteriaceae , e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia: escherichia