Tagged microorganisms and methods of tagging

• US 20080124725A1
• Filed: 05/18/2007
• Published: 05/29/2008
• Est. Priority Date: 05/19/2006
• Status: Active Grant

• Current Assignee: International N&H Denmark APS (International Flavors & Fragrances Incorporated)
• Original Assignee: DuPont Nutrition Biosciences ApS (International Flavors & Fragrances Incorporated)
• Inventors: Barrangou, Rodolphe, Fremaux, Christophe, Romero, Dennis, Horvath, Philippe
• Granted Patent: US 9,399,801 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer

Abstract

The present invention provides methods for tagging and/or identifying microorganisms. In some preferred embodiments, the microorganisms are bacteria. In some particularly preferred embodiments, the bacteria are members of the genus Streptococcus, while in other embodiments, the bacteria are members of other genera. The present invention also provides microorganisms tagged using the methods set forth herein. In some preferred embodiments, the tagged microorganisms are bacteria. In some particularly preferred embodiments, the tagged bacteria are members of the genus Streptococcus, while in other embodiments, the tagged bacteria are members of other genera.

Claims

1. A method for tagging a bacterium comprising the steps of:

(a) exposing at least one parent bacterium comprising at least a portion of a CRISPR locus to at least one exogenous nucleic acid sequence to produce at least one tagged bacterium comprising a modified CRISPR locus, wherein said modified CRISPR locus comprises at least one additional repeat-spacer unit than said CRISPR locus of said parent bacterium and wherein said additional repeat-spacer unit comprises a tag; and

(b) comparing at least a portion of said CRISPR locus of said parent bacterium and said modified CRISPR locus of said tagged bacterium.

2. The method of claim 1, wherein said exogenous nucleic acid sequence is selected from bacteriophages, plasmids, megaplasmids, transposable elements, transposons, and insertion sequences.

3. The method of claim 1, wherein said tagged bacterium is a bacteriophage insensitive mutant.

4. The method of claim 1, wherein the 5′

end and/or the 3′

end of said CRISPR locus of the parent bacterium is compared with said modified CRISPR locus of said tagged bacterium.

5. The method of claim 1, wherein the 5′

and/or the 3′

end of the at least the first CRISPR repeat or at least the first CRISPR spacer of said CRISPR locus of said parent bacterium is compared with said modified CRISPR locus of said tagged bacterium.

6. The method of claim 1, further comprising the step of selecting said at least one tagged bacterium.

7. The method of claim 2, wherein said parent bacterium is simultaneously or sequentially exposed to two or more bacteriophages.

8. The method of claim 7, wherein said tagged bacterium comprises at least one additional repeat-spacer unit.

9. The method of claim 1, wherein said at least a portion of said CRISPR locus of said parent bacterium and at least a portion of said modified CRISPR locus of said tagged bacterium are compared by amplifying at least a portion of said CRISPR locus and at least a portion of said modified CRISPR locus, to produce an amplified CRISPR locus sequence and an amplified modified CRISPR locus sequence.

10. The method of claim 9, wherein said amplifying comprises polymerase chain reaction.

11. The method of claim 1, wherein said at least a portion of said CRISPR locus of said parent bacterium and at least a portion of said modified CRISPR locus of said tagged bacterium are compared by sequencing at least a portion of said CRISPR locus and at least a portion of said modified CRISPR locus.

12. The method of claim 9, further comprising the step of sequencing said amplified CRISPR locus sequence and said amplified modified CRISPR sequence locus.

13. The method of claim 1, wherein said additional repeat-spacer unit comprises at least about 44 nucleotides.

14. The method of claim 1, wherein said additional repeat-spacer unit comprises at least one nucleotide sequence that has at least 95% identity to a CRISPR repeat in said CRISPR locus of said parent bacterium.

15. The method of claim 1, wherein said additional repeat-spacer unit comprises at least one nucleotide sequence that has at least 95% identity to a nucleotide sequence in the genome of at least one bacteriophage.

16. The method of claim 15, wherein said tagged bacterium further comprises at least one additional nucleotide sequence that has at least 95% identity to a nucleotide sequence in the genome of said at least one bacteriophage.

17. The method of claim 1, wherein said parent bacterium is an industrially useful culture.

18. The method of claim 17, wherein said parent bacterium comprises a culture selected from starter cultures, probiotic cultures, and dietary supplement cultures.

19. The method of claim 1, wherein said parent bacterium is selected from Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, Brucella, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Lactobacillus, Pediococcus, Leuconostoc and Oenococcus.

20. The method of claim 2, wherein said at least one bacteriophage is selected from the group of virus families consisting of:

Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, and Tectiviridae.

21. A tagged bacterium obtained using the method of claim 1.

22. The tagged bacterium of claim 21, wherein said tagged bacterium is an industrially useful culture.

23. A cell culture comprising the tagged bacterium of claim 21.

24. The cell culture of claim 23, wherein said tagged bacterium comprises an industrially useful culture.

25. The cell culture of claim 24, wherein said tagged bacterium comprises a culture selected from starter cultures, probiotic cultures and dietary supplement cultures.

26. A food or feed comprising the tagged bacterium obtained using the method of claim 1.

27. A method for preparing a food or feed comprising the use of said tagged bacterium, wherein said tagged bacterium is added to said food or feed.

28. The method of claim 27, wherein said tagged bacterium comprises an industrially useful culture.

29. The method of claim 28, wherein said culture is selected from starter cultures, probiotic cultures, and dietary supplement cultures.

30. A food product or feed obtained using the method of claim 27.

31. A method for generating at least one CRISPR variant comprising a tag, comprising the steps of:

(a) exposing a parent bacterium comprising at least a portion of a CRISPR locus to at least one bacteriophage to produce a culture of bacteriophage resistant variant bacteria comprising a modified CRISPR locus;

(b) selecting said bacteriophage resistant variant bacteria;

(c) comparing said CRISPR locus or a portion thereof of said parent bacterium and said modified CRISPR locus of said bacteriophage insensitive variant, to identify bacteriophage insensitive variants comprising at least one tag, wherein said at least one tag comprises at least one additional nucleic acid fragment in said modified CRISPR locus that is absent from said CRISPR locus of said parent bacterium;

(d) selecting said bacteriophage insensitive variants comprising a tag; and

(e) analyzing said at least one tag.

32. The method of claim 31, wherein said analyzing is selected from isolating, cloning and sequencing.

33. At least one CRISPR variant obtained using the method of claim 31.

34. A cell culture comprising said at least one CRISPR variant of claim 33.

35. A food or feed comprising said at least one CRISPR variant of claim 33.

36. The method of claim 31, wherein said at least one tag is integrated into the CRISPR locus of said parent bacterium to produce said CRISPR variant.

38. The method of claim 31, wherein said database comprises nucleotide sequences of tagged bacteria.

39. The method of claim 31, wherein said database is selected from bacteriophage sequence databases and bacterial sequence databases.

37. A method for identifying a tagged bacterium comprising at least one CRISPR locus, comprising the steps of:

(a) screening said tagged bacterium for the presence of a tag in said CRISPR locus;

(b) determining the nucleotide sequence said tag;

(c) comparing said nucleotide sequence of said tag with nucleotide sequences present in at least one database; and

(d) identifying a nucleotide sequence in said database that shares homology with said tag.