Tagged Microorganisms and Methods of Tagging

• US 20100104690A1
• Filed: 05/18/2007
• Published: 04/29/2010
• 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, Horvath, Philippe
• Granted Patent: US 9,816,140 B2
• Time in Patent Office: Days
• US Class Current: 426/7
• 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 labelling a bacterium comprising the steps of:

(a) exposing a parent bacterium to a bacteriophage;

(b) selecting a bacteriophage insensitive mutant;

(c) comparing a CRISPR locus or a portion thereof from the parent bacterium and the bacteriophage insensitive mutant; and

(d) selecting a labelled bacterium comprising an additional repeat-spacer unit in the CRISPR locus that is not present in the parent bacterium.

2. The method according to claim 1, wherein the 5′

end and/or the 3′

end of the CRISPR locus of the parent bacterium is compared.

3. The method according to claim 1 or claim 2, wherein at least the first CRISPR repeat or the first CRISPR spacer at the 5′

end of the CRISPR locus is compared.

4. The method according to any of the preceding claims, wherein at least the last CRISPR repeat or the last CRISPR spacer at the 3′

end of the CRISPR locus is compared.

5. The method according to any of the preceding claims, wherein the method comprises the step of selecting a labelled bacterium comprising an additional repeat-spacer unit at the 5′

end and/or at the 3′

end of the CRISPR locus that is not present in the parent bacterium.

6. The method according to any of the preceding claims, wherein said method comprises exposing the parent bacterium to two or more bacteriophage either simultaneously or sequentially.

7. The method according to any of the preceding claims, wherein the CRISPR locus or a portion thereof from the parent bacterium and the bacteriophage insensitive mutant are compared by amplifying said CRISPR locus or a portion thereof from said parent bacterium and/or said bacteriophage insensitive mutant.

8. The method according to claim 7, wherein the amplification is performed using PCR.

9. The method according to any of the preceding claims, wherein the CRISPR locus or a portion thereof from the parent bacterium and the bacteriophage insensitive mutant are compared by sequencing said CRISPR locus or a portion thereof from said parent bacterium and/or said bacteriophage insensitive mutant.

10. The method according to any of the preceding claims, wherein the CRISPR locus or a portion thereof from the parent bacterium and the bacteriophage insensitive mutant are compared by amplifying and then sequencing said CRISPR locus or a portion thereof from said parent bacterium and/or said bacteriophage insensitive mutant.

11. The method according to any of the preceding claims, wherein the additional repeat-spacer unit is at least 44 nucleotides in length.

12. The method according to any of the preceding claims, wherein a labelled bacterium comprising two or three or more additional repeat-spacer units is selected.

13. The method according to any of the preceding claims, wherein the additional repeat-spacer unit comprises at least one nucleotide sequence that has at least 95% identity, preferably, 100% identity to a CRISPR repeat in the CRISPR locus of the parent bacterium.

14. The method according to any of the preceding claims, wherein the additional repeat-spacer unit comprises at least one nucleotide sequence that has at least 95% identity, preferably, 100% identity to a nucleotide sequence in the genome of the bacteriophage used for the selection of the labelled bacterium.

15. The method according to any of the preceding claims, wherein the additional repeat-spacer unit comprises a first nucleotide sequence that has at least 95% identity, preferably, 100% identity to a CRISPR repeat in the CRISPR locus of the parent bacterium and a second nucleotide sequence that has at least one nucleotide sequence that has at least 95% identity, preferably, 100% identity to a nucleotide sequence in the genome of the bacteriophage used for the selection of the labelled bacterium.

16. The method according to any of the preceding claims wherein the parent bacterium is suitable for use as a starter culture, a probiotic culture or a dietary supplement.

17. The method according to any of the preceding claims wherein the parent bacterium is selected from the group of genera consisting of:

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.

18. The method according to any of preceding claims, wherein the bacteriophage is selected from the group of virus families consisting of:

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

19. A labelled bacterium obtained or obtainable by the method according to any of claims 1-18.

20. A cell culture comprising a labelled bacterium according to claim 19.

21. The cell culture according to claim 20, wherein said cell culture is a starter culture, a probiotic culture or a dietary supplement.

22. A food product or feed comprising the labelled bacterium according to claim 19 or the cell culture according to claim 20 or claim 21.

23. A process for preparing a food product or feed comprising the use of the labelled bacterium according to claim 19 or the cell culture according to claim 20 or claim 21.

24. A process for preparing a food product or feed comprising the step of adding the labelled bacterium according to claim 19 or the cell culture according to claim 20 or claim 21 to said food product or feed.

25. A food product or feed obtained or obtainable by the process of claim 23 or 24.

28. Use of the labelled bacterium according to claim 19 or the cell culture according to claim 20 or claim 21 for preparing a food product.

33. A method for identifying a labelled bacterium comprising the steps of:

(a) screening the bacterium for an additional repeat-spacer unit in a CRISPR locus;

(b) determining the nucleotide sequence of the additional repeat-spacer unit;

(c) comparing said nucleotide sequence of said additional repeat-spacer unit with a database of labelled bacteria obtained or obtainable by the method according to any of claims 1-18; and

(d) identifying a nucleotide sequence in the database of labelled bacteria that matches the additional repeat-spacer unit.

26. A method for generating a CRISPR variant comprising the steps of:

(a) exposing a parent bacterium to a bacteriophage;

(b) selecting a bacteriophage resistant bacterium;

(c) comparing the CRISPR locus or a portion thereof from the parent bacterium and the bacteriophage insensitive mutant;

(d) selecting a labelled bacterium comprising an additional repeat-spacer unit in the CRISPR locus that is not present in the parent bacterium; and

(e) isolating and/or cloning and/or sequencing the additional repeat-spacer unit.

27. A CRISPR variant obtained or obtainable by the method according to claim 26.

29. Use of a nucleotide sequence obtained or obtainable from a bacteriophage for tagging and/or identifying a bacterium, wherein said nucleotide sequence is integrated within the CRISPR locus of the parent bacterium.

30. Use of a nucleotide sequence for labelling and/or identifying a bacterium, wherein said nucleotide sequence is obtained or obtainable by:

(a) exposing a parent bacterium to a bacteriophage;

(b) selecting a bacteriophage insensitive mutant;

(c) comparing a CRISPR locus or a portion thereof from the parent bacterium and the bacteriophage insensitive mutant; and

(d) selecting a labelled bacterium comprising an additional repeat-spacer unit in the CRISPR locus that is not present in the parent bacterium.

31. A method for identifying a labelled bacterium comprising the step of screening the bacterium for an additional repeat-spacer unit within a CRISPR locus of said bacterium.

32. The method according to claim 31, wherein said method comprises the additional step of:

comparing said additional repeat-spacer unit with a bacteriophage sequence database and/or a bacterial sequence database.

34. 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.

35. The method of claim 34, wherein said exogenous nucleic acid sequence is selected from bacteriophages, plasmids, megaplasmids, transposable elements, transposons, and insertion sequences.

36. The method of claim 34, wherein said tagged bacterium is a bacteriophage insensitive mutant.

37. The method of claim 34, 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.

38. The method of claim 34, 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.

39. The method of claim 34, further comprising the step of selecting said at least one tagged bacterium.

40. The method of claim 35, wherein said parent bacterium is simultaneously or sequentially exposed to two or more bacteriophages.

41. The method of claim 40, wherein said tagged bacterium comprises at least one additional repeat-spacer unit.

42. The method of claim 34, 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.

43. The method of claim 42, wherein said amplifying comprises polymerase chain reaction.

44. The method of claim 34, 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.

45. The method of claim 42, further comprising the step of sequencing said amplified CRISPR locus sequence and said amplified modified CRISPR sequence locus.

46. The method of claim 34, wherein said additional repeat-spacer unit comprises at least about 44 nucleotides.

47. The method of claim 34, 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.

48. The method of claim 34, 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.

50. The method of claim 48, 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.

51. The method of claim 34, wherein said parent bacterium is an industrially useful culture.

52. The method of claim 51, wherein said parent bacterium comprises a culture selected from starter cultures, probiotic cultures, and dietary supplement cultures.

53. The method of claim 34, 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.

54. The method of claim 35, 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.

55. A tagged bacterium obtained using the method of claim 34.

56. The tagged bacterium of claim 55, wherein said tagged bacterium is an industrially useful culture.

57. A cell culture comprising the tagged bacterium of claim 55.

58. The cell culture of claim 57, wherein said tagged bacterium comprises an industrially useful culture.

59. The cell culture of claim 58, wherein said tagged bacterium comprises a culture selected from starter cultures, probiotic cultures and dietary supplement cultures.

60. A food or feed comprising the tagged bacterium obtained using the method of claim 34.

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

62. The method of claim 61, wherein said tagged bacterium comprises an industrially useful culture.

63. The method of claim 62, wherein said culture is selected from starter cultures, probiotic cultures, and dietary supplement cultures.

64. A food product or feed obtained using the method of claim 61.

65. 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.

66. The method of claim 65, wherein said analyzing is selected from isolating, cloning and sequencing.

67. At least one CRISPR variant obtained using the method of claim 65.

68. A cell culture comprising said at least one CRISPR variant of claim 67.

69. A food or feed comprising said at least one CRISPR variant of claim 67.

70. The method of claim 65, wherein said at least one tag is integrated into the CRISPR locus of said parent bacterium to produce said CRISPR variant.

71. 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.

72. The method of claim 71, wherein said database comprises nucleotide sequences of tagged bacteria.

73. The method of claim 71, wherein said database is selected from bacteriophage sequence databases and bacterial sequence databases.