CULTURES WITH IMPROVED PHAGE RESISTANCE

• US 20150093473A1
• Filed: 12/15/2014
• Published: 04/02/2015
• Est. Priority Date: 03/02/2007
• 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, Phillippe, Romero, Dennis, Boyaval, Patrick
• Granted Patent: US 9,951,342 B2
• Time in Patent Office: Days
• US Class Current: 426/43
• CPC Class Codes: A23C 2220/202 Genetic engineering of microorganisms used in dairy technology
• CPC Class Codes: A23C 9/123 using only microorganisms of the genus lactobacteriaceae; Yoghurt A23C9/13 takes precedence
• CPC Class Codes: A23C 9/1238 using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
• CPC Class Codes: A23V 2400/249 Thermophilus
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 2795/00011 Details
• 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 and compositions related to modulating the resistance of a cell against a target nucleic acid or a transcription product thereof. In some preferred embodiments, the present invention provides compositions and methods for the use of one or more cas genes or proteins for modulating the resistance of a cell against a target nucleic acid or a transcription product thereof. In some embodiments, the present invention provides methods and compositions that find use in the development and use of strain combinations and starter culture rotations. In additional embodiments, the present invention provides methods for labelling and/or identifying bacteria. In some preferred embodiments, the present invention provides methods for the use of CRISPR loci to determine the potential virulence of a phage against a cell and the use of CRISPR-cas to modulate the genetic sequence of a phage for increased virulence level. In still further embodiments, the present invention provides means and compositions for the development and use of phages as biocontrol agents.

Claims

1-64. -64. (canceled)

65. A method for generating a mixed bacterial starter culture comprising at least two different bacteriophage resistant variant strains, comprising the steps of:

(a) exposing a parent bacterial strain comprising at least a portion of a CRISPR locus to at least one bacteriophage to produce a mixture of bacteria comprising at least one bacteriophage resistant variant strain comprising a modified CRISPR locus comprising at least one additional spacer in said modified CRISPR locus;

(b) exposing a parent bacterial strain comprising at least a portion of a CRISPR locus, different to the parent bacterial strain of step (a), to a different at least one bacteriophage to that of step (a) to produce a mixture of bacteria comprising at least another bacteriophage resistant variant strain comprising a modified CRISPR locus comprising at least one additional spacer in said modified CRISPR locus;

(c) selecting said bacteriophage resistant variant strains from said mixtures of bacteria;

(d) selecting said bacteriophage resistant variant strains comprising an additional spacer in said modified CRISPR locus from said bacteriophage resistant strains selected in step (c); and

(e) isolating said bacteriophage resistant variant strains, wherein said strains comprise an additional spacer in said modified CRISPR locus.

66. The method of claim 65, wherein the method comprises the steps of:

(a) exposing a parent bacterial strain comprising at least a portion of a CRISPR locus to a mixture of two or more bacteriophage to produce a mixture of bacteria comprising at least one bacteriophage resistant variant strain comprising a modified CRISPR locus comprising at least one additional spacer in said modified CRISPR locus;

(b) exposing a parent bacterial strain comprising at least a portion of a CRISPR locus, different to the parent bacterial strain of step (a), to a different mixture of two or more bacteriophage to that of step (a) to produce a mixture of bacteria comprising at least another bacteriophage resistant variant strain comprising a modified CRISPR locus comprising at least one additional spacer in said modified CRISPR locus;

(c) selecting said bacteriophage resistant variant strains from said mixtures of bacteria;

(d) selecting said bacteriophage resistant variant strains comprising an additional spacer in said modified CRISPR locus from said bacteriophage resistant strains selected in step (c); and

(e) isolating said bacteriophage resistant variant strains, wherein said strains comprise an additional spacer in said modified CRISPR locus.

67. The method of claim 65, wherein said method further comprises the step of comparing said CRISPR locus or a portion thereof of said parent bacterial strain and said modified CRISPR locus of said bacteriophage resistant variant strain to identify bacteriophage resistant variant strains comprising at least one additional spacer in said modified CRISPR locus that is absent from said CRISPR locus of said parent bacterial strain.

68. The method of claim 67, further comprising the step of selecting said bacteriophage resistant variant strains comprising an additional spacer in said modified CRISPR locus.

69. The method of claim 65, wherein said parent bacterial strain is exposed to two or more bacteriophages.

70. The method of claim 65, wherein said parent bacterial strain is simultaneously exposed to two or more bacteriophages.

71. The method of claim 65, wherein said parent bacterial strain is sequentially exposed to two or more bacteriophages.

72. The method of claim 65, wherein said parent bacterial strain is infected by the bacteriophage.

73. The method of claim 72, 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.

74. The method of claim 72, wherein said at least one bacteriophage is a naturally occurring bacteriophage.

75. The method of claim 72, wherein said at least one bacteriophage is a mutated bacteriophage obtained through selective pressure using a bacteriophage resistant bacterium.

76. The method of claim 65, wherein the 5′

end and/or the 3′

end of said CRISPR locus of the parent bacterial strain is compared with said modified CRISPR locus of said bacteriophage resistant variant strain.

77. The method of claim 65, wherein the 5′

and/or the 3′

end of the first CRISPR spacer of said CRISPR locus of said parent bacterial strain is compared with said modified CRISPR locus of said bacteriophage resistant variant strain.

78. The method of claim 65, wherein said at least a portion of said CRISPR locus of said parent bacterial strain and at least a portion of said modified CRISPR locus of said bacteriophage resistant variant strain 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.

79. The method of claim 78, wherein said amplifying is conducted using the polymerase chain reaction.

80. The method of claim 65, wherein said at least a portion of said CRISPR locus of said parent bacterial strain and at least a portion of said modified CRISPR locus of said bacteriophage resistant variant strain are compared by sequencing at least a portion of said CRISPR locus and at least a portion of said modified CRISPR locus.

81. The method of claim 78, further comprising the step of sequencing said amplified CRISPR locus sequence and said amplified modified CRISPR sequence locus.

82. The method of claim 65, wherein said additional spacer in said modified CRISPR locus forms part of a repeat-spacer unit.

83. The method of claim 82, wherein said repeat-spacer unit comprises at least about 44 nucleotides.

84. The method of claim 82, wherein said repeat-spacer unit comprises between about 44 and about 119 nucleotides.

85. The method of claim 82, wherein said repeat-spacer unit comprises at least one nucleotide sequence that has at least about 95% identity to a CRISPR repeat in said CRISPR locus of said parent bacterial strain.

86. The method of claim 82, wherein said repeat-spacer unit comprises at least one nucleotide sequence that has at least about 95% identity to a nucleotide sequence in the genome of at least one bacteriophage.

87. The method of claim 65, wherein said parent bacterial strain is an industrially useful strain.

88. The method of claim 87, wherein said parent bacterial strain is susceptible to infection by at least one bacteriophage.

89. The method of claim 87, wherein said parent bacterial strain is suitable for use as a starter cultures, a probiotic culture, and/or a dietary supplement culture.

90. The method of claim 65, wherein said parent bacterial strain 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, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Lactobacillus, Pediococcus, Leuconostoc, and Oenococcus.

91. The method of claim 65, wherein the parental bacterial strain is sensitive to each of the bacteriophages that it is exposed to in the mixture.

92. The method of claim 65, wherein the parental bacterial strain is sensitive to some of the bacteriophages that it is exposed to in the mixture.

93. The method of claim 65, wherein two or more difference spacers each integrate into two or more different CRISPR loci.

94. The method of claim 65, wherein two or more different spacers integrate into one CRISPR locus.

95. A starter culture obtained by the method of claim 65.

96. The starter culture according to claim 95, in the form of a dried starter culture, a dehydrated starter culture, a frozen starter culture, or a concentrated starter culture.

97. The starter culture according to claim 96 in the form or a tablet, pellet, capsule, dust, granule or powder.

98. The starter culture according to claim 96 in which the dried starter culture is wettable, spray dried, freeze-dried or lyophilized.

99. The starter culture according to claim 96 in the form of a deep frozen pellet or a freeze-dried powder.

100. The starter culture according to claim 96 in the form of a concentrate.

101. A fermentation method comprising adding the starter culture of claim 95 to a fermentation medium under conditions such that fermentation of the components of said fermentation medium occur.

102. The method of claim 101, wherein said fermentation is unaffected by the presence of bacteriophages.

103. The method of claim 101, wherein said fermentation medium is a food product.

104. The method of claim 103, wherein said food product is a dairy product.

105. The method of claim 104, wherein said dairy product is milk.