Cultures with Improved Phage Resistance

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.

1 . A method for generating at least one bacteriophage resistant variant strain, comprising the steps of:
 (a) exposing a parent bacterial strain comprising at least a portion of a CRISPR locus to at least one nucleic acid sequence to produce a mixture of bacteria comprising at least one bacteriophage resistant variant strain comprising a modified CRISPR locus;   (b) selecting said bacteriophage resistant variant strain from said mixture of bacteria;   (c) selecting said bacteriophage resistant variant strains comprising an additional nucleic acid fragment in said modified CRISPR locus from said bacteriophage resistant strains selected in step (b); and   (d) isolating said at least one bacteriophage resistant variant strain, wherein said strain comprises an additional nucleic acid fragment in said modified CRISPR locus.   
     
     
         2 . The method of  claim 1 , 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 nucleic acid fragment in said modified CRISPR locus that is absent from said CRISPR locus of said parent bacterial strain. 
     
     
         3 . The method of  claim 2 , further comprising the step of selecting said bacteriophage resistant variant strains comprising an additional nucleic acid fragment in said modified CRISPR locus. 
     
     
         4 . The method of  claim 1 , wherein said parent bacterial strain is exposed to two or more nucleic acid sequences. 
     
     
         5 . The method of  claim 1 , wherein said parent bacterial strain is simultaneously exposed to two or more nucleic acid sequences. 
     
     
         6 . The method of  claim 1 , wherein said parent bacterial strain is sequentially exposed to two or more nucleic acid sequences. 
     
     
         7 . The method of  claim 1 , wherein said parent bacterial strain is exposed to said nucleic acid sequence through infection by at least one bacteriophage comprising said nucleic acid sequence 
     
     
         8 . The method of  claim 7 , 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. 
     
     
         9 . The method of  claim 7 , wherein said at least one bacteriophage is a naturally occurring bacteriophage. 
     
     
         10 . The method of  claim 7 , wherein said at least one bacteriophage is a mutated bacteriophage obtained through selective pressure using a bacteriophage resistant bacterium. 
     
     
         11 . The method of  claim 1 , wherein said parent bacterial strain is exposed to said nucleic acid through a natural mechanism of nucleic acid uptake. 
     
     
         12 . The method of  claim 11 , wherein said natural mechanism of nucleic acid uptake comprises natural competence. 
     
     
         13 . The method of  claim 11 , wherein said natural mechanism of nucleic acid uptake parent bacterial strain by conjugation or transformation. 
     
     
         14 . The method of  claim 1 , wherein said bacteriophage resistant strain is a bacteriophage insensitive mutant. 
     
     
         15 . The method of  claim 1 , wherein said parent bacterial strain is a bacteriophage insensitive mutant. 
     
     
         16 . The method of  claim 1 , 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. 
     
     
         17 . 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 bacterial strain is compared with said modified CRISPR locus of said bacteriophage resistant variant strain. 
     
     
         18 . The method of  claim 1 , wherein said bacteriophage resistant variant strain comprises at least one additional nucleic acid fragment in said modified CRISPR locus. 
     
     
         19 . The method of  claim 1 , 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. 
     
     
         20 . The method of  claim 19 , wherein said amplifying is conducted using the polymerase chain reaction. 
     
     
         21 . The method of  claim 1 , 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. 
     
     
         22 . The method of  claim 19 , further comprising the step of sequencing said amplified CRISPR locus sequence and said amplified modified CRISPR sequence locus. 
     
     
         23 . The method of  claim 1 , wherein said additional nucleic acid fragment in said modified CRISPR locus is an additional repeat-spacer unit. 
     
     
         24 . The method of  claim 1 , wherein said additional repeat-spacer unit comprises at least about 44 nucleotides. 
     
     
         25 . The method of  claim 1 , wherein said additional repeat-spacer unit comprises between about 44 and about 119 nucleotides. 
     
     
         26 . The method of  claim 1 , wherein said additional 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. 
     
     
         27 . The method of  claim 1 , wherein said additional 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. 
     
     
         28 . The method of  claim 1 , wherein said parent bacterial strain is an industrially useful strain. 
     
     
         29 . The method of  claim 28 , wherein said parent bacterial strain is susceptible to infection by at least one bacteriophage. 
     
     
         30 . The method of  claim 28 , wherein said parent bacterial strain comprises a strain obtained from culture selected from starter cultures, probiotic cultures, and dietary supplement cultures. 
     
     
         31 . The method of  claim 1 , 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.    
     
     
         32 . At least one bacteriophage resistant variant strain obtained using the method of  claim 1 . 
     
     
         33 . The bacteriophage resistant variant strain of  claim 1 , wherein said bacteriophage resistant variant strain is an industrially useful strain. 
     
     
         34 . The bacteriophage resistant variant strain of  claim 1 , wherein said bacteriophage resistant variant strain is selected from starter cultures, probiotic cultures and dietary supplement cultures. 
     
     
         35 . A composition comprising a bacteriophage resistant variant strain produced using the method set forth in  claim 1 . 
     
     
         36 . A composition comprising at least two bacteriophage resistant variant strains produced using the method set forth in  claim 1 . 
     
     
         37 . A food or feed comprising the composition of  claim 35  or  36 . 
     
     
         38 . A method for preparing a food or feed comprising adding said composition of  claim 35  or  36  to said food or feed. 
     
     
         39 . A starter culture, probiotic culture, or dietary supplement culture comprising the composition of  claim 35  or  36 . 
     
     
         40 . A fermentation method comprising adding the composition of  claim 35  or  36  to a starter culture. 
     
     
         41 . A fermentation method comprising adding the composition of  claim 35  or  36  to a fermentation medium, under conditions such that fermentation of the components of said fermentation medium occur. 
     
     
         42 . The method of  claim 41 , wherein said fermentation is unaffected by the presence of bacteriophages. 
     
     
         43 . The method of  claim 41 , wherein said fermentation medium is a food product. 
     
     
         44 . The method of  claim 43 , wherein said food product is a dairy product. 
     
     
         45 . The method of  claim 44 , wherein said dairy product is milk 
     
     
         46 . The method of  claim 41 , wherein at least two different compositions comprising two or more bacteriophage resistant variant strains are sequentially exposed to said fermentation medium. 
     
     
         47 . A method for reducing the detrimental bacteriophage population in a fermentation medium comprising exposing a fermentation medium to at least one bacteriophage resistant variant strain produced using the method of  claim 1 , under conditions such that the bacteriophage population is reduced. 
     
     
         48 . A method for generating at least one bacteriophage resistant variant strain, comprising the steps of:
 (a) exposing a parent bacterial strain comprising at least a portion of a CRISPR locus to at least one nucleic acid sequence to produce a mixture of bacteria comprising at least one bacteriophage resistant variant strain comprising a modified CRISPR locus;   (b) selecting said bacteriophage resistant variant strain from said mixture of bacteria;   (c) 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 nucleic acid fragment in said modified CRISPR locus that is absent from said CRISPR locus of said parent bacterial strain;   (d) selecting said bacteriophage resistant variant strains comprising an additional nucleic acid fragment in said modified CRISPR locus;   (e) analyzing said at least one additional nucleic acid fragment in said modified CRISPR locus to identify said at least one bacteriophage resistant variant strain; and   (f) isolating said at least one bacteriophage resistant variant strain.   
     
     
         49 . A method for generating CRISPR-escape phage mutants comprising:
 (a) obtaining: at least one parent phage and a phage-resistant bacterial strain comprising at least one CRISPR locus, wherein said CRISPR locus comprises a nucleic acid sequence that is at least about 95% identical to at least one protospacer sequence in the genome of said at least one parent phage;   (b) exposing said at least one parent phage to said phage-resistant bacterial strain, under conditions such that at least one phage variant is produced; and   (c) selecting said at least one phage variant, wherein said at least one phage variant exhibits the ability to infect the said phage-resistant bacterial strain and is a CRISPR-escape phage mutant.   
     
     
         50 . The method of  claim 49 , wherein said phage-resistant bacterial strain is a bacteriophage-resistant variant strain obtained using the method set forth in  claim 48 . 
     
     
         51 . The method of  claim 49 , further comprising the step of comparing at least a portion of said at least one protospacer sequence and a CRISPR motif positioned near said at least one protospacer sequence in said phage variant with the at least one protospacer sequence and CRISPR motif of said parent phage. 
     
     
         52 . The method of  claim 51 , further comprising the step of selecting said variant phages that infect said phage resistant bacterial strain, wherein said variant phages comprise said CRISPR-escape phage mutants, and wherein said CRISPR-escape phages comprise at least one mutation in said at least one protospacer sequence and/or in the CRISPR motif of said CRISPR-escape phage mutants. 
     
     
         53 . The method of  claim 49 , wherein said method is iteratively repeated one or more times using the said CRISPR-escape phage mutants and different CRISPR phage-resistant bacterial strain comprising at least one CRISPR locus, wherein said CRISPR locus comprises a nucleic acid sequence that is at least about 95% identical to at least one protospacer sequence in the genome of said CRISPR-escape phage mutants. 
     
     
         54 . The method of  claim 49 , 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 . The method of  claim 49 , wherein said phage-resistant bacterial strain is selected from  Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, 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, Streptococcus , and  Oenococcus.    
     
     
         56 . A CRISPR-escape phage mutant obtained using the method of  claim 49 . 
     
     
         57 . The CRISPR-escape phage mutant of  claim 56 , wherein two or more mutations are present in at least two protospacer sequences and/or in said CRISPR motif. 
     
     
         58 . A CRISPR-escape phage mutant, wherein the genome of said CRISPR-escape phage mutant is genetically engineered to comprise mutations in at least one protospacer and/or said CRISPR motif. 
     
     
         59 . A CRISPR-escape phage mutant, wherein at least one said CRISPR motif is mutated. 
     
     
         60 . A CRISPR-escape phage mutant, wherein at least one said CRISPR motif is deleted. 
     
     
         61 . A composition comprising at least one CRISPR-escape phage mutant of  claim 56 . 
     
     
         62 . A method for controlling bacterial populations in a product comprising exposing said composition of  claim 61  to a fermentation medium, wherein said fermentation medium contains at least one population of undesirable bacteria, under conditions such that the population of said undesirable bacteria is reduced, and said fermentation medium is used to generate said product. 
     
     
         63 . The method of  claim 62 , wherein said product is selected from foods, feeds, cosmetics, personal care products, health care products, veterinary products, and dietary supplements. 
     
     
         64 . The method of  claim 62 , wherein said method is repeated at least once and said different compositions of  claim 61  are used in rotation.