Characterization and comparison of CRISPR Loci in Streptococcus thermophilus

  • Tong Hu
  • Yanhua CuiEmail author
  • Xiaojun Qu
Original Paper


Clustered regularly interspaced short palindromic repeats (CRISPR) consists of a series of regular repeat-spacer sequences. It can not only act as a natural immune system in most prokaryotes, but also be utilized as the tool of newly developed genome modification and evolutionary researches. Streptococcus thermophilus is an important model organism for the study and application of CRISPR systems. In present study, the occurrence and diversity of CRISPR–Cas systems in the genomes of S. thermophilus were investigated including 4 new sequenced strains CS5, CS9, CS18, CS20, and other 23 strains downloaded from NCBI website. 66 CRISPR/Cas systems were identified among these 27 strains and could divided into four subsystems according to the arrangement of Cas proteins, notably I-E, II-A, II-C and III-A. Overall, 26 type II-C systems, 18 type II-A systems, 13 type III-A systems, 9 type I-E systems were identified. It was mentioned that CS20 contained two type II-C systems which had not been identified in the other 26 S. thermophilus strains. Overall, 1,080 spacers were analyzed and blasted. Sequence identity searches of spacers implied that most spacers derived from partial sequences of exogenous DNA, including various bacteriophages and plasmids. Of note, a large number of novel spacers were found in this study, indicating the unique phage environment they have undergone, especially CS20 strain. In addition, the analysis of the cas1 and cas9 genes revealed the genetic relationship among CRISPR–Cas system in these strains. Furthermore, the analysis of CRISPR spacers also indicated protospacer adjacent motif (PAM) sequences. Summary of PAM sequences could lay the foundations for the application of S. thermophilus CRISPR–Cas system. Our results suggested CS5 and CS18 can be used as model strains in the research of CRISPR–Cas system, and CS20 might have greater application potential in gene editing.


CRISPR–Cas systems Diversity Streptococcus thermophilus Probiotics Spacer 



This work was supported by National Natural Science Foundation of China (Grant nos. 31471712; 31371827).

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interests to declare.

Supplementary material

203_2019_1780_MOESM1_ESM.docx (28 kb)
Supplementary file1 (DOCX 27 kb)
203_2019_1780_MOESM2_ESM.docx (94 kb)
Supplementary file2 (DOCX 94 kb)


  1. Achigar R, Magadán AH, Tremblay DM, Pianzzola MJ, Moineau S (2017) Phage-host interactions in Streptococcus thermophilus: genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array. Sci Rep 7:43438PubMedPubMedCentralCrossRefGoogle Scholar
  2. Ali Y, Koberg S, Heßner S, Sun X, Rabe B, Back A, Neve H, Heller KJ (2014) Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the ltp type. Front Microbiol 5:98PubMedPubMedCentralCrossRefGoogle Scholar
  3. Allison GE, Klaenhammer TR (1998) Phage resistance mechanisms in lactic acid bacteria. Int Dairy J 8:207–226CrossRefGoogle Scholar
  4. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedPubMedCentralCrossRefGoogle Scholar
  5. Barrangou R, Doudna JA (2016) Applications of CRISPR technologies in research and beyond. Nat Biotechnol 34(9):933–941PubMedCrossRefGoogle Scholar
  6. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712PubMedCrossRefGoogle Scholar
  7. Barrangou R, Horvath P (2017) A decade of discovery: CRISPR functions and applications. Nat Microbiol 2(7):17092PubMedCrossRefGoogle Scholar
  8. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151(8):2551–2561PubMedCrossRefGoogle Scholar
  9. Carte J, Christopher RT, Smith JT, Olson S, Barrangou R, Moineau S, Glover CV, Graveley BR, Terns RM, Terns MP (2014) The three major types of CRISPR–Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus. Mol Microbiol 93:98–112PubMedPubMedCentralCrossRefGoogle Scholar
  10. Chopin MC, Chopin A, Bidnenko E (2005) Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8:473–479PubMedCrossRefPubMedCentralGoogle Scholar
  11. Chylinski K, Makarova KS, Charpentier E, Koonin EV (2014) Classification and evolution of type II CRISPR–Cas systems. Nucleic Acids Res 42:6091–6105PubMedPubMedCentralCrossRefGoogle Scholar
  12. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190PubMedPubMedCentralCrossRefGoogle Scholar
  13. Cui Y, Xu T, Qu X, Hu T, Xu J, Zhao C (2016) New insights into various production characteristics of Streptococcus thermophilus strains. Int J Mol Sci 17(10):1701PubMedCentralCrossRefPubMedGoogle Scholar
  14. Cusack S (1999) RNA–protein complexes. Curr Opin Struct Biol 9:66–73PubMedCrossRefGoogle Scholar
  15. Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3:945PubMedCrossRefGoogle Scholar
  16. Deng K, Huo G (2013) Detection and homology analysis of CRISPR in Streptococcus thermophilus. Food Sci 34:153–157 (In Chinese) Google Scholar
  17. Deng W, Wang Y, Liu Z, Cheng H, Xue Y (2014) Hemi: a toolkit for illustrating heatmaps. PLoS One 9(11):e111988PubMedPubMedCentralCrossRefGoogle Scholar
  18. Deveau H, Barrangou R, Garneau JE, Labonte J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190:1390–1400PubMedCrossRefGoogle Scholar
  19. Fernandez MA, Picard-Deland É, Le Barz M, Daniel N, Marette A (2017) Chap. 13 Yogurt and health. In: Frías J, Martínez-Villaluenga C, Peñas E (eds) Fermented foods in health and disease prevention. Academic Press, Cambridge, pp 305–338CrossRefGoogle Scholar
  20. Freitas M (2017) Chapter 24. The benefits of yogurt, cultures, and fermentation. In: Floch MH, Ringel Y, Walker WA (eds) The microbiota in gastrointestinal pathophysiology. Implications for human health, prebiotics, probiotics, and dysbiosis. Academic Press, Cambridge, pp 209–223CrossRefGoogle Scholar
  21. Fujii W, Kakuta S, Yoshioka S, Kyuwa S, Sugiura K, Naito K (2016) Zygote-mediated generation of genome-modified mice using Streptococcus thermophilus 1-derived CRISPR/Cas system. Biochem Biophys Res Commun 477(3):473–476PubMedCrossRefGoogle Scholar
  22. Garneau JE, Dupuis M, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468(7320):67–71PubMedCrossRefGoogle Scholar
  23. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 109:15539–15540CrossRefGoogle Scholar
  24. Gasiunas G, Sinkunas T, Siksnys V (2014) Molecular mechanisms of CRISPR-mediated microbial immunity. Cell Mol Life Sci 71:449–465PubMedCrossRefGoogle Scholar
  25. Godde JS, Bickerton A (2006) The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes. J Mol Evol 62:718–729PubMedCrossRefGoogle Scholar
  26. Goh YJ, Goin C, O’Flaherty S, Altermann E, Hutkins R (2011) Specialized adaptation of a lactic acid bacterium to the milk environment: the comparative genomics of Streptococcus thermophilus LMD-9. Microb Cell Fact 10:S22PubMedPubMedCentralCrossRefGoogle Scholar
  27. Grissa I, Vergnaud G, Pourcel C (2007a) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:1–6CrossRefGoogle Scholar
  28. Grissa I, Vergnaud G, Pourcel C (2007b) The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinform 8:172–182CrossRefGoogle Scholar
  29. Haft DH, Selengut J, Mongodin EF, Nelson KE (2005) A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 1(6):e60PubMedPubMedCentralCrossRefGoogle Scholar
  30. Hao M, Cui Y, Qu X (2018) Analysis of CRISPR–Cas system in Streptococcus thermophilus and its application. Front Microbiol 9:257PubMedPubMedCentralCrossRefGoogle Scholar
  31. Hatmaker EA, Riley LA, O'Dell KB, Papanek B, Graveley B, Garrett SC, Wei Y, Terns MP, Guss AM (2018) Complete genome sequence of industrial dairy strain Streptococcus thermophilus DGCC 7710. Genome Announc 6(6):e01587–e1617PubMedPubMedCentralCrossRefGoogle Scholar
  32. He Y, Cheng A, Wang M, Zhu D, Wang X, Zhang X (2013) Sequence analysis of the cas2 gene in riemerella anatipestifer. Adv Mater Res 647(3):570–576CrossRefGoogle Scholar
  33. Hidalgo-Cantabrana C, Crawley AB, Sanchez B, Barrangou R (2017) Characterization and exploitation of CRISPR loci in Bifidobacterium longum. Front Microbiol 8:1851PubMedPubMedCentralCrossRefGoogle Scholar
  34. Hofacker IL, Fontana W, Stadler PF, Bonhoeffer LS, Tacker M, Schuster P (1994) Fast folding and comparison of RNA secondary structures. Monatsh Chem 125:167–188CrossRefGoogle Scholar
  35. Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170PubMedCrossRefGoogle Scholar
  36. Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R (2008) Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol 190:1401–1412PubMedCrossRefGoogle Scholar
  37. Hrle A, Maier LK, Sharma K, Ebert J, Basquin C, Urlaub H, Marchfelder A, Conti E (2014) Structural analyses of the crispr protein csc2 reveal the RNA-binding interface of the type I-D cas7 family. RNA Biol 11(8):1072–1082PubMedPubMedCentralCrossRefGoogle Scholar
  38. Hu T, Zhang Y, Cui Y, Zhao C, Jiang X, Zhu X, Wang Y, Qu X (2018) Technological properties assessment and two component systems distribution of Streptococcus thermophilus strains isolated from fermented milk. Arch Microbiol 200(4):567–580PubMedCrossRefGoogle Scholar
  39. Hynes AP, Labrie SJ, Moineau S (2016a) Programming native CRISPR arrays for the generation of targeted immunity. MBio 7:e00202–00216PubMedPubMedCentralCrossRefGoogle Scholar
  40. Hynes AP, Lemay ML, Moineau S (2016b) Applications of CRISPR–Cas in its natural habitat. Curr Opin Chem Biol 34:30–36PubMedCrossRefGoogle Scholar
  41. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821PubMedPubMedCentralCrossRefGoogle Scholar
  42. Kala S, Cumby N, Sadowski PD, Hyder BZ, Kanelis V, Davidson AR, Maxwell KL (2014) HNH proteins are a widespread component of phage DNA packaging machines. Proc Natl Acad Sci USA 111:6022–6027PubMedCrossRefGoogle Scholar
  43. Koo Y, Jung D, Bae E (2012) Crystal structure of Streptococcus pyogenes csn2 reveals calcium-dependent conformational changes in its tertiary and quaternary structure. PLoS One 7(3):e33401PubMedPubMedCentralCrossRefGoogle Scholar
  44. Koonin EV, Makarova KS, Zhang F (2017) Diversity, classification and evolution of CRISPR–Cas systems. Curr Opin Microbiol 37:67–78PubMedPubMedCentralCrossRefGoogle Scholar
  45. Kumar S, Stecher G, Tamura K (2016) Mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1897PubMedPubMedCentralCrossRefGoogle Scholar
  46. Kunin V, Sorek R, Hugenholtz P (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 8:1–7CrossRefGoogle Scholar
  47. Labrie SJ, Tremblay DM, Plante PL, Wasserscheid J, Dewar K, Corbeil J, Moineau S (2015) Complete genome sequence of Streptococcus thermophilus SMQ-301, a model strain for phage-host interactions. Genome Announc 3:e480–e1415CrossRefGoogle Scholar
  48. Li B, Ding X, Evivie SE, Jin D, Meng Y, Huo G, Liu F (2017) Short communication: genomic and phenotypic analyses of exopolysaccharides produced by, Streptococcus thermophilus KLDS SM. J Dairy Sci 101(1):106–112PubMedCrossRefGoogle Scholar
  49. Lillestøl RK, Redder P, Garrett RA, Brügger K (2006) A putative viral defence mechanism in archaeal cells. Archaea 2:59–72PubMedPubMedCentralCrossRefGoogle Scholar
  50. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV (2011) Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol 9:467–477PubMedCrossRefPubMedCentralGoogle Scholar
  51. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV (2015) An updated evolutionary classification of CRISPR–Cas systems. Nat Rev Microbiol 13:722–736PubMedPubMedCentralCrossRefGoogle Scholar
  52. Marraffini LA, Sontheimer EJ (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11:181–190PubMedPubMedCentralCrossRefGoogle Scholar
  53. McGinn J, Marraffini LA (2016) CRISPR–Cas systems optimize their immune response by specifying the site of spacer integration. Mol Cell 64:616–623PubMedPubMedCentralCrossRefGoogle Scholar
  54. Mills S, Griffin C, Coffey A, Meijer WC, Hafkamp B, Ross RP (2010) CRISPR analysis of bacteriophage insensitive mutants (BIMs) of industrial Streptococcus thermophilus implications for starter design. J Appl Microbiol 108(3):945–955PubMedCrossRefPubMedCentralGoogle Scholar
  55. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Almendros C (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155(Pt 3):733–740PubMedPubMedCentralGoogle Scholar
  56. Paezespino D, Sharon I, Morovic W, Stahl B, Thomas BC, Barrangou R, Banfield JF (2015) CRISPR immunity drives rapid phage genome evolution in Streptococcus thermophilus. Mbio 6(2):e00262–e315Google Scholar
  57. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011) The Streptococcus thermophilus crispr/cas system provides immunity in Escherichia coli. Nucleic Acids Res 39(21):9275–9282PubMedPubMedCentralCrossRefGoogle Scholar
  58. Shi Y, Chen Y, Li Z, Yang L, Chen W, Mu Z (2015) Complete genome sequence of Streptococcus thermophilus MN-BM-A02, a rare strain with a high acid-producing rate and low post-acidification ability. Genome Announc 3(5):e00979–e1015PubMedPubMedCentralCrossRefGoogle Scholar
  59. Sinkunas T, Gasiunas G, Waghmare SP, Dickman MJ, Barrangou R, Horvath P, Siksnys V (2013) In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. Embo J 32:385–394PubMedPubMedCentralCrossRefGoogle Scholar
  60. Stern A, Mick E, Tirosh I, Sagy O, Sorek R (2012) CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome. Genome Res 22:1985–1994PubMedPubMedCentralCrossRefGoogle Scholar
  61. Stranges PB, Esvelt KM, Moosburner M (2013) Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838PubMedPubMedCentralCrossRefGoogle Scholar
  62. Sun L, Zhang X, Gao S, Rao PA, Padilla-Sanchez V, Chen Z, Sun S, Xiang Y, Subramaniam S, Rao VB, Rossmann MG (2015) Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution. Nat Commun 6:7548PubMedPubMedCentralCrossRefGoogle Scholar
  63. Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H, Drungowski M, Elge T, Brosius J, Hüttenhofer A (2002) Identification of 86 candidates for small non-messenger rnas from the archaeon archaeoglobus fulgidus. Proc Natl Acad Sci USA 99(11):7536–7541PubMedCrossRefGoogle Scholar
  64. Tremblay DM, Moineau S (1999) Complete genomic sequence of the lytic bacteriophage DT1 of Streptococcus thermophilus. Virology 255(1):63–76PubMedCrossRefGoogle Scholar
  65. Turgeon N, Frenette M, Moineau S (2004) Characterization of a theta-replicating plasmid from Streptococcus thermophilus. Plasmid 51:24–36PubMedCrossRefGoogle Scholar
  66. Uriot O, Denis S, Junjua M, Roussel Y, Dary-Mourot A, Blanquet-Diot S (2017) Streptococcus thermophilus: from yogurt starter to a new promising probiotic candidate? J Funct Foods 37:74–89CrossRefGoogle Scholar
  67. van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJ (2009) CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci 34:401–407PubMedCrossRefGoogle Scholar
  68. Wei Y, Chesne MT, Terns RM, Terns MP (2015) Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus. Nucleic Acids Res 43:1749–1758PubMedPubMedCentralCrossRefGoogle Scholar
  69. Wu Q, Tun HM, Leung CC, Shah NP (2014) Genomic insights into high exopolysaccharide-producing dairy starter bacterium Streptococcus thermophilus ASCC 1275. Sci Rep UK 4(7500):4974–4974Google Scholar
  70. Young JC, Dill BD, Pan C, Hettich RL, Banfield JF, Shah M, Fremaux C, Horvath P, Barrangou R, Verberkmoes NC (2012) Phage-induced expression of CRISPR-associated proteins is revealed by shotgun proteomics in Streptococcus thermophilus. PLoS One 7:e38077PubMedPubMedCentralCrossRefGoogle Scholar

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© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Food Science and Engineering, School of Chemistry and Chemical EngineeringHarbin Institute of TechnologyHarbinPeople’s Republic of China
  2. 2.Institute of MicrobiologyHeilongjiang Academy of SciencesHarbinPeople’s Republic of China

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