1887

Abstract

Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system. Although the primary outcome of the assay was efficient interference, a small proportion of the transformed population overcame targeting. Mutants containing plasmids that had escaped were recovered to investigate the genetic routes of escape and their relative frequencies. Deletion of the targeting spacer in the native CRISPR array was the dominant pattern of escape, accounting for 52–70 % of the mutants from two L. gasseri strains. We repeatedly observed internal deletions in the chromosomal CRISPR array, characterized by polarized excisions from the leader end that spanned 1–15 spacers, and systematically included the leader-proximal targeting spacer. This study shows that deletions of spacers within CRISPR arrays constitute a key escape mechanism to evade CRISPR targeting, while preserving the functionality of the CRISPR-Cas system. This mechanism enables cells to maintain an active immune system, but allows the uptake of potentially beneficial plasmids. Our study revealed the co-occurrence of other genomic mutations associated with various phenotypes, showing how this selection process uncovers population diversification.

Keyword(s): Cas , Cas9 , CRISPR , interference , Lactobacillus and spacer
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000689
2018-07-19
2024-11-12
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/9/1098.html?itemId=/content/journal/micro/10.1099/mic.0.000689&mimeType=html&fmt=ahah

References

  1. Selle K, Klaenhammer TR. Genomic and phenotypic evidence for probiotic influences of Lactobacillus gasseri on human health. FEMS Microbiol Rev 2013; 37:915–935 [View Article][PubMed]
    [Google Scholar]
  2. Lauer E, Kandler O. Lactobacillus gasseri sp. nov., a new species of the subgenus Thermobacterium. Zentralblatt fur Bakteriologie Mikrobiologie und Hygiene: I Abt Originale C 1980; 1:75–78
    [Google Scholar]
  3. Azcarate-Peril MA, Altermann E, Goh YJ, Tallon R, Sanozky-Dawes RB et al. Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism. Appl Environ Microbiol 2008; 74:4610–4625 [View Article][PubMed]
    [Google Scholar]
  4. Food and Agricultural Organization of the United Nations and World Health Organization Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food. Food and Agricultural Organization of the United Nations 2002
    [Google Scholar]
  5. Stoeker L, Nordone S, Gunderson S, Zhang L, Kajikawa A et al. Assessment of Lactobacillus gasseri as a candidate oral vaccine vector. Clin Vaccine Immunol 2011; 18:1834–1844 [View Article][PubMed]
    [Google Scholar]
  6. Baugher JL, Durmaz E, Klaenhammer TR. Spontaneously induced prophages in Lactobacillus gasseri contribute to horizontal gene transfer. Appl Environ Microbiol 2014; 80:3508–3517 [View Article][PubMed]
    [Google Scholar]
  7. Ismail EA, Neve H, Geis A, Heller KJ. Characterization of temperate Lactobacillus gasseri phage LgaI and its impact as prophage on autolysis of its lysogenic host strains. Curr Microbiol 2009; 58:648–653 [View Article][PubMed]
    [Google Scholar]
  8. Raya RR, Kleeman EG, Luchansky JB, Klaenhammer TR. Characterization of the temperate bacteriophage phi adh and plasmid transduction in Lactobacillus acidophilus ADH. Appl Environ Microbiol 1989; 55:2206–2213[PubMed]
    [Google Scholar]
  9. Sanozky-Dawes R, Selle K, O'Flaherty S, Klaenhammer T, Barrangou R. Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri. Microbiology 2015; 161:1752–1761 [View Article][PubMed]
    [Google Scholar]
  10. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007; 315:1709–1712 [View Article][PubMed]
    [Google Scholar]
  11. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321:960–964 [View Article][PubMed]
    [Google Scholar]
  12. Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 2008; 322:1843–1845 [View Article][PubMed]
    [Google Scholar]
  13. Barrangou R. Diversity of CRISPR-Cas immune systems and molecular machines. Genome Biol 2015; 16:247 [View Article][PubMed]
    [Google Scholar]
  14. Briner AE, Barrangou R. Deciphering and shaping bacterial diversity through CRISPR. Curr Opin Microbiol 2016; 31:101–108 [View Article][PubMed]
    [Google Scholar]
  15. Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 2014; 54:234–244 [View Article][PubMed]
    [Google Scholar]
  16. Hale C, Kleppe K, Terns RM, Terns MP. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA 2008; 14:2572–2579 [View Article][PubMed]
    [Google Scholar]
  17. Garneau JE, Dupuis , Villion M, Romero DA, Barrangou R et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 2010; 468:67–71 [View Article][PubMed]
    [Google Scholar]
  18. Sun Z, Harris HM, McCann A, Guo C, Argimón S et al. Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. Nat Commun 2015; 6:8322 [View Article][PubMed]
    [Google Scholar]
  19. Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nat Biotechnol 2016; 34:933–941 [View Article][PubMed]
    [Google Scholar]
  20. Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 2008; 190:1390–1400 [View Article][PubMed]
    [Google Scholar]
  21. Paez-Espino D, Morovic W, Sun CL, Thomas BC, Ueda K et al. Strong bias in the bacterial CRISPR elements that confer immunity to phage. Nat Commun 2013; 4:1430 [View Article][PubMed]
    [Google Scholar]
  22. Marraffini LA, Sontheimer EJ. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 2010; 463:568–571 [View Article][PubMed]
    [Google Scholar]
  23. Heler R, Samai P, Modell JW, Weiner C, Goldberg GW et al. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature 2015; 519:199–202 [View Article][PubMed]
    [Google Scholar]
  24. Hale CR, Majumdar S, Elmore J, Pfister N, Compton M et al. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol Cell 2012; 45:292–302 [View Article][PubMed]
    [Google Scholar]
  25. Carte J, Christopher RT, Smith JT, Olson S, Barrangou R et al. The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus. Mol Microbiol 2014; 93:98–112 [View Article][PubMed]
    [Google Scholar]
  26. Briner AE, Donohoue PD, Gomaa AA, Selle K, Slorach EM et al. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell 2014; 56:333–339 [View Article][PubMed]
    [Google Scholar]
  27. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011; 471:602–607 [View Article][PubMed]
    [Google Scholar]
  28. van der Oost J, Westra ER, Jackson RN, Wiedenheft B. Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat Rev Microbiol 2014; 12:479–492 [View Article][PubMed]
    [Google Scholar]
  29. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 2014; 507:62–67 [View Article][PubMed]
    [Google Scholar]
  30. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 2012; 109:E2579E2586 [View Article][PubMed]
    [Google Scholar]
  31. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337:816–821 [View Article][PubMed]
    [Google Scholar]
  32. Vercoe RB, Chang JT, Dy RL, Taylor C, Gristwood T et al. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet 2013; 9:e1003454 [View Article][PubMed]
    [Google Scholar]
  33. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 2013; 31:233–239 [View Article][PubMed]
    [Google Scholar]
  34. Stern A, Keren L, Wurtzel O, Amitai G, Sorek R. Self-targeting by CRISPR: gene regulation or autoimmunity?. Trends Genet 2010; 26:335–340 [View Article][PubMed]
    [Google Scholar]
  35. Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci USA 2011; 108:10098–10103 [View Article][PubMed]
    [Google Scholar]
  36. Wiedenheft B, van Duijn E, Bultema JB, Bultema J, Waghmare SP et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci USA 2011; 108:10092–10097 [View Article][PubMed]
    [Google Scholar]
  37. Selle K, Klaenhammer TR, Barrangou R. CRISPR-based screening of genomic island excision events in bacteria. Proc Natl Acad Sci USA 2015; 112:8076–8081 [View Article][PubMed]
    [Google Scholar]
  38. Jiang W, Maniv I, Arain F, Wang Y, Levin BR et al. Dealing with the evolutionary downside of CRISPR immunity: bacteria and beneficial plasmids. PLoS Genet 2013; 9:e1003844 [View Article][PubMed]
    [Google Scholar]
  39. Pawluk A, Davidson AR, Maxwell KL. Anti-CRISPR: discovery, mechanism and function. Nat Rev Microbiol 2018; 16:12–17 [View Article][PubMed]
    [Google Scholar]
  40. Paez-Espino D, Sharon I, Morovic W, Stahl B, Thomas BC et al. CRISPR immunity drives rapid phage genome evolution in Streptococcus thermophilus. MBio 2015; 6:e00262-15 [View Article][PubMed]
    [Google Scholar]
  41. Walker DC, Aoyama K, Klaenhammer TR. Electrotransformation of Lactobacillus acidophilus group A1. FEMS Microbiol Lett 1996; 138:233–237 [View Article][PubMed]
    [Google Scholar]
  42. Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol 2008; 190:1401–1412 [View Article][PubMed]
    [Google Scholar]
  43. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 2011; 17:10–12 [View Article]
    [Google Scholar]
  44. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article][PubMed]
    [Google Scholar]
  45. Langmead B. Aligning short sequencing reads with Bowtie. Curr Protoc Bioinformatics 2010; 32:11.7.1–11.711 [View Article][PubMed]
    [Google Scholar]
  46. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012; 28:1647–1649 [View Article][PubMed]
    [Google Scholar]
  47. Ruiz L, Margolles A, Sánchez B. Bile resistance mechanisms in Lactobacillus and Bifidobacterium. Front Microbiol 2013; 4:396 [View Article][PubMed]
    [Google Scholar]
  48. Chatfield CH, Koo H, Quivey RG. The putative autolysin regulator LytR in Streptococcus mutans plays a role in cell division and is growth-phase regulated. Microbiology 2005; 151:625–631 [View Article][PubMed]
    [Google Scholar]
  49. Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005; 151:653–663 [View Article][PubMed]
    [Google Scholar]
  50. Tyson GW, Banfield JF. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ Microbiol 2008; 10:200–207 [View Article][PubMed]
    [Google Scholar]
  51. Briner AE, Barrangou R. Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity. Appl Environ Microbiol 2014; 80:994–1001 [View Article][PubMed]
    [Google Scholar]
  52. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 2009; 139:945–956 [View Article][PubMed]
    [Google Scholar]
  53. Babu M, Beloglazova N, Flick R, Graham C, Skarina T et al. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol Microbiol 2011; 79:484–502 [View Article][PubMed]
    [Google Scholar]
  54. Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 2013; 497:254–257 [View Article][PubMed]
    [Google Scholar]
  55. Westra ER, Buckling A, Fineran PC. CRISPR-Cas systems: beyond adaptive immunity. Nat Rev Microbiol 2014; 12:317–326 [View Article][PubMed]
    [Google Scholar]
  56. Kullen MJ, Sanozky-Dawes RB, Crowell DC, Klaenhammer TR. Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex. J Appl Microbiol 2000; 89:511–516 [View Article][PubMed]
    [Google Scholar]
  57. Kok J, van der Vossen JM, Venema G. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol 1984; 48:726–731[PubMed]
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.000689
Loading
/content/journal/micro/10.1099/mic.0.000689
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error