Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Pervasive acquisition of CRISPR memory driven by inter-species mating of archaea can limit gene transfer and influence speciation

Nature Microbiology volume 4, pages 177–186 (2019)Cite this article

Subjects

Abstract

CRISPR–Cas systems provide prokaryotes with sequence-specific immunity against viruses and plasmids based on DNA acquired from these invaders, known as spacers. Surprisingly, many archaea possess spacers that match chromosomal genes of related species, including those encoding core housekeeping genes. By sequencing genomes of environmental archaea isolated from a single site, we demonstrate that inter-species spacers are common. We show experimentally, by mating Haloferax volcanii and Haloferax mediterranei, that spacers are indeed acquired chromosome-wide, although a preference for integrated mobile elements and nearby regions of the chromosome exists. Inter-species mating induces increased spacer acquisition and may result in interactions between the acquisition machinery of the two species. Surprisingly, many of the spacers acquired following inter-species mating target self-replicons along with those originating from the mating partner, indicating that the acquisition machinery cannot distinguish self from non-self under these conditions. Engineering the chromosome of one species to be targeted by the other’s CRISPR–Cas reduces gene exchange between them substantially. Thus, spacers acquired during inter-species mating could limit future gene transfer, resulting in a role for CRISPR–Cas systems in microbial speciation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Simplified representation of perfect inter-species matches between CRISPR spacers and DNA sequences in the genomes of environmental Atlit isolates.
Fig. 2: Number of spacers acquired during mating between H. volcanii and H. mediterranei.
Fig. 3: Spacers acquired from the H. mediterranei chromosome by either H. volcanii or H. mediterranei CRISPR–Cas.
Fig. 4: PAM preference for each of the species against different replicons.
Fig. 5: Inter-species mating is reduced by CRISPR–Cas targeting.

Similar content being viewed by others

Data availability

Haloarchaeal environmental isolates genomes are available at GenBank under accession numbers PSYS00000000, PSYT00000000, PSYU00000000, PSYV00000000, PSYW00000000, PSYX00000000, PSYY00000000, QEQI00000000, QEQJ00000000, QPLN00000000, QPLO00000000, QPLP00000000, QPLQ00000000, QPLR00000000, QPLS00000000, QPLT00000000, QPLU00000000, QXIJ00000000, QXIK00000000 and QPLV00000000.

References

  1. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Marraffini, L. A. & Sontheimer, E. J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Shah, S. A., Hansen, N. R. & Garrett, R. A. Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism. Biochem. Soc. Trans. 37, 23–28 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Held, N. L., Herrera, A., Quiroz, H. C. & Whitaker, R. J. CRISPR associated diversity within a population of Sulfolobus islandicus. PLoS ONE 5, e12988 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Shmakov, S. A. et al. The CRISPR spacer space is dominated by sequences from species-specific mobilomes. mBio 8, e01397–17 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Brodt, A., Lurie-Weinberger, M. N. & Gophna, U. CRISPR loci reveal networks of gene exchange in archaea. Biol. Direct 6, 65 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rosenshine, I., Tchelet, R. & Mevarech, M. The mechanism of DNA transfer in the mating system of an archaebacterium. Science 245, 1387–1389 (1989).

    Article  CAS  PubMed  Google Scholar 

  9. Naor, A., Lapierre, P., Mevarech, M., Papke, R. T. & Gophna, U. Low species barriers in halophilic archaea and the formation of recombinant hybrids. Curr. Biol. 22, 1444–1448 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Schleper, C., Holz, I., Janekovic, D., Murphy, J. & Zillig, W. A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating. J. Bacteriol. 177, 4417–4426 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kuwabara, T. et al. Thermococcus coalescens sp. nov., a cell-fusing hyperthermophilic archaeon from Suiyo Seamount. Int. J. Syst. Evol. Microbiol. 55, 2507–2514 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Burstein, D. et al. New CRISPR–Cas systems from uncultivated microbes. Nature 542, 237–241 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Haft, D. H., Selengut, J., Mongodin, E. F., Nelson, K. E. & White, O. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, e60 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 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 151, 653–663 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Stern, A., Keren, L., Wurtzel, O., Amitai, G. & Sorek, R. Self-targeting by CRISPR: gene regulation or autoimmunity? Trends Genet. 26, 335–340 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yosef, I., Goren, M. G., Kiro, R., Edgar, R. & Qimron, U. High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. Proc. Natl Acad. Sci. USA 108, 20136–20141 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Vercoe, R. B. et al. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet. 9, e1003454 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Selle, K., Klaenhammer, T. R. & Barrangou, R. CRISPR-based screening of genomic island excision events in bacteria. Proc. Natl Acad. Sci. USA 112, 8076–8081 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Li, Y. et al. Harnessing type I and type III CRISPR-Cas systems for genome editing. Nucleic Acids Res. 44, e34 (2016).

    Article  PubMed  Google Scholar 

  22. Fischer, S. et al. An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA. J. Biol. Chem. 287, 33351–33365 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, M., Wang, R. & Xiang, H. Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation. Nucleic Acids Res. 42, 7226–7235 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mojica, F. J. M., Juez, G. & Rodriguez-Valera, F. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Mol. Microbiol. 9, 613–621 (1993).

    Article  CAS  PubMed  Google Scholar 

  25. Brendel, J. et al. A complex of cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii. J. Biol. Chem. 289, 7164–7177 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li, M. et al. Characterization of CRISPR RNA biogenesis and Cas6 cleavage-mediated inhibition of a provirus in the haloarchaeon Haloferax mediterranei. J. Bacteriol. 195, 867–875 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Artieri, C. G. et al. Cis-regulatory evolution in prokaryotes revealed by interspecific archaeal hybrids. Sci. Rep. 7, 3986 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yang, H. et al. Activation of a dormant replication origin is essential for Haloferax mediterranei lacking the primary origins. Nat. Commun. 6, 8321 (2015).

    Article  PubMed  Google Scholar 

  30. Levy, A. et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505–510 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Staals, R. H. J. et al. Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR–cas system. Nat. Commun. 7, 12853 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Shiimori, M. et al. Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus. Nucleic Acids Res 45, 11281–11294 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chimileski, S., Dolas, K., Naor, A., Gophna, U. & Papke, R. T. Extracellular DNA metabolism in Haloferax volcanii. Front. Microbiol. 5, 57 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Erdmann, S., Le Moine Bauer, S. & Garrett, R. A. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Mol. Microbiol. 91, 900–917 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Maier, L.-K. et al. Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B. RNA Biol. 10, 865–874 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29, 1072–1075 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Modell, J. W., Jiang, W. & Marraffini, L. A. CRISPR–Cas systems exploit viral DNA injection to establish and maintain adaptive immunity. Nature 544, 101–104 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lopez-Sanchez, M.-J. et al. The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol. Microbiol. 85, 1057–1071 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Grissa, I., Vergnaud, G. & Pourcel, C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8, 172 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Chimileski, S., Franklin, M. J. & Papke, R. Biofilms formed by the archaeon Haloferax volcanii exhibit cellular differentiation and social motility, and facilitate horizontal gene transfer. BMC Biol. 12, 65 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Naor, A. et al. Impact of a homing intein on recombination frequency and organismal fitness. Proc. Natl Acad. Sci. USA 113, 4654–4661 (2016).

    Article  Google Scholar 

  43. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal 17, 10 (2011).

    Article  Google Scholar 

  44. Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bland, C. et al. CRISPR Recognition Tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics 8, 209 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Allers, T., Barak, S., Liddell, S., Wardell, K. & Mevarech, M. Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii. Appl. Environ. Microbiol. 76, 1759–1769 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Allers, T., Ngo, H. P., Mevarech, M. & Lloyd, R. G. Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA Genes. Appl. Environ. Microbiol. 70, 943–953 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Bitan-Banin, G., Ortenberg, R. & Mevarech, M. Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE Gene. J. Bacteriol. 185, 772–778 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rognes, T., Flouri, T., Nichols, B., Quince, C. & Mahé, F. VSEARCH: a versatile open source tool for metagenomics. PeerJ 4, e2584 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Hall, T. BioEdit Sequence Alignment Editor for Windows 95/98/NT/XP/Vista/7 (Ibis Therapeutics, 2013); http://www.mbio.ncsu.edu/BioEdit/bioedit.html.

  53. Leenay, R. T. et al. Identifying and visualizing functional PAM diversity across CRISPR–Cas systems. Mol. Cell 62, 137–147 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Hartman, A. L. et al. The complete genome sequence of Haloferax volcanii DS2, a model archaeon. PLoS ONE 5, e9605 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Han, J. et al. Complete genome sequence of the metabolically versatile halophilic archaeon Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) producer. J. Bacteriol. 194, 4463–4464 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016).

    Article  PubMed  Google Scholar 

  57. Zimmermann, L. et al. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J. Mol. Biol. 430, 2237–2243 (2018).

    Article  CAS  PubMed  Google Scholar 

  58. Makarova, K. S. et al. Dark matter in archaeal genomes: a rich source of novel mobile elements, defense systems and secretory complexes. Extremophiles 18, 877–893 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank R. Sorek and A. Herskovits for their helpful comments and insights, and H. Xiang for providing sequence data and provirus annotations. The authors thank S. Green of the University of Illinois at Chicago for his continued expert help in challenging sequencing projects and E. Koonin (NIH) for helpful discussions. Funding was provided by Deutsche Forschungsgemeinschaft (MA1538/16-2), the Israel Science Foundation (535/15), the Binational Science Foundation (2013061) with partial support by the Constantiner Institute, European Research Council (grant ERC-AdG 787514).

Author information

Author notes

  1. Adit Naor

    Present address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

Authors and Affiliations

  1. Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    Israela Turgeman-Grott, Shirley Joseph, Sam Marton, Kim Eizenshtein, Adit Naor, Yarden Shalev, Mor Zarkor, Leah Reshef, Neta Altman-Price & Uri Gophna

  2. Department of Biological Sciences, Dartmouth College, Hanover, NH, USA

    Shannon M. Soucy

  3. Department of Biology II, Ulm University, Ulm, Germany

    Aris-Edda Stachler & Anita Marchfelder

Authors
  1. Israela Turgeman-Grott
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shirley Joseph
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sam Marton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kim Eizenshtein
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Adit Naor
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shannon M. Soucy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Aris-Edda Stachler
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yarden Shalev
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mor Zarkor
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Leah Reshef
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Neta Altman-Price
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Anita Marchfelder
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Uri Gophna
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

U.G. and I.T.-G. conceived the study. U.G., I.T.-G. and A.M. designed the experiments. S.S. assembled and annotated genome sequences. I.T.-G., A.N. and N.A.-P. designed and constructed strains. I.T.-G., S.J., K.E., Y.S., A.-E.S. and M.Z. performed experiments. L.R., S.M., I.T.G. and U.G. analysed data. U.G. and I.T.-G. wrote the manuscript. L.R., S.S. and A.M. commented and made critical revisions to the manuscript.

Corresponding author

Correspondence to Uri Gophna.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–10, Supplementary Tables 1–12, Supplementary References.

Reporting Summary

Supplementary Table 13

Complete spacer acquisition data from H. volcanii arrays obtained following three independent inter-species mating experiments.

Supplementary Table 14

Complete spacer acquisition data from H. mediterranei arrays obtained following three independent inter-species mating experiments.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Turgeman-Grott, I., Joseph, S., Marton, S. et al. Pervasive acquisition of CRISPR memory driven by inter-species mating of archaea can limit gene transfer and influence speciation. Nat Microbiol 4, 177–186 (2019). https://doi.org/10.1038/s41564-018-0302-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-018-0302-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing