RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation1,2. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference1,2. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence3. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5′-phosphorylated DNA guides, 13–25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5′-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference.

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Gene Expression Omnibus

Data deposits

The RNA-seq data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus under accession number GSE52738. The siDNA sequence data discussed in this publication have been deposited in NCBI s BioSample database and are accessible under accession number SAMN02593821.


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We want to thank A. Hidalgo, C. E. César, M. Davids and R. H. J. Staals for advice on experimental procedures. Furthermore, we would like to thank R. Engelhart, B. van Genugten, G. Göertz and R. Stolk for experimental contributions. This work was financially supported by grants from the Netherlands Organization of Scientific Research (NWO) to J.O. (NWO-TOP, 854.10.003), and to S.J.J.B. (NWO Vidi , 864.11.005), and by project BIO2010-18875 from the Spanish Ministry of Science and Innovation, and an Institutional Grant from the Fundación Ramón Areces to CBMSO (J.B.).

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Author notes

    • Daan C. Swarts
    •  & Matthijs M. Jore

    These authors contributed equally to this work.


  1. Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, the Netherlands

    • Daan C. Swarts
    • , Matthijs M. Jore
    • , Edze R. Westra
    • , Yifan Zhu
    • , Jorijn H. Janssen
    • , Stan J. J. Brouns
    •  & John van der Oost
  2. Clare Hall Laboratories, Cancer Research UK, London Research Institute, South Mimms EN6 3LD, UK

    • Ambrosius P. Snijders
  3. Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

    • Yanli Wang
  4. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA

    • Dinshaw J. Patel
  5. Centro de Biología Molecular Severo Ochoa, UAM-CSIC, Campus de Cantoblanco, 28049 Madrid, Spain

    • José Berenguer


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M.M.J. and J.H.J. made genomic T. thermophilus mutants under the supervision of J.v.d.O. T. thermophilus experiments were performed by D.C.S., M.M.J. and J.H.J. under the supervision of J.B., S.J.J.B. and J.v.d.O. D.C.S. and E.R.W. purified RNA for RNA-seq, and D.C.S. analysed RNA-seq data under the supervision of S.J.J.B. and J.v.d.O. D.C.S. and A.P.S. performed experiments in which TtAgo expression in T. thermophilus was shown using mass spectrometry. D.C.S., M.M.J. and J.H.J. made all plasmid constructs under the supervision of S.J.J.B., J.B. and J.v.d.O. D.C.S., E.R.W. and Y.Z. purified and analysed TtAgo guides. In vitro activity assays were designed and analysed by D.C.S., S.J.J.B., Y.W., D.J.P. and J.v.d.O., and performed by D.C.S. and Y.Z. under the supervision of S.J.J.B. and J.v.d.O. All authors read and approved the submitted manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to John van der Oost.

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