Skip to main content

Thank you for visiting 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.

Prophage-mediated defence against viral attack and viral counter-defence


Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host–virus dynamics, and counter-defence promotes phage co-evolution.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Cluster N phage genotypes and morphotypes.
Figure 2: Genomic organization of Cluster N mycobacteriophages.
Figure 3: Transcription in Cluster N lysogens of M. smegmatis.
Figure 4: Cluster N prophage-mediated defence against phage infection.
Figure 5: Genetics of Cluster N prophage-mediated defences.
Figure 6: Mechanisms of prophage-mediated defence against viral attack.


  1. 1

    Hambly, E. & Suttle, C. A. The viriosphere, diversity, and genetic exchange within phage communities. Curr. Opin. Microbiol. 8, 444–450 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Abedon, S. T. Phage evolution and ecology. Adv. Appl. Microbiol. 67, 1–45 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Jacobs-Sera, D. et al. On the nature of mycobacteriophage diversity and host preference. Virology 434, 187–201 (2012).

    CAS  Article  Google Scholar 

  4. 4

    Hendrix, R. W. Bacteriophages: evolution of the majority. Theor. Popul. Biol. 61, 471–480 (2002).

    Article  Google Scholar 

  5. 5

    Suttle, C. A. Marine viruses—major players in the global ecosystem. Nat. Rev. Microbiol. 5, 801–812 (2007).

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Arber, W. & Dussoix, D. Host specificity of DNA produced by Escherichia coli. I. Host controlled modification of bacteriophage lambda. J. Mol. Biol. 5, 18–36 (1962).

    CAS  Article  Google Scholar 

  8. 8

    Dussoix, D. & Arber, W. Host specificity of DNA produced by Escherichia coli. II. Control over acceptance of DNA from infecting phage lambda. J. Mol. Biol. 5, 37–49 (1962).

    CAS  Article  Google Scholar 

  9. 9

    Tock, M. R. & Dryden, D. T. The biology of restriction and anti-restriction. Curr. Opin. Microbiol. 8, 466–472 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Swarts, D. C. et al. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258–261 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Goldfarb, T. et al. BREX is a novel phage resistance system widespread in microbial genomes. EMBO J. 34, 169–183 (2015).

    CAS  Article  Google Scholar 

  12. 12

    Fineran, P. C. et al. The phage abortive infection system, ToxIN, functions as a protein–RNA toxin–antitoxin pair. Proc. Natl Acad. Sci. USA 106, 894–899 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Chopin, M.-C., Chopin, A. & Bidnenko, E. Phage abortive infection in lactococci: variations on a theme. Curr. Opin. Microbiol. 8, 473–479 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Bondy-Denomy, J., Pawluk, A., Maxwell, K. L. & Davidson, A. R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Blower, T. R., Evans, T. J., Przybilski, R., Fineran, P. C. & Salmond, G. P. C. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism. PLoS Genet. 8, e1003023 (2012).

    CAS  Article  Google Scholar 

  16. 16

    Casjens, S. Prophages and bacterial genomics: what have we learned so far? Mol. Microbiol. 49, 277–300 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Knowles, B. et al. Lytic to temperate switching of viral communities. Nature 531, 466–470 (2016).

    CAS  Article  Google Scholar 

  18. 18

    Fortier, L.-C. & Sekulovic, O. Importance of prophages to evolution and virulence of bacterial pathogens. Virulence 4, 354–365 (2013).

    Article  Google Scholar 

  19. 19

    Kita, K., Kawakami, H. & Tanaka, H. Evidence for horizontal transfer of the EcoT38I restriction-modification gene to chromosomal DNA by the P2 phage and diversity of defective P2 prophages in Escherichia coli TH38 strains. J. Bacteriol. 185, 2296–2305 (2003).

    CAS  Article  Google Scholar 

  20. 20

    Hofer, B., Ruge, M. & Dreiseikelmann, B. The superinfection exclusion gene (sieA) of bacteriophage P22: identification and overexpression of the gene and localization of the gene product. J. Bacteriol. 177, 3080–3086 (1995).

    CAS  Article  Google Scholar 

  21. 21

    Cumby, N., Edwards, A. M., Davidson, A. R. & Maxwell, K. L. The bacteriophage HK97 gp15 moron element encodes a novel superinfection exclusion protein. J. Bacteriol. 194, 5012–5019 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Pope, W. H. et al. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4, e06416 (2015).

    Article  Google Scholar 

  23. 23

    Hatfull, G. F. et al. Comparative genomic analysis of 60 mycobacteriophage genomes: genome clustering, gene acquisition, and gene size. J. Mol. Biol. 397, 119–143 (2010).

    CAS  Article  Google Scholar 

  24. 24

    Hendrix, R. W., Smith, M. C., Burns, R. N., Ford, M. E. & Hatfull, G. F. Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage. Proc. Natl Acad. Sci. USA 96, 2192–2197 (1999).

    CAS  Article  Google Scholar 

  25. 25

    Pedulla, M. L. et al. Origins of highly mosaic mycobacteriophage genomes. Cell 113, 171–182 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Hatfull, G. F. The secret lives of mycobacteriophages. Adv. Virus Res. 82, 179–288 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Jordan, T. C. et al. A broadly implementable research course in phage discovery and genomics for first-year undergraduate students. mBio 5, e01051-13 (2014).

    Article  Google Scholar 

  28. 28

    Hatfull, G. F. et al. Complete genome sequences of 63 mycobacteriophages. Genome Announc. 1, e00847-13 (2013).

    Article  Google Scholar 

  29. 29

    Hatfull, G. F. et al. Complete genome sequences of 61 mycobacteriophages. Genome Announc. 4, e00389-16 (2016).

    Article  Google Scholar 

  30. 30

    Broussard, G. W. et al. Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches. Mol. Cell. 49, 237–248 (2013).

    CAS  Article  Google Scholar 

  31. 31

    Oliveira, P. H., Touchon, M. & Rocha, E. P. The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts. Nucleic Acids Res. 42, 10618–10631 (2014).

    CAS  Article  Google Scholar 

  32. 32

    Pham, T. T., Jacobs-Sera, D., Pedulla, M. L., Hendrix, R. W. & Hatfull, G. F. Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria. Microbiology 153, 2711–2723 (2007).

    CAS  Article  Google Scholar 

  33. 33

    Tahar, R., Ringwald, P. & Basco, L. K. Heterogeneity in the circumsporozoite protein gene of Plasmodium malariae isolates from sub-Saharan Africa. Mol. Biochem. Parasitol. 92, 71–78 (1998).

    CAS  Article  Google Scholar 

  34. 34

    Booth, H. A. & Holland, P. W. H. Annotation, nomenclature and evolution of four novel homeobox genes expressed in the human germ line. Gene 387, 7–14 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Soding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005).

    Article  Google Scholar 

  36. 36

    Hogg, T., Mechold, U., Malke, H., Cashel, M. & Hilgenfeld, R. Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]. Cell 117, 57–68 (2004).

    CAS  Article  Google Scholar 

  37. 37

    Steinchen, W. & Bange, G. The magic dance of the alarmones (p)ppGpp. Mol. Microbiol. 101, 531–544 (2016).

    CAS  Article  Google Scholar 

  38. 38

    Yang, J. & Zhang, Y. Protein structure and function prediction using I-TASSER. Curr. Protoc. Bioinformatics 52, 5.8.1–5.8.15 (2015).

    Google Scholar 

  39. 39

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    CAS  Article  Google Scholar 

  40. 40

    Potrykus, K. & Cashel, M. (P)ppGpp still magical? Annu. Rev. Microbiol. 62, 35–51 (2008).

    CAS  Article  Google Scholar 

  41. 41

    Bondy-Denomy, J. et al. Prophages mediate defense against phage infection through diverse mechanisms. ISME J. 10, 2854–2866 (2016).

    Article  Google Scholar 

  42. 42

    Pope, W. H. et al. Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes. J. Virol. 88, 2461–2480 (2014).

    Article  Google Scholar 

  43. 43

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    CAS  Article  Google Scholar 

  44. 44

    Cresawn, S. G. et al. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12, 395 (2011).

    Article  Google Scholar 

  45. 45

    Marinelli, L. J. et al. BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS ONE 3, e3957 (2008).

    Article  Google Scholar 

  46. 46

    Lee, M. H., Pascopella, L., Jacobs, W. R. Jr & Hatfull, G. F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. Proc. Natl Acad. Sci. USA 88, 3111–3115 (1991).

    CAS  Article  Google Scholar 

  47. 47

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  48. 48

    Li, H. et al. The sequence alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  49. 49

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  Article  Google Scholar 

  50. 50

    Thorvaldsdottir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013).

    CAS  Article  Google Scholar 

  51. 51

    Krumsiek, J., Arnold, R. & Rattei, T. Gepard: a rapid and sensitive tool for creating dotplots on genome scale. Bioinformatics 23, 1026–1028 (2007).

    CAS  Article  Google Scholar 

  52. 52

    Huson, D. H. SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics 14, 68–73 (1998).

    CAS  Article  Google Scholar 

  53. 53

    Hatfull, G. F. et al. Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet. 2, e92 (2006).

    Article  Google Scholar 

Download references


The authors thank the many students in the SEA-PHAGES programme that contributed to the isolation, annotation and characterization of the phages described here. Specific contributions are noted at The authors thank J. Schiebel, A. Jonas, T. Stoner, D. Green, R. Rush and L. Lin for help with escape mutant isolation, C.-C. Ko for help with plasmid construction and D. Asai, V. Sivanathan, K. Bradley and L. Barker for support of the SEA-PHAGES programme. This work was supported by grants from the National Institutes of Health (GM116884) and the Howard Hughes Medical Institute (54308198) to G.F.H. and a National Science Foundation pre-doctoral fellowship to T.N.M. (no. 1247842).

Author information




R.M.D., D.J.-S., C.A.G.B., T.N.M., W.H.P., V.C.W., J.W. and G.F.H. conceived and designed the experiments. R.M.D., D.J.-S., C.A.G.B., R.A.G., W.H.P., J.C.C.R., D.A.R., B.R.B., C.F.H., C.M.M., M.T.M. and J.N.T. performed the experiments. R.M.D., D.J.-S., C.A.G.B., R.A.G., T.N.M., W.H.P., B.R.B., J.C.C.R., D.A.R., T.A., R.A., J.A.B., J.S.B., D.B., S.G.C., W.B.D., L.A.D., N.P.E., A.M.F., U.G., J.H.G., C.F.H., L.E.H., K.W.H., S.I., A.A.J., M.A.K., K.K.K., C.M.M., S.F.M., S.D.M., M.T.M., J.N., S.T.P., M.C.P., M.K.P., C.A.R., C.J.R., M.R.R., J.N.T., E.V., V.C.W., J.W. and G.F.H. analysed the data. S.G.C. contributed material/analysis tools. R.M.D., D.J.-S., T.N.M., W.H.P., D.A.R., T.A., R.A., J.A.B., J.S.B., D.B., S.G.C., W.B.D., L.A.D., N.P.E., A.M.F., U.G., J.H.G., L.E.H., K.W.H., S.I., A.A.J., M.A.K., K.K.K., C.M.M., S.F.M., S.D.M., J.N., S.T.P., M.C.P., M.K.P., C.A.R., C.J.R., M.R.R., E.V., V.C.W., J.W. and G.F.H. wrote the paper.

Corresponding author

Correspondence to Graham F. Hatfull.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Discussion; Supplementary References; Supplementary Tables 1–4; Supplementary Figures 1–22 (PDF 10476 kb)

Supplementary Table 5

Oligonucleotides used in this study (XLSX 30 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dedrick, R., Jacobs-Sera, D., Bustamante, C. et al. Prophage-mediated defence against viral attack and viral counter-defence. Nat Microbiol 2, 16251 (2017).

Download citation

Further reading


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