#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

A PNPase Dependent CRISPR System in


The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in “CRISPRology”.


Vyšlo v časopise: A PNPase Dependent CRISPR System in. PLoS Genet 10(1): e32767. doi:10.1371/journal.pgen.1004065
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004065

Souhrn

The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in “CRISPRology”.


Zdroje

1. CossartP (2011) Illuminating the landscape of host-pathogen interactions with the bacterium Listeria monocytogenes. Proc Natl Acad Sci USA 108: 19484–19491.

2. MellinJR, CossartP (2012) The non-coding RNA world of the bacterial pathogen Listeria monocytogenes. RNABiol 9: 372–378.

3. SestoN, WurtzelO, ArchambaudC, SorekR, CossartP (2013) The excludon: a new concept in bacterial antisense RNA-mediated gene regulation. Nat Rev Microbiol 11: 75–82.

4. Toledo-AranaA, DussurgetO, NikitasG, SestoN, Guet-RevilletH, et al. (2009) The Listeria transcriptional landscape from saprophytism to virulence. Nature 459: 950–956.

5. StorzG, VogelJ, WassarmanKM (2011) Regulation by Small RNAs in Bacteria: Expanding Frontiers. Mol Cell 43: 880–891.

6. MakarovaKS, HaftDH, BarrangouR, BrounsSJJ, CharpentierE, et al. (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9: 467–477.

7. SangalV, FineranPC, HoskissonPA (2013) Novel configurations of Type I and II CRISPR/Cas systems in Corynebacterium diphtheriae. Microbiology Epub.

8. BolotinA, QuinquisB, SorokinA, EhrlichSD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology (Reading, Engl) 151: 2551–2561.

9. MojicaFJ, Diez-VillasenorC, Garcia-MartinezJ, SoriaE (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60: 174–182.

10. PourcelC, SalvignolG, VergnaudG (2005) CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology (Reading, Engl) 151: 653–663.

11. BarrangouR, FremauxC, DeveauH, RichardsM, BoyavalP, et al. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315: 1709–1712.

12. BrounsSJJ, JoreMM, LundgrenM, WestraER, SlijkhuisRJH, et al. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321: 960–964.

13. MarraffiniLA, SontheimerEJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322: 1843–1845.

14. BarrangouR (2013) CRISPR-Cas systems and RNA-guided interference. Wiley interdisciplinary reviews RNA 4: 267–278.

15. WiedenheftB, SternbergSH, DoudnaJA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482: 331–338.

16. KuenneC, VogetS, PischimarovJ, OehmS, GoesmannA, et al. (2010) Comparative Analysis of Plasmids in the Genus Listeria. PLoS ONE 5: e12511.

17. DorschtJ, KlumppJ, BielmannR, SchmelcherM, BornY, et al. (2009) Comparative genome analysis of Listeria bacteriophages reveals extensive mosaicism, programmed translational frameshifting, and a novel prophage insertion site. J Bacteriol 191: 7206–7215.

18. KuenneC, BillionA, MraheilMA, StrittmatterA, DanielR, et al. (2013) Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome. BMC Genomics 14: 47.

19. GlaserP, FrangeulL, BuchrieserC, RusniokC, AmendA, et al. (2001) Comparative genomics of Listeria species. Science 294: 849–852.

20. NelsonKE, FoutsDE, MongodinEF, RavelJ, DeBoyRT, et al. (2004) Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res 32: 2386–2395.

21. HainT, GhaiR, BillionA, KuenneCT, SteinwegC, et al. (2012) Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes. BMC Genomics 13: 144.

22. MandinP, RepoilaF, VergassolaM, GeissmannT, CossartP (2007) Identification of new noncoding RNAs in Listeria monocytogenes and prediction of mRNA targets. Nucleic Acids Res 35: 962–974.

23. CondonC (2007) Maturation and degradation of RNA in bacteria. Curr Opin Microbiol 10: 271–278.

24. SchmukiMM, ErneD, LoessnerMJ, KlumppJ (2012) Bacteriophage p70: unique morphology and unrelatedness to other listeria bacteriophages. J Virol 86: 13099–13102.

25. KuninV, SorekR, HugenholtzP (2007) Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 8: R61.

26. ArraianoCM, AndradeJM, DominguesS, GuinoteIB, MaleckiM, et al. (2010) The critical role of RNA processing and degradation in the control of gene expression. FEMS Microbiol Rev 34: 883–923.

27. BhayaD, DavisonM, BarrangouR (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45: 273–297.

28. BikardD, MarraffiniLA (2012) Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages. Curr Opin Immunol 24: 15–20.

29. AlmendrosC, GuzmanNM, Diez-VillasenorC, Garcia-MartinezJ, MojicaFJ (2012) Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli. PloS ONE 7: e50797.

30. WestraER, SwartsDC, StaalsRH, JoreMM, BrounsSJ, et al. (2012) The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity. Annu Rev Genet 46: 311–339.

31. FineranPC, CharpentierE (2012) Memory of viral infections by CRISPR-Cas adaptive immune systems Acquisition of new information. Virology 434: 202–209.

32. DeltchevaE, ChylinskiK, SharmaCM, GonzalesK, ChaoY, et al. (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471: 602–607.

33. ZhangY, HeidrichN, AmpattuBJ, GundersonCW, SeifertHS, et al. (2013) Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis. Mol Cell 50: 488–503.

34. FarrrG, OussenkoI, BechhhoferD (1999) Protection against 3′-to-5′ RNA Decay in Bacillus subtilis. J Bacteriol 23: 1–9.

35. Lehnik-HabrinkM, LewisRJ, MäderU, StülkeJ (2012) RNA degradation in Bacillus subtilis: an interplay of essential endo- and exoribonucleases. Mol Microbiol 84: 1005–1017.

36. CardenasPP, CarrascoB, SanchezH, DeikusG, BechhoferDH, et al. (2009) Bacillus subtilis polynucleotide phosphorylase 3′-to-5′ DNase activity is involved in DNA repair. Nucleic Acids Res 37: 4157–4169.

37. CardenasPP, CarzanigaT, ZangrossiS, BrianiF, Garcia-TiradoE, et al. (2011) Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins. Nucleic Acids Res 39: 9250–9261.

38. AndradeJM, PobreV, MatosAM, ArraianoCM (2012) The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq. RNA 18: 844–855.

39. De LayN, GottesmanS (2011) Role of polynucleotide phosphorylase in sRNA function in Escherichia coli. RNA 17: 1172–1189.

40. KuoC-H, OchmanH (2010) The extinction dynamics of bacterial pseudogenes. PLoS Genet 6: e1001050.

41. WurtzelO, SestoN, MellinJR, KarunkerI, EdelheitS, et al. (2012) Comparative transcriptomics of pathogenic and non-pathogenic Listeria species. Mol Syst Biol 8: 583.

42. DengL, GarrettRA, ShahSA, PengX, SheQ (2013) A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus. Mol Microbiol 87: 1088–1099.

43. ZegansME, WagnerJC, CadyKC, MurphyDM, HammondJH, et al. (2009) Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J Bacteriol 191: 210–219.

44. ViswanathanP, MurphyK, JulienB, GarzaAG, KroosL (2007) Regulation of dev, an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats. J Bacteriol 189: 3738–3750.

45. SampsonTR, SarojSD, LlewellynAC, TzengYL, WeissDS (2013) A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 497: 254–257.

46. RoossinckMJ (2011) The good viruses: viral mutualistic symbioses. Nat Rev Microbiol 9: 99–108.

47. BoydEF, BrussowH (2002) Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol 10: 521–529.

48. RabinovichL, SigalN, BorovokI, Nir-PazR, HerskovitsAA (2012) Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence. Cell 150: 792–802.

49. ArnaudM, ChastanetA, DebarbouilleM (2004) New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. App Environ Microbiol 70: 6887–6891.

50. BalestrinoD, HamonMA, DortetL, NahoriMA, Pizarro-CerdaJ, et al. (2010) Single-cell techniques using chromosomally tagged fluorescent bacteria to study Listeria monocytogenes infection processes. App Environ Microbiol 76: 3625–3636.

51. LauerP, ChowMY, LoessnerMJ, PortnoyDA, CalendarR (2002) Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J Bacteriology 184: 4177–4186.

52. JestinJL, DèmeE, JacquierA (1997) Identification of structural elements critical for inter-domain interactions in a group II self-splicing intron. EMBO J 16: 2945–2954.

53. SaidN, RiederR, HurwitzR, DeckertJ, UrlaubH, et al. (2009) In vivo expression and purification of aptamer-tagged small RNA regulators. Nucleic Acids Res 37: e133.

54. ChevalierC, GeissmannT, HelferA-C, RombyP (2009) Probing mRNA structure and sRNA-mRNA interactions in bacteria using enzymes and lead(II). Methods Mol Biol 540: 215–232.

55. BobayL-M, RochaEPC, TouchonM (2013) The Adaptation of Temperate Bacteriophages to Their Host Genomes. Mol Biol Evol 30: 737–757.

56. EddySR (2011) Accelerated Profile HMM Searches. PLoS Comput Biol 7: e1002195.

57. TouchonM, HoedeC, TenaillonO, BarbeV, BaeriswylS, et al. (2009) Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet 5: e1000344.

58. EdgarRC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.

59. SchmidtHA, StrimmerK, VingronM, von HaeselerA (2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18: 502–504.

60. GascuelO (1997) BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol 14: 685–695.

61. BlandC, RamseyTL, SabreeF, LoweM, BrownK, et al. (2007) CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics 8: 209.

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2014 Číslo 1
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#