Natural Diversity of CRISPR Spacers Sofia Medvedeva To cite this version: Sofia Medvedeva. Natural Diversity of CRISPR Spacers. Microbiology and Parasitology. Sor- bonne Université; Skolkovo Institute of Science and Technology (Moscou), 2019. English. NNT : 2019SORUS538. tel-03139813 HAL Id: tel-03139813 https://tel.archives-ouvertes.fr/tel-03139813 Submitted on 12 Feb 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Natural Diversity of CRISPR Spacers ABSTRACT ................................................................................................................................................. 3 PUBLICATIONS ........................................................................................................................................ 5 INTRODUCTION ....................................................................................................................................... 7 List of acronyms ........................................................................................................................................ 9 1. Defense systems of prokaryotes .......................................................................................................... 11 2. CRISPR array ..................................................................................................................................... 13 3. Adaptation module ............................................................................................................................. 15 4. Interference modules ........................................................................................................................... 19 5. Distribution of CRISPR-Cas systems .................................................................................................. 22 6. CRISPR-Cas immunity of Sulfolobales .............................................................................................. 23 7. Strain subtyping ................................................................................................................................... 27 8. CRISPR in metagenomics ................................................................................................................... 27 9. Anti-CRISPR proteins ......................................................................................................................... 28 10. Alternative functions of CRISPR-Cas systems ................................................................................. 29 AIMS OF THE STUDY ............................................................................................................................ 31 RESULTS................................................................................................................................................... 35 CHAPTER I Dynamics of Escherichia coli type I-E CRISPR spacers over 42 000 years ..................................... 37 CHAPTER II Metagenomic Analysis of Bacterial Communities of Antarctic Surface Snow ................................ 53 CHAPTER III Natural diversity of CRISPR spacers of Thermus: evidence of local spacer acquisition and global spacer exchange ......................................................................................................................................... 67 CHAPTER IV Virus-borne mini-CRISPR arrays promote interviral conflicts and virus speciation .................... 89 CHAPTER V Integrated Mobile Genetic Elements in Thaumarchaeota ............................................................... 113 CHAPTER VI Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation ...................................................... 139 CONCLUSIONS AND FUTURE PERSPECTIVES ........................................................................... 155 ANNEX..................................................................................................................................................... 165 REFERNCES ........................................................................................................................................... 175 ACKNOWLEDGEMENTS .................................................................................................................... 193 RÉSUMÉ .................................................................................................................................................. 195 ABSTRACT CRISPR-Cas is a prokaryotic immunity system against mobile genetic elements, such as viruses and plasmids. The system consists of two components: the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). In the CRISPR array, short fragments of foreign DNA, called spacers, are interleaved with palindromic repeats. During the adaptation stage of the CRISPR-Cas immunity, new spacers are inserted into the CRISPR array, whereas during the expression and interference stages, spacers are transcribed, processed and complexed with Cas proteins to target the complementary foreign DNA or RNA molecules for degradation. CRISPR array is a fast-evolving part of the genome, with acquisition, duplication, and loss of spacers occurring concurrently to point mutations in the CRISPR repeat and spacer sequences. Thus, sequences of CRISPR arrays can be used to differentiate closely related bacterial lineages. Moreover, analysis of CRISPR spacers is a valuable source of information about virus-host interactions, particularly powerful when applied to metagenomic data. In this work, we explored the diversity of CRISPR spacers in different natural prokaryotic communities, including extinct Escherichia coli community from a mammoth intestine, Flavobacterium communities from Antarctic surface snow, Thermus communities from four distant hot springs in Italy and Chile, and Sulfolobales community from a Japanese thermal field. The comparison of obtained environmental spacer sets with each other and with spacers from public databases as well as with sequences of viruses allowed us to reach several non-trivial conclusions and to gain insights into virus-host and virus-virus interactions in natural microbial communities. PUBLICATIONS 1. Savitskaya E, Lopatina A, Medvedeva S, Kapustin M, Shmakov S, Tikhonov A, Artamonova I, Logacheva M, Severinov K. Dynamics of Escherichia coli type I-E CRISPR spacers over 42,000 years. Mol Ecol. 2016 Dec 20; 26(7):2019-2026. 2. Lopatina A, Medvedeva S, Shmakov S, Logacheva MD, Krylenkov V and Severinov K. Metagenomic Analysis of Bacterial Communities of Antarctic Surface Snow. Front. Microbiol. 2016 Mar 31;7:398 3. Lopatina A#, Medvedeva S#, Artamonova D, Sitnik V, Ispolatov J and Severinov K. Natural Diversity of CRISPR Spacers of Thermus: Evidence of Local Adaptation and Global Spacer Exchange. Philos Trans R Soc Lond B Biol Sci. 2018 Mar 25. 4. Medvedeva S, Liu Y, Koonin EV, Severinov K, Prangishvili D, Krupovic M. Virus-borne mini-CRISPR arrays promote interviral conflicts and virus speciation. Submitted. 5. Krupovic M, Makarova KS, Wolf YI, Medvedeva S, Prangishvili D, Forterre P, Koonin EV. Integrated Mobile Genetic Elements in Thaumarchaeota. Environmental Microbiology. 2019 Feb 17, doi: 10.1111/1462-2920.14564. 6. Musharova O, Vyhovskyi D, Medvedeva S, Guzina J, Zhitnyuk Y, Djordjevic M, Severinov K, Savitskaya E. Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation. mBio Dec 2018, 9 (6) e02169-18 #equal contribution INTRODUCTION LIST OF ACRONYMS ATP – adenosine triphosphate BLAST – Basic Local Alignment Search Tool BREX system – Bacteriophage Exclusion system bp – base pairs Cas – CRISPR-associated Cascade – CRISPR-associated complex for anti-viral defense CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats crRNA – CRISPR RNA dsDNA – double-stranded DNA IHF – integration host factor HTS – High Throughput Sequencing MGE – mobile genetic element mRNA – messenger RNA NGS – Next Generation Sequencing nt – nucleotides PAM – Protospacer Adjacent Motif PCR – polymerase chain reaction R-M system – restriction-modification system RNase – ribonuclease RT – reverse transcriptase ssDNA – single-stranded DNA TA system – toxin-antitoxin system tracrRNA – trans-activating crRNA INTRODUCTION 1. Defense systems of prokaryotes Bacteria and Archaea developed a wide range of immune mechanisms to defend themselves against foreign DNA. The restriction-modification (R-M), CRISPR-Cas, pAgos (prokaryotic Argonaute proteins) (1), and BREX systems are all based on self vs non-self DNA (or RNA) discrimination. By contrast, the abortive infection, and toxin-antitoxin systems induce programmed cell death or cell dormancy upon virus infection. Different defense mechanisms often coexist within one genome. Moreover, they are colocalized in genomic regions called “defense islands” (2). Restriction-modification systems The R-M systems