1887

Abstract

Bacteriophage defences are divided into innate and adaptive systems. sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the genome and predicted its toxin–antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to ‘shut down’, limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in and shown the consequences of AbiE induction.

Funding
This study was supported by the:
  • University of Otago Doctoral Scholarship
    • Principle Award Recipient: Leah M. Smith
  • University of Otago Doctoral Scholarship
    • Principle Award Recipient: Hannah G. Hampton
  • Tertiary Education Commission
    • Principle Award Recipient: Peter C Fineran
  • H2020 Marie Skłodowska-Curie Actions (Award MSCA - 842656)
    • Principle Award Recipient: Sean Meaden
  • Ministry for Business Innovation and Employment (Award C10X1308)
    • Principle Award Recipient: Shaun Ferguson
  • University of Otago
    • Principle Award Recipient: Peter C Fineran
  • Marsden Fund
    • Principle Award Recipient: Peter C Fineran
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000458
2020-10-19
2024-10-05
Loading full text...

Full text loading...

/deliver/fulltext/mgen/6/11/mgen000458.html?itemId=/content/journal/mgen/10.1099/mgen.0.000458&mimeType=html&fmt=ahah

References

  1. Hampton HG, Watson BNJ, Fineran PC. The arms race between bacteria and their phage foes. Nature 2020; 577:327–336 [View Article][PubMed]
    [Google Scholar]
  2. Marraffini LA. CRISPR-Cas immunity in prokaryotes. Nature 2015; 526:55–61 [View Article][PubMed]
    [Google Scholar]
  3. Chopin MC, Chopin A, Bidnenko E. Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 2005; 8:473–479 [View Article][PubMed]
    [Google Scholar]
  4. Lopatina A, Tal N, Sorek R. Abortive infection: bacterial suicide as an antiviral immune strategy. Annu Rev Virol 2020; 7:371–384 [View Article][PubMed]
    [Google Scholar]
  5. Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS et al. The phage abortive infection system, toxin, functions as a protein-RNA toxin-antitoxin pair. Proc Natl Acad Sci USA 2009; 106:894–899 [View Article][PubMed]
    [Google Scholar]
  6. Samson JE, Spinelli S, Cambillau C, Moineau S. Structure and activity of AbiQ, a lactococcal endoribonuclease belonging to the type III toxin-antitoxin system. Mol Microbiol 2013; 87:756–768 [View Article][PubMed]
    [Google Scholar]
  7. Dy RL, Przybilski R, Semeijn K, Salmond GPC, Fineran PC. A widespread bacteriophage abortive infection system functions through a type IV toxin-antitoxin mechanism. Nucleic Acids Res 2014; 42:4590–4605 [View Article][PubMed]
    [Google Scholar]
  8. Garvey P, Fitzgerald GF, Hill C. Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40. Appl Environ Microbiol 1995; 61:4321–4328 [View Article][PubMed]
    [Google Scholar]
  9. Hazan R, Engelberg-Kulka H. Escherichia coli mazEF-mediated cell death as a defense mechanism that inhibits the spread of phage P1. Mol Genet Genomics 2004; 272:227–234 [View Article][PubMed]
    [Google Scholar]
  10. Pecota DC, Wood TK. Exclusion of T4 phage by the hok/sok killer locus from plasmid R1. J Bacteriol 1996; 178:2044–2050 [View Article][PubMed]
    [Google Scholar]
  11. Sberro H, Leavitt A, Kiro R, Koh E, Peleg Y et al. Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning. Mol Cell 2013; 50:136–148 [View Article][PubMed]
    [Google Scholar]
  12. Otsuka Y, Koga M, Iwamoto A, Yonesaki T. A role of RnlA in the RNase LS activity from Escherichia coli . Genes Genet Syst 2007; 82:291–299 [View Article][PubMed]
    [Google Scholar]
  13. Ogura T, Hiraga S. Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci USA 1983; 80:4784–4788 [View Article][PubMed]
    [Google Scholar]
  14. Page R, Peti W. Toxin-antitoxin systems in bacterial growth arrest and persistence. Nat Chem Biol 2016; 12:208–214 [View Article][PubMed]
    [Google Scholar]
  15. Li Y, Liu X, Tang K, Wang W, Guo Y et al. Prophage encoding toxin/antitoxin system PfiT/PfiA inhibits Pf4 production in Pseudomonas aeruginosa . Microb Biotechnol 2020; 13:1132–1144 [View Article][PubMed]
    [Google Scholar]
  16. Gerdes K, Maisonneuve E. Bacterial persistence and toxin-antitoxin loci. Annu Rev Microbiol 2012; 66:103–123 [View Article][PubMed]
    [Google Scholar]
  17. Song S, Wood TK. Post-segregational killing and phage inhibition are not mediated by cell death through toxin/antitoxin systems. Front Microbiol 2018; 9:814 [View Article][PubMed]
    [Google Scholar]
  18. Jankevicius G, Ariza A, Ahel M, Ahel I. The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA. Mol Cell 2016; 64:1109–1116 [View Article][PubMed]
    [Google Scholar]
  19. Makarova KS, Wolf YI, Snir S, Koonin EV. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems. J Bacteriol 2011; 193:6039–6056 [View Article][PubMed]
    [Google Scholar]
  20. Dupuis M-È, Villion M, Magadán AH, Moineau S. CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance. Nat Commun 2013; 4:2087 [View Article][PubMed]
    [Google Scholar]
  21. Koonin EV, Makarova KS. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol 2013; 10:679–686 [View Article][PubMed]
    [Google Scholar]
  22. Patterson AG, Jackson SA, Taylor C, Evans GB, Salmond GPC et al. Quorum sensing controls adaptive immunity through the regulation of multiple CRISPR-Cas systems. Mol Cell 2016; 64:1102–1108 [View Article][PubMed]
    [Google Scholar]
  23. Malone LM, Warring SL, Jackson SA, Warnecke C, Gardner PP et al. A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity. Nat Microbiol 2020; 5:48–55 [View Article][PubMed]
    [Google Scholar]
  24. Jackson SA, Birkholz N, Malone LM, Fineran PC. Imprecise spacer acquisition generates CRISPR-Cas immune diversity through primed adaptation. Cell Host Microbe 2019; 25:250–260 [View Article][PubMed]
    [Google Scholar]
  25. Gerdes K, Rasmussen PB, Molin S. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Proc Natl Acad Sci USA 1986; 83:3116–3120 [View Article][PubMed]
    [Google Scholar]
  26. Aakre CD, Phung TN, Huang D, Laub MT. A bacterial toxin inhibits DNA replication elongation through a direct interaction with the β sliding clamp. Mol Cell 2013; 52:617–628 [View Article][PubMed]
    [Google Scholar]
  27. Marimon O, Teixeira JM, Cordeiro TN, Soo VW, Wood TL et al. An oxygen-sensitive toxin-antitoxin system. Nat Commun 2016; 7:13634 [View Article][PubMed]
    [Google Scholar]
  28. Cai Y, Usher B, Gutierrez C, Tolcan A, Mansour M et al. A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems. Sci Adv 2020; 6:eabb6651 [View Article][PubMed]
    [Google Scholar]
  29. Bycroft BW, Maslen C, Box SJ, Brown AG, Tyler JW. The isolation and characterisation of (3R,5R)- and (3S,5R)- carbapenem-3-carboxylic acid from Serratia and Erwinia species and their putative biosynthetic role. J Chem Soc Chem Commun 1987; 21:1623–1625
    [Google Scholar]
  30. Thomson NR, Crow MA, McGowan SJ, Cox A, Salmond GPC. Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control. Mol Microbiol 2000; 36:539–556 [View Article][PubMed]
    [Google Scholar]
  31. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119 [View Article][PubMed]
    [Google Scholar]
  32. Arndt D, Grant JR, Marcu A, Sajed T, Pon A et al. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res 2016; 44:W16–W21 [View Article][PubMed]
    [Google Scholar]
  33. Bertelli C, Laird MR, Williams KP, Lau BY et al. Simon Fraser University Research Computing Group IslandViewer 4: expanded prediction of genomic islands for larger-scale datasets. Nucleic Acids Res 2017; 45:W30–W35 [View Article][PubMed]
    [Google Scholar]
  34. Sevin EW, Barloy-Hubler F. RASTA-Bacteria: a web-based tool for identifying toxin-antitoxin loci in prokaryotes. Genome Biol 2007; 8:R155 [View Article][PubMed]
    [Google Scholar]
  35. Akarsu H, Bordes P, Mansour M, Bigot DJ, Genevaux P et al. TASmania: a bacterial Toxin-Antitoxin Systems database. PLoS Comput Biol 2019; 15:e1006946 [View Article][PubMed]
    [Google Scholar]
  36. Zhang Y, Zhang Z, Zhang H, Zhao Y, Zhang Z et al. PADS Arsenal: a database of prokaryotic defense systems related genes. Nucleic Acids Res 2020; 48:D590–D598 [View Article][PubMed]
    [Google Scholar]
  37. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011; 7:539 [View Article][PubMed]
    [Google Scholar]
  38. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014; 30:2114–2120 [View Article][PubMed]
    [Google Scholar]
  39. Barquist L, Mayho M, Cummins C, Cain AK, Boinett CJ et al. The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 2016; 32:1109–1111 [View Article][PubMed]
    [Google Scholar]
  40. R Core team R: a language and environment for statistical computing Vienna: R Foundation for Statistical Computing; 2019 http://www.r-project.org/index.html
  41. Barquist L, Langridge GC, Turner DJ, Phan MD, Turner AK et al. A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium. Nucleic Acids Res 2013; 41:4549–4564 [View Article][PubMed]
    [Google Scholar]
  42. Langridge GC, Phan MD, Turner DJ, Perkins TT, Parts L et al. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res 2009; 19:2308–2316 [View Article][PubMed]
    [Google Scholar]
  43. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012; 9:357–359 [View Article][PubMed]
    [Google Scholar]
  44. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009; 25:2078–2079 [View Article][PubMed]
    [Google Scholar]
  45. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; 15:550 [View Article][PubMed]
    [Google Scholar]
  46. Carver T, Harris SR, Berriman M, Parkhill J, McQuillan JA. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics 2012; 28:464–469 [View Article][PubMed]
    [Google Scholar]
  47. Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 1995; 177:4121–4130 [View Article][PubMed]
    [Google Scholar]
  48. Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene 1990; 96:23–28 [View Article][PubMed]
    [Google Scholar]
  49. Fineran PC, Iglesias Cans MC, Ramsay JP, Wilf NM, Cossyleon D et al. Draft genome sequence of Serratia sp. strain ATCC 39006, a model bacterium for analysis of the biosynthesis and regulation of prodigiosin, a carbapenem, and gas vesicles. Genome Announc 2013; 1:e01039-13 [View Article][PubMed]
    [Google Scholar]
  50. Harms A, Brodersen DE, Mitarai N, Gerdes K. Toxins, targets, and triggers: an overview of toxin-antitoxin biology. Mol Cell 2018; 70:768–784 [View Article][PubMed]
    [Google Scholar]
  51. Lobato-Márquez D, Moreno-Córdoba I, Figueroa V, Díaz-Orejas R, García-del Portillo F. Distinct type I and type II toxin-antitoxin modules control Salmonella lifestyle inside eukaryotic cells. Sci Rep 2015; 5:9374 [View Article][PubMed]
    [Google Scholar]
  52. Wozniak RAF, Waldor MK. A toxin-antitoxin system promotes the maintenance of an integrative conjugative element. PLoS Genet 2009; 5:e1000439 [View Article][PubMed]
    [Google Scholar]
  53. Chao MC, Abel S, Davis BM, Waldor MK. The design and analysis of transposon insertion sequencing experiments. Nat Rev Microbiol 2016; 14:119–128 [View Article][PubMed]
    [Google Scholar]
  54. LeRoux M, Culviner PH, Liu YJ, Littlehale ML, Laub MT. Stress can induce transcription of toxin-antitoxin systems without activating toxin. Mol Cell 2020; 79:280–292 [View Article][PubMed]
    [Google Scholar]
  55. Christen B, Abeliuk E, Collier JM, Kalogeraki VS, Passarelli B et al. The essential genome of a bacterium. Mol Syst Biol 2011; 7:528 [View Article][PubMed]
    [Google Scholar]
  56. Hutchison CA, Merryman C, Sun L, Assad-Garcia N, Richter RA et al. Polar effects of transposon insertion into a minimal bacterial genome. J Bacteriol 2019; 201:e00185-19 [View Article][PubMed]
    [Google Scholar]
  57. Kawano M, Aravind L, Storz G. An antisense RNA controls synthesis of an SOS-induced toxin evolved from an antitoxin. Mol Microbiol 2007; 64:738–754 [View Article][PubMed]
    [Google Scholar]
  58. Manna D, Porwollik S, McClelland M, Tan R, Higgins NP. Microarray analysis of Mu transposition in Salmonella enterica, serovar Typhimurium: transposon exclusion by high-density DNA binding proteins. Mol Microbiol 2007; 66:315–328 [View Article][PubMed]
    [Google Scholar]
  59. Aizenman E, Engelberg-Kulka H, Glaser G. An Escherichia coli chromosomal "addiction module" regulated by guanosine 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci USA 1996; 93:6059–6063 [View Article][PubMed]
    [Google Scholar]
  60. Ramisetty BCM, Santhosh RS. Endoribonuclease type II toxin-antitoxin systems: functional or selfish?. Microbiology 2017; 163:931–939 [View Article][PubMed]
    [Google Scholar]
  61. Moyed HS, Bertrand KP. HipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 1983; 155:768–775 [View Article][PubMed]
    [Google Scholar]
  62. Germain E, Castro-Roa D, Zenkin N, Gerdes K. Molecular mechanism of bacterial persistence by HipA. Mol Cell 2013; 52:248–254 [View Article][PubMed]
    [Google Scholar]
  63. Anantharaman V, Aravind L. New connections in the prokaryotic toxin-antitoxin network: relationship with the eukaryotic nonsense-mediated RNA decay system. Genome Biol 2003; 4:R81 [View Article][PubMed]
    [Google Scholar]
  64. Leplae R, Geeraerts D, Hallez R, Guglielmini J, Drèze P et al. Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families. Nucleic Acids Res 2011; 39:5513–5525 [View Article][PubMed]
    [Google Scholar]
  65. Masuda H, Tan Q, Awano N, Wu KP, Inouye M. YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli . Mol Microbiol 2012; 84:979–989 [View Article][PubMed]
    [Google Scholar]
  66. Yu X, Gao X, Zhu K, Yin H, Mao X et al. Characterization of a toxin-antitoxin system in Mycobacterium tuberculosis suggests neutralization by phosphorylation as the antitoxicity mechanism. Commun Biol 2020; 3:216 [View Article][PubMed]
    [Google Scholar]
  67. Aravind L, Koonin EV. DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history. Nucleic Acids Res 1999; 27:1609–1618 [View Article][PubMed]
    [Google Scholar]
  68. Kuchta K, Knizewski L, Wyrwicz LS, Rychlewski L, Ginalski K. Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human. Nucleic Acids Res 2009; 37:7701–7714 [View Article][PubMed]
    [Google Scholar]
  69. Hampton HG, Jackson SA, Fagerlund RD, Vogel AIM, Dy RL et al. AbiEi binds cooperatively to the type IV abiE toxin-antitoxin operator via a positively-charged surface and causes DNA bending and negative autoregulation. J Mol Biol 2018; 430:1141–1156 [View Article][PubMed]
    [Google Scholar]
  70. Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 2005; 12:291–299 [View Article][PubMed]
    [Google Scholar]
  71. Svenningsen SL, Kongstad M, Stenum TS, Muñoz-Gómez AJ, Sørensen MA. Transfer RNA is highly unstable during early amino acid starvation in Escherichia coli . Nucleic Acids Res 2017; 45:793–804 [View Article][PubMed]
    [Google Scholar]
  72. Walling LR, Butler JS. Toxins targeting transfer RNAs: translation inhibition by bacterial toxin-antitoxin systems. Wiley Interdiscip Rev RNA 2019; 10:e1506 [View Article][PubMed]
    [Google Scholar]
  73. Winther KS, Gerdes K. Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA. Proc Natl Acad Sci USA 2011; 108:7403–7407 [View Article][PubMed]
    [Google Scholar]
  74. Lopes AP, Lopes LM, Fraga TR, Chura-Chambi RM, Sanson AL et al. VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA. PLoS One 2014; 9:e101678 [View Article][PubMed]
    [Google Scholar]
  75. Rycroft JA, Gollan B, Grabe GJ, Hall A, Cheverton AM et al. Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection. Nat Commun 2018; 9:1993 [View Article][PubMed]
    [Google Scholar]
  76. Cheverton AM, Gollan B, Przydacz M, Wong CT, Mylona A et al. A Salmonella toxin promotes persister formation through acetylation of tRNA. Mol Cell 2016; 63:86–96 [View Article][PubMed]
    [Google Scholar]
  77. Schifano JM, Cruz JW, Vvedenskaya IO, Edifor R, Ouyang M et al. tRNA is a new target for cleavage by a MazF toxin. Nucleic Acids Res 2016; 44:1256–1270 [View Article][PubMed]
    [Google Scholar]
  78. Zhang Y, Zhang J, Hoeflich KP, Ikura M, Qing G et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli . Mol Cell 2003; 12:913–923 [View Article][PubMed]
    [Google Scholar]
  79. Schuster CF, Park JH, Prax M, Herbig A, Nieselt K et al. Characterization of a mazEF toxin-antitoxin homologue from Staphylococcus equorum . J Bacteriol 2013; 195:115–125 [View Article][PubMed]
    [Google Scholar]
  80. Bailly-Bechet M, Vergassola M, Rocha E. Causes for the intriguing presence of tRNAs in phages. Genome Res 2007; 17:1486–1495 [View Article][PubMed]
    [Google Scholar]
/content/journal/mgen/10.1099/mgen.0.000458
Loading
/content/journal/mgen/10.1099/mgen.0.000458
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error