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Essential Genes and Genomes pp 237–258Cite as

Identification and Analysis of Essential Genes in Streptococcus mutans with Transposon Sequencing

Part of the Methods in Molecular Biology book series (MIMB,volume 2377)

Abstract

Transposon sequencing (Tn-seq) has greatly accelerated the rate at which gene function can be profiled in microbial organisms. This technique has been applied to the study of the dental caries pathogen Streptococcus mutans where it has been used to generate large transposon mutant libraries. Coupled with high-throughput sequencing and bioinformatics tools, culture of these transposon mutant libraries has facilitated the identification of essential and conditional essential genes. In this chapter, we describe a procedure for performing Tn-seq studies in S. mutans that covers pooled transposon mutant construction, in vitro culture, and DNA library sequencing and data analysis.

Key words

  • Transposon mutagenesis
  • Essential genes
  • Streptococcus mutans
  • Tn-seq
  • Himar1
  • In vitro transposition

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References

  1. van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6(10):767–772

    CrossRef  CAS  Google Scholar 

  2. Langridge GC, Phan M-D, Turner DJ, Perkins TT, Parts L, Haase J et al (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res 19:2308–2316. https://doi.org/10.1101/gr.097097.109

    CrossRef  PubMed  PubMed Central  CAS  Google Scholar 

  3. Goodman AL, McNulty NP, Zhao Y, Leip D, Mitra RD, Lozupone CA et al (2009) Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6(3):279–289

    CrossRef  CAS  Google Scholar 

  4. Wetmore KM, Price MN, Waters RJ, Lamson JS, He J, Hoover CA et al (2015) Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 6(3):e00306–e00315

    CrossRef  CAS  Google Scholar 

  5. Gawronski JD, Wong SMS, Giannoukos G, Ward DV, Akerley BJ (2009) Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc Natl Acad Sci U S A 106(38):16422–16427

    CrossRef  Google Scholar 

  6. Dale JL, Beckman KB, Willett JLE, Nilson JL, Palani NP, Baller JA et al (2018) Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF. mSystems 3(5):e00062–e00018

    CrossRef  CAS  Google Scholar 

  7. Gallagher LA, Shendure J, Manoil C (2011) Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq. mBio 2(1):e00315–e00310

    CrossRef  CAS  Google Scholar 

  8. Shields RC, O’Brien G, Maricic N, Kesterson A, Grace M, Hagen SJ et al (2018) Genome-wide screens reveal new gene products that influence genetic competence in Streptococcus mutans. J Bacteriol 200(2):16 e00508-17

    CrossRef  Google Scholar 

  9. Shields RC, Zeng L, Culp DJ, Burne RA (2018) Genomewide identification of essential genes and fitness determinants of Streptococcus mutans UA159. mSphere 3(1):e00031-18

    CrossRef  Google Scholar 

  10. van Opijnen T, Lazinski DW, Camilli A (2014) Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms. Curr Protoc Mol Biol 106:7.16.1–7.16.24

    Google Scholar 

  11. Lampe DJ, Akerley BJ, Rubin EJ, Mekalanos JJ, Robertson HM (1999) Hyperactive transposase mutants of the Himar1 mariner transposon. Proc Natl Acad Sci U S A 96(20):11428–11433

    CrossRef  CAS  Google Scholar 

  12. Lampe DJ, Churchill ME, Robertson HM (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J 15:5470–5479

    CrossRef  CAS  Google Scholar 

  13. Somervuo P, Koskinen P, Mei P, Holm L, Auvinen P, Paulin L (2018) BARCOSEL: a tool for selecting an optimal barcode set for high-throughput sequencing. BMC Bioinformatics 19(1):257

    CrossRef  CAS  Google Scholar 

  14. Gordon A, Hannon GJ (2014) Gordon. FASTX-Toolkit. http://hannonlab.cshl.edu/fastx_toolkit

  15. Andrews S. (2015) FASTQC: a quality control tool for high throughput sequence data. Babraham Institute

    Google Scholar 

  16. Li H, Durbin R (2009) Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25(14):1754–1760

    CrossRef  CAS  Google Scholar 

  17. Anders S, Pyl PT, Huber W (2015) HTSeq-A Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169

    CrossRef  CAS  Google Scholar 

  18. Team RDC. (2010) R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing

    Google Scholar 

  19. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G et al (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26

    CrossRef  CAS  Google Scholar 

  20. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079

    CrossRef  CAS  Google Scholar 

  21. DeJesus MA, Ambadipudi C, Baker R, Sassetti C, Ioerger TR (2015) TRANSIT - a software tool for Himar1 TnSeq analysis. PLoS Comput Biol 11(10):e1004401

    CrossRef  CAS  Google Scholar 

  22. Valentino MD, Foulston L, Sadaka A, Kos VN, Villet RA, Santa Maria J et al (2014) Genes contributing to Staphylococcus aureus fitness in abscess- and infection-related ecologies. mBio 5(5):e01729–e01714

    CrossRef  CAS  Google Scholar 

  23. Shields RC, Walker AR, Maricic N, Chakraborty B, Underhill SAM, Burne RA (2020) Repurposing the Streptococcus mutans CRISPR-Cas9 system to understand essential gene function. PLoS Pathog 16:e1008344. https://doi.org/10.1371/journal.ppat.1008344

    CrossRef  PubMed  PubMed Central  CAS  Google Scholar 

  24. Morrison DA, Khan R, Junges R, Åmdal HA, Petersen FC (2015) Genome editing by natural genetic transformation in Streptococcus mutans. J Microbiol Methods 119:134–141. https://doi.org/10.1016/j.mimet.2015.09.023

    CrossRef  PubMed  CAS  Google Scholar 

  25. Chao MC, Abel S, Davis BM, Waldor MK (2016) The design and analysis of transposon insertion sequencing experiments. Nat Rev Microbiol 14(2):119–128

    CrossRef  CAS  Google Scholar 

  26. Aird D, Ross MG, Chen W-S, Danielsson M, Fennell T, Russ C et al (2011) Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 12(2):R18

    CrossRef  CAS  Google Scholar 

  27. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D, Čech M et al (2018) The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46(W1):W537–W544

    CrossRef  CAS  Google Scholar 

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Acknowledgements

We thank Nick Jakubovics at Newcastle University for help with the genomic DNA isolation protocol; Andrew Camilli at Tufts University for providing us the Tn-seq plasmids (pMalC9 and pMagellan6); Lin Zeng at the University of Florida for assistance with adapting the original Tn-seq protocol to work with S. mutans and help with editing this chapter; and David Moraga at the Interdisciplinary Center for Biotechnology Research at the University of Florida for help with troubleshooting and optimization of Tn-seq DNA sequencing.

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Authors and Affiliations

  1. Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL, USA

    Alejandro R. Walker & Robert C. Shields

  2. Department of Biological Sciences, College of Sciences and Mathematics, Arkansas State University, Jonesboro, AR, USA

    Robert C. Shields

Authors
  1. Alejandro R. Walker
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  2. Robert C. Shields
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Corresponding author

Correspondence to Robert C. Shields .

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Editors and Affiliations

  1. Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA

    Ph.D. Ren Zhang

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Walker, A.R., Shields, R.C. (2022). Identification and Analysis of Essential Genes in Streptococcus mutans with Transposon Sequencing. In: Zhang, R. (eds) Essential Genes and Genomes. Methods in Molecular Biology, vol 2377. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1720-5_13

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  • DOI: https://doi.org/10.1007/978-1-0716-1720-5_13

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  • Publisher Name: Humana, New York, NY

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  • Online ISBN: 978-1-0716-1720-5

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