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.
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References
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
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
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
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
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
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
Gallagher LA, Shendure J, Manoil C (2011) Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq. mBio 2(1):e00315–e00310
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
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
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
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
Lampe DJ, Churchill ME, Robertson HM (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J 15:5470–5479
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
Gordon A, Hannon GJ (2014) Gordon. FASTX-Toolkit. http://hannonlab.cshl.edu/fastx_toolkit
Andrews S. (2015) FASTQC: a quality control tool for high throughput sequence data. Babraham Institute
Li H, Durbin R (2009) Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25(14):1754–1760
Anders S, Pyl PT, Huber W (2015) HTSeq-A Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169
Team RDC. (2010) R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing
Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G et al (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26
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
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
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
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
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
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
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
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
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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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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