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Defining Essential Genes and Identifying Virulence Factors of Porphyromonas gingivalis by Massively Parallel Sequencing of Transposon Libraries (Tn-seq)

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Gene Essentiality

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1279))

Abstract

Porphyromonas gingivalis is a keystone pathogen in the development and progression of periodontal disease. Obstacles to the development of saturated transposon libraries have previously limited transposon mutant-based screens as well as essential gene studies. We have developed a system for efficient transposon mutagenesis of P. gingivalis using a modified mariner transposon. Tn-seq is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon–genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify nonessential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures.

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Abbreviations

BAM:

Binary alignment/map format

Pg :

Porphyromonas gingivalis

PCR:

Polymerase chain reaction

BHIHKSbcStgC:

Supplemented brain–heart infusion

BAPHK:

Supplemented blood agar plate

BROP:

Bioinformatics resource oral pathogens

BED:

Browser extensible data format

SAM:

Sequence alignment/map format

BLAST:

Basic local alignment search tool

DEG:

Database of essential genes

WT:

Wild-type

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Acknowledgements

We would like to thank Dr. Andrew Goodman (Yale University School of Medicine) for providing mutagenesis strains and plasmids. We are grateful to Dr. Andrew Camilli, Dr. David Lazinski, and the Tufts University Core Facility (Tufts University Sackler School of Sciences and Howard Hughes Medical Institute) for technical assistance with Illumina sequencing and analysis. We would also like to thank Drs. Michael Malamy (Tufts University Sackler School of Biomedical Sciences) and Pamela Baker (Bates College) for their insightful discussions pertaining to anaerobic bacteria, mutagenesis, and genetics.

Funding

This project was supported by a Grant from the National Institute of Dental and Craniofacial Research, F31 DE022491 (BAK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Dental and Craniofacial Research or the National Institutes of Health.

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Authors and Affiliations

  1. Graduate Program of Molecular Microbiology, Tufts University School of Medicine, Boston, MA, USA

    Brian A. Klein & Linden T. Hu

  2. Department of Microbiology, The Forsyth Institute, Cambridge, MA, USA

    Margaret J. Duncan

  3. Department of Geographical Medicine and Infectious Disease, Tufts Medical Center, 171 Harrison Avenue, Boston, MA, 02111, USA

    Linden T. Hu

Authors
  1. Brian A. Klein
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  2. Margaret J. Duncan
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  3. Linden T. Hu
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Corresponding author

Correspondence to Linden T. Hu .

Editor information

Editors and Affiliations

  1. Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA

    Long Jason Lu

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Klein, B.A., Duncan, M.J., Hu, L.T. (2015). Defining Essential Genes and Identifying Virulence Factors of Porphyromonas gingivalis by Massively Parallel Sequencing of Transposon Libraries (Tn-seq). In: Lu, L. (eds) Gene Essentiality. Methods in Molecular Biology, vol 1279. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2398-4_3

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  • DOI: https://doi.org/10.1007/978-1-4939-2398-4_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2397-7

  • Online ISBN: 978-1-4939-2398-4

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