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

  • Brian A. Klein
  • Margaret J. Duncan
  • Linden T. Hu
Part of the Methods in Molecular Biology book series (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.

Key words

Porphyromonas gingivalis Transposon mutagenesis Essential genes Mariner Tn-seq 

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

Notes

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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Brian A. Klein
    • 1
  • Margaret J. Duncan
    • 2
  • Linden T. Hu
    • 1
    • 3
  1. 1.Graduate Program of Molecular MicrobiologyTufts University School of MedicineBostonUSA
  2. 2.Department of MicrobiologyThe Forsyth InstituteCambridgeUSA
  3. 3.Department of Geographical Medicine and Infectious DiseaseTufts Medical CenterBostonUSA

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