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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
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
References
Holt SC, Ebersole J, Felton J, Brunsvold M, Kornman KS (1988) Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis. Science 239:55–57
Hajishengallis G et al (2011) Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 10:497–506
Hajishengallis G, Darveau RP, Curtis MA (2012) The keystone-pathogen hypothesis. Nat Rev Microbiol 10:717–725
Holt SC, Kesavalu L, Walker S, Genco CA (1999) Virulence factors of Porphyromonas gingivalis. Periodontol 2000 20:168–238
Duncan MJ (2003) Genomics of oral bacteria. Crit Rev Oral Biol Med 14:175–187
Kuramitsu HK (2003) Molecular genetic analysis of the virulence of oral bacterial pathogens: an historical perspective. Crit Rev Oral Biol Med 14:331–344
Nakayama K (2003) Molecular genetics of Porphyromonas gingivalis: gingipains and other virulence factors. Curr Protein Pept Sci 4:389–395
Genco CA, Simpson W, Forng RY, Egal M, Odusanya BM (1995) Characterization of a Tn4351-generated hemin uptake mutant of Porphyromonas gingivalis: evidence for the coordinate regulation of virulence factors by hemin. Infect Immun 63:2459–2466
Chen T et al (2000) Identification and cloning of genes from Porphyromonas gingivalis after mutagenesis with a modified Tn4400 transposon from Bacteroides fragilis. Infect Immun 68:420–423
BĂ©langer M, Rodrigues P, Progulske-Fox A (2007) Genetic manipulation of Porphyromonas gingivalis. Curr Protoc Microbiol. Chapter 13
Bryan G, Garza D, Hartl D (1990) Insertion and excision of the transposable element mariner in Drosophila. Genetics 125:103–114
Lampe DJ, Churchill MEA, Robertson HM (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J 15:5470–5479
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 96:11428–11433
Barquist L, Boinett CJ, Cain AK (2013) Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol 10:1161–1169
Van Opijnen T, Camilli A (2013) Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11:435–442
Goodman AL et al (2009) Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6:279–289
Gawronski JD, Wong SM, 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 106:16422–16427
van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767–772
Klein BA et al (2012) Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis. BMC Genomics 13
Lazinski DW, Camilli A (2013) Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction. Biotechniques 54:25–34
Dong X, Stothard P, Forsythe IJ, Wishart DS (2004) PlasMapper: a web server for drawing and auto-annotating plasmid maps. Nucleic Acids Res 32:W660–W664
Goecks J, Nekrutenko A, Taylor J, Galaxy Team (2010) Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol: 11, R86-2010-11-8-r86. Epub 2010 Aug 25
Abeel T, Van Parys T, Saeys Y, Galagan J, Van De Peer Y (2012) GenomeView: a next-generation genome browser. Nucleic Acids Res 40(2):e12
Luo H, Lin Y, Gao F, Zhang C-T, Zhang R (2014) DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements. Nucleic Acids Res 42:D574–D580
Goodman AL, Wu M, Gordon JI (2011) Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat Protoc 6:1969–1980
Kobayashi K et al (2003) Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100:4678–4683
Baba T et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008
Glass JI et al (2006) Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A 103:425–430
Van Opijnen T, Camilli A (2012) A fine scale phenotype-genotype virulence map of a bacterial pathogen. Genome Res 22:2541–2551
Wei W, Ning L-W, Ye Y-N, Guo F-B (2013) Geptop: a gene essentiality prediction tool for sequenced bacterial genomes based on orthology and phylogeny. PLoS ONE 8
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2398-4_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2397-7
Online ISBN: 978-1-4939-2398-4
eBook Packages: Springer Protocols