Advertisement

Identifying Essential Streptococcus sanguinis Genes Using Genome-Wide Deletion Mutation

  • Lei Chen
  • Xiuchun Ge
  • Ping Xu
Part of the Methods in Molecular Biology book series (MIMB, volume 1279)

Abstract

Essential genes in pathogens are important for the development of antibacterial drugs. In this report, we described a protocol to identify essential genes in the Streptococcus sanguinis SK36 strain using genome-wide deletion mutation. A fusion PCR-based method is used to construct gene deletion fragments, which contain kanamycin resistance cassettes with two flanking arms of DNA upstream and downstream of the target gene. The linear fused PCR amplicons were transformed into S. sanguinis SK36 cells. No kanamycin-resistant transformants suggested the gene essentiality because the deletion of the essential gene renders a lethal phenotype of the transformants. The putative essential genes were further confirmed by independent transformations up to five attempts. The false nonessential genes were also identified by removing double-band mutants.

Key words

Essential genes Streptococcus sanguinis Fusion PCR Deletion mutation Genome-wide 

Notes

Acknowledgment

The authors thank Xiaojing Wang, Yuetan Dou, Jerry Z. Xu, Jenishkumar R Patel, Victoria Stone, My Trinh, Karra Evans, Todd Kitten, Danail Bonchev, and Gregory A. Buck for their contributions in this research. The research is supported by grants R01DE018138 (PX) and R01DE023078 (PX) from the National Institutes of Health.

References

  1. 1.
    Juhas M, Eberl L, Church GM (2012) Essential genes as antimicrobial targets and cornerstones of synthetic biology. Trends Biotechnol 30:601–607PubMedCrossRefGoogle Scholar
  2. 2.
    Xu P, Ge X, Chen L, Wang X, Dou Y et al (2011) Genome-wide essential gene identification in Streptococcus sanguinis. Sci Rep 1:125PubMedCentralPubMedGoogle Scholar
  3. 3.
    Chen L, Ge X, Wang X, Patel JR, Xu P (2012) SpxA1 involved in hydrogen peroxide production, stress tolerance and endocarditis virulence in Streptococcus sanguinis. PLoS One 7(6):e40034PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Nyvad B, Kilian M (1987) Microbiology of the early colonization of human enamel and root surfaces in vivo. Scand J Dent Res 95:369–380PubMedGoogle Scholar
  5. 5.
    Mylonakis E, Calderwood SB (2001) Infective endocarditis in adults. N Engl J Med 345:1318–1330PubMedCrossRefGoogle Scholar
  6. 6.
    Paik S, Senty L, Das S, Noe JC, Munro CL et al (2005) Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis. Infect Immun 73:6064–6074PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Xu P, Alves JM, Kitten T, Brown A, Chen Z et al (2007) Genome of the opportunistic pathogen Streptococcus sanguinis. J Bacteriol 189:3166–3175PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Ge X, Xu P (2012) Genome-wide gene deletions in Streptococcus sanguinis by high throughput PCR. J Vis Exp 69:e4356Google Scholar
  9. 9.
    Gaustad P, Havardstein LS (1997) Competence-pheromone in Streptococcus sanguis. Identification of the competence gene comC and the competence pheromone. Adv Exp Med Biol 418:1019–1021PubMedCrossRefGoogle Scholar
  10. 10.
    Das S, Kanamoto T, Ge X, Xu P, Unoki T et al (2009) Contribution of lipoproteins and lipoprotein processing to endocarditis virulence in Streptococcus sanguinis. J Bacteriol 191:4166–4179PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Turner LS, Das S, Kanamoto T, Munro CL, Kitten T (2009) Development of genetic tools for in vivo virulence analysis of Streptococcus sanguinis. Microbiology 155:2573–2582PubMedCrossRefGoogle Scholar
  12. 12.
    De Berardinis V, Vallenet D, Castelli V, Besnard M, Pinet A et al (2008) A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1. Mol Syst Biol 4:174PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Philips Institute, Virginia Commonwealth UniversityRichmondUSA
  2. 2.The Philips Institute for Oral Health Research, Virginia Commonwealth UniversityRichmondUSA

Personalised recommendations