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Construction of Fluorescent Pneumococci for In Vivo Imaging and Labeling of the Chromosome

  • Morten KjosEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1968)

Abstract

Advances in fluorescence imaging techniques and development and optimization of fluorescent proteins recent years have made major impacts on different fields of pneumococcal research. This chapter provides methodology for construction of fluorescent pneumococcal strains using fusions to DNA-binding proteins. By expressing fluorescent proteins fused to HlpA, a pneumococcal nucleoid binding protein, brightly fluorescent pneumococci are generated. HlpA fusions may be used both for in vivo imaging of pneumococci as well as for marking the nucleoid in cell biology studies. Furthermore, it also explains how to construct strains for imaging of specific chromosomal loci in pneumococci, using a heterologous ParBS system.

Key words

GFP mKate2 HlpA Fluorescent fusions ParB 

References

  1. 1.
    Beilharz K, van Raaphorst R, Kjos M, Veening JW (2015) Red fluorescent proteins for gene expression and protein localization studies in Streptococcus pneumoniae and efficient transformation with DNA assembled via the Gibson assembly method. Appl Environ Microbiol 81:7244–7252CrossRefGoogle Scholar
  2. 2.
    Eberhardt A, Wu LJ, Errington J, Vollmer W, Veening JW (2009) Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems. Mol Microbiol 74:395–408CrossRefGoogle Scholar
  3. 3.
    Kjos M, Veening JW (2014) Tracking of chromosome dynamics in live Streptococcus pneumoniae reveals that transcription promotes chromosome segregation. Mol Microbiol 91:1088–1105CrossRefGoogle Scholar
  4. 4.
    Overkamp W, Beilharz K, Detert Oude Weme R, Solopova A, Karsens H, Kovacs A, Kok J, Kuipers OP, Veening JW (2013) Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl Environ Microbiol 79:6481–6490CrossRefGoogle Scholar
  5. 5.
    Henriques MX, Catalao MJ, Figueiredo J, Gomes JP, Filipe SR (2013) Construction of improved tools for protein localization studies in Streptococcus pneumoniae. PLoS One 8:e55049CrossRefGoogle Scholar
  6. 6.
    van Raaphorst R, Kjos M, Veening JW (2017) Chromosome segregation drives division site selection in Streptococcus pneumoniae. Proc Natl Acad Sci U S A 114:E5959–E5968CrossRefGoogle Scholar
  7. 7.
    Jacq M, Adam V, Bourgeois D, Moriscot C, Di Guilmi AM, Vernet T, Morlot C (2015) Remodeling of the Z-ring nanostructure during the Streptococcus pneumoniae cell cycle revealed by photoactivated localization microscopy. MBio 6:e01108–e01115CrossRefGoogle Scholar
  8. 8.
    Kjos M, Aprianto R, Fernandes VE, Andrew PW, van Strijp JA, Nijland R, Veening JW (2015) Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions. J Bacteriol 197:807–818CrossRefGoogle Scholar
  9. 9.
    Ercoli G, Fernandes VE, Chung WY, Wanford JJ, Thomson S, Bayliss CD, Straatman K, Crocker PR, Dennison A, Martinez-Pomares L et al (2018) Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia. Nat Microbiol 3:600–610CrossRefGoogle Scholar
  10. 10.
    Jim KK, Engelen-Lee J, van der Sar AM, Bitter W, Brouwer MC, van der Ende A, Veening JW, van de Beek D, Vandenbroucke-Grauls CM (2016) Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model. J Neuroinflammation 13:188CrossRefGoogle Scholar
  11. 11.
    Reddinger RM, Luke-Marshall NR, Sauberan SL, Håkansson AP, Campagnari AA (2018) Streptococcus pneumoniae modulates Staphylococcus aureus biofilm dispersion and the transition from colonization to invasive disease. MBio 9:e02089–e02017CrossRefGoogle Scholar
  12. 12.
    Nourikyan J, Kjos M, Mercy C, Cluzel C, Morlot C, Noirot-Gros MF, Guiral S, Lavergne JP, Veening JW, Grangeasse C (2015) Autophosphorylation of the bacterial tyrosine-kinase CpsD connects capsule synthesis with the cell cycle in Streptococcus pneumoniae. PLoS Genet 11:e1005518CrossRefGoogle Scholar
  13. 13.
    Mercy C, Lavergne J-P, Slager J, Ducret A, Garcia PS, Noirot-Gros M-F, Dubarry N, Nourikyan J, Veening J-W, Grangeasse C (2018) RocS drives chromosome segregation and nucleoid occlusion in Streptococcus pneumoniae. bioRxiv doi: 10.1101/359943Google Scholar
  14. 14.
    Attaiech L, Minnen A, Kjos M, Gruber S, Veening JW (2015) The ParB-parS chromosome segregation system modulates competence development in Streptococcus pneumoniae. MBio 6:e00662CrossRefGoogle Scholar
  15. 15.
    Lacks S, Hotchkiss RD (1960) A study of the genetic material determining an enzyme in pneumococcus. Biochim Biophys Acta 39:508–518CrossRefGoogle Scholar
  16. 16.
    Martin B, Garcia P, Castanie MP, Claverys JP (1995) The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. Mol Microbiol 15:367–379CrossRefGoogle Scholar
  17. 17.
    Aprianto R, Slager J, Holsappel S, Veening J-W (2018) High-resolution analysis of the pneumococcal transcriptome under a wide range of infection-relevant conditions. Nucleic Acids Res 46:9990-10006PubMedPubMedCentralGoogle Scholar
  18. 18.
    Straume D, Stamsås GA, Berg KH, Salehian Z, Håvarstein LS (2017) Identification of pneumococcal proteins that are functionally linked to penicillin-binding protein 2b (PBP2b). Mol Microbiol 103:99–116CrossRefGoogle Scholar
  19. 19.
    Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T (2001) Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 14:529–532CrossRefGoogle Scholar
  20. 20.
    Higuchi R, Krummel B, Saiki RK (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16:7351–7367CrossRefGoogle Scholar
  21. 21.
    Irwin CR, Farmer A, Willer DO, Evans DH (2012) In-fusion(R) cloning with vaccinia virus DNA polymerase. Methods Mol Biol 890:23–35CrossRefGoogle Scholar
  22. 22.
    Liu X, Gallay C, Kjos M, Domenech A, Slager J, van Kessel SP, Knoops K, Sorg RA, Zhang JR, Veening JW (2017) High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Mol Syst Biol 13:931CrossRefGoogle Scholar
  23. 23.
    Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:349–361CrossRefGoogle Scholar
  24. 24.
    Slager J, Kjos M, Attaiech L, Veening JW (2014) Antibiotic-induced replication stress triggers bacterial competence by increasing gene dosage near the origin. Cell 157:395–406CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Faculty of Chemistry, Biotechnology and Food ScienceNorwegian University of Life SciencesÅsNorway

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