Skip to main content
SpringerLink
Log in

Fluorescence Polarization (FP) Assay for Measuring Staphylococcus aureus Membrane Fluidity

  • Protocol
  • First Online:
Staphylococcus aureus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2341))

Abstract

Fluorescence polarization is a method to determine membrane fluidity using a hydrophobic fluorescent dye that intercalates into the fatty acid bilayer. A spectrofluorometer is used to polarize UV light as a vertical excitation beam which passes through the dye-labeled membrane where the dye fluoresces. The beams perpendicular and horizontal to the excitation light are then collected and analyzed. Membrane structural properties are largely due to the packing of the fatty acids in the lipid bilayer that determines the membrane biophysical parameters. Staphylococcus aureus contains straight-chain (SCFAs) and branched-chain (BCFAs) fatty acids in the membrane and alters the proportion of membrane fluidizing BCFAs and stabilizing SCFAs as a response to a variety of stresses. Herein, we describe a method for determination of membrane fluidity in S. aureus using diphenylhexatriene, one of the most used fluorescent dyes for this purpose.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Zhang Y-M, Rock CO (2008) Membrane lipid homeostasis in bacteria. Nat Rev Microbiol 6:222–233

    Article  Google Scholar 

  2. Dowhan W (1997) Molecular basis for membrane phospholipids diversity, why are there so many lipids. Annu Rev Biochem 66:199–232

    Article  CAS  Google Scholar 

  3. Mykytczuk NCS, Trevors JT, Leduc LG et al (2007) Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress. Prog Biophys Mol Biol 95:60–82

    Article  CAS  Google Scholar 

  4. Parsons JB, Frank MW, Jackson P et al (2014) Incorporation of extracellular fatty acids by a fatty acid kinase-dependent pathway in Staphylococcus aureus. Mol Microbiol 92:234–245

    Article  CAS  Google Scholar 

  5. Sen S, Sirobhushanam S, Johnson SR et al (2016) Growth-environment dependent modulation of Staphylococcus aureus branched-chain to straight-chain fatty acid ratio and incorporation of unsaturated fatty acids. PLoS One 11:e0165300

    Article  Google Scholar 

  6. Mishra NN, Liu GY, Yeaman MR et al (2011) Carotenoid-related alteration of cell membrane fluidity impacts Staphylococcus aureus susceptibility to host defense peptides. Antimicrob Agents Chemother 55:526–531

    Article  CAS  Google Scholar 

  7. Wisniewska A, Widomska J, Subczynski WK (2006) Carotenoid-membrane interactions in liposomes: effect of dipolar, monopolar, and nonpolar carotenoids. Acta Biochim Pol 53:475–484

    Article  CAS  Google Scholar 

  8. Denich TJ, Beaudette LA, Lee H et al (2003) Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes. J Microbiol Methods 52:149–182

    Article  CAS  Google Scholar 

  9. Trevors JT (2003) Fluorescent probes for bacterial cytoplasmic membrane research. J Biochem Biophys Methods 57:87–103

    Article  CAS  Google Scholar 

  10. Adler M, Trirron TR (1988) Fluorescence depolarization measurements in oriented membranes. Biophys J 53:989–1005

    Article  CAS  Google Scholar 

  11. Litman BJ, Barenholz Y (1982) Fluorescent probe: diphenylhexatriene. Methods Enzymol 81:678–685

    Article  CAS  Google Scholar 

  12. Borenstain V, Barenholz Y (1993) Characterization of liposomes and other lipid assemblies by multiprobe fluorescence polarization. Chem Phys Lipids 64:117–127

    Article  CAS  Google Scholar 

  13. Shinitzky M, Barenholz Y (1978) Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim Biophys Acta 515:387–394

    Article  Google Scholar 

  14. Singh VK, Hattangady DS, Giotis ES et al (2008) Insertional inactivation of branched-chain α-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses. Appl Environ Microbiol 74:5882–5890

    Article  CAS  Google Scholar 

  15. Chamberlain NR, Mehrtens BG, Xiong Z et al (1991) Correlation of carotenoid production, decreased membrane fluidity, and resistance to oleic acid killing in Staphylococcus aureus 18Z. Infect Immun 59:4332–4337

    Article  CAS  Google Scholar 

  16. Bayer AS, Prasad R, Chandra J et al (2000) In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity. Infect Immun 68:3548–3553

    Article  CAS  Google Scholar 

  17. Oku Y, Kurokawa K, Ichihashi N et al (2004) Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol. Microbiology 150:45–51

    Article  CAS  Google Scholar 

  18. Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  19. Liu GY, Essex A, Buchanan JT et al (2005) Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. J Exp Med 202:209–215

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Biological Sciences, Illinois State University, Normal, IL, USA

    Kiran B. Tiwari, Craig Gatto & Brian J. Wilkinson

  2. Loyola University Chicago, Chicago, IL, USA

    Suranjana Sen

Authors
  1. Kiran B. Tiwari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suranjana Sen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Craig Gatto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brian J. Wilkinson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brian J. Wilkinson .

Editor information

Editors and Affiliations

  1. IFAS, Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA

    Kelly C. Rice

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Tiwari, K.B., Sen, S., Gatto, C., Wilkinson, B.J. (2021). Fluorescence Polarization (FP) Assay for Measuring Staphylococcus aureus Membrane Fluidity. In: Rice, K.C. (eds) Staphylococcus aureus. Methods in Molecular Biology, vol 2341. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1550-8_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1550-8_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1549-2

  • Online ISBN: 978-1-0716-1550-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation