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Volume 168, Issue 9

A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes Open Access

Abstract

Transmembrane potential is one of the main bioenergetic parameters of bacterial cells, and is directly involved in energizing key cellular processes such as transport, ATP synthesis and motility. The most common approach to measure membrane potential levels is through use of voltage-sensitive fluorescent dyes. Such dyes either accumulate or are excluded from the cell in a voltage-dependent manner, which can be followed by means of fluorescence microscopy, flow cytometry, or fluorometry. Since the cell’s ability to maintain transmembrane potential relies upon low and selective membrane ion conductivity, voltage-sensitive dyes are also highly sensitive reporters for the activity of membrane-targeting antibacterials. However, the presence of an additional membrane layer in Gram-negative (diderm) bacteria complicates their use significantly. In this paper, we provide guidance on how membrane potential and its changes can be monitored reliably in Gram-negatives using the voltage-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide [DiSC(5)]. We also discuss the confounding effects caused by the presence of the outer membrane, or by measurements performed in buffers rather than growth medium. We hope that the discussed methods and protocols provide an easily accessible basis for the use of voltage-sensitive dyes in Gram-negative organisms, and raise awareness of potential experimental pitfalls associated with their use.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): depolarization , Escherichia coli , membrane potential , Salmonella enterica and voltage-sensitive dyes
© 2022 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2022-09-27
2024-05-09
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References

  1. Spellberg B, Blaser M, Guidos RJ, Boucher HW, Bradley JS et al. Combating antimicrobial resistance: policy recommendations to save lives. Clin Infect Dis 2011; 52 Suppl 5:S397–428 [View Article]
     [Google Scholar]
  2. Mingeot-Leclercq M-P, Décout J-L. Bacterial lipid membranes as promising targets to fight antimicrobial resistance, molecular foundations and illustration through the renewal of aminoglycoside antibiotics and emergence of amphiphilic aminoglycosides. Med Chem Commun 2016; 7:586–611 [View Article]
     [Google Scholar]
  3. Silhavy TJ, Kahne D, Walker S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2010; 2:a000414 [View Article] [PubMed]
     [Google Scholar]
  4. Sochacki KA, Barns KJ, Bucki R, Weisshaar JC. Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37. Proc Natl Acad Sci U S A 2011; 108:E77–81 [View Article] [PubMed]
     [Google Scholar]
  5. Henzler Wildman KA, Lee D-K, Ramamoorthy A. Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37. Biochemistry 2003; 42:6545–6558 [View Article] [PubMed]
     [Google Scholar]
  6. Riool M, de Breij A, Kwakman PHS, Schonkeren-Ravensbergen E, de Boer L et al. Thrombocidin-1-derived antimicrobial peptide TC19 combats superficial multi-drug resistant bacterial wound infections. Biochim Biophys Acta Biomembr 2020; 1862:183282 [View Article] [PubMed]
     [Google Scholar]
  7. Sabnis A, Hagart KL, Klöckner A, Becce M, Evans LE et al. Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane. Elife 2021; 10:e65836 [View Article] [PubMed]
     [Google Scholar]
  8. Sauermann R, Rothenburger M, Graninger W, Joukhadar C. Daptomycin: A review 4 years after first approval. Pharmacology 2008; 81:79–91 [View Article] [PubMed]
     [Google Scholar]
  9. Vaara M. Polymyxins and their potential next generation as therapeutic antibiotics. Front Microbiol 2019; 10:1689 [View Article]
     [Google Scholar]
  10. Gray DA, Wenzel M. More than a pore: A current perspective on the in vivo mode of action of the lipopeptide antibiotic Daptomycin. Antibiotics (Basel) 2020; 9:E17 [View Article]
     [Google Scholar]
  11. Strahl H, Hamoen LW. Membrane potential is important for bacterial cell division. Proc Natl Acad Sci U S A 2010; 107:12281–12286 [View Article] [PubMed]
     [Google Scholar]
  12. Müller A, Wenzel M, Strahl H, Grein F, Saaki TNV et al. Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains. Proc Natl Acad Sci U S A 2016; 113:E7077–E7086 [View Article] [PubMed]
     [Google Scholar]
  13. Scheinpflug K, Wenzel M, Krylova O, Bandow JE, Dathe M et al. Antimicrobial peptide cWFW kills by combining lipid phase separation with autolysis. Sci Rep 2017; 7:44332 [View Article] [PubMed]
     [Google Scholar]
  14. Wenzel M, Rautenbach M, Vosloo JA, Siersma T, Aisenbrey CHM et al. The multifaceted antibacterial mechanisms of the pioneering peptide antibiotics tyrocidine and gramicidin S. mBio 2018; 9:e00802-18 [View Article]
     [Google Scholar]
  15. Wiedemann I, Benz R, Sahl H-G. Lipid II-mediated pore formation by the peptide antibiotic nisin: a black lipid membrane study. J Bacteriol 2004; 186:3259–3261 [View Article] [PubMed]
     [Google Scholar]
  16. Roth BL, Poot M, Yue ST, Millard PJ. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 1997; 63:2421–2431 [View Article] [PubMed]
     [Google Scholar]
  17. Stiefel P, Schmidt-Emrich S, Maniura-Weber K, Ren Q. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiol 2015; 15:1–9 [View Article] [PubMed]
     [Google Scholar]
  18. Bruni GN, Kralj JM. Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides. Elife 2020; 9:e58706 [View Article] [PubMed]
     [Google Scholar]
  19. Jolliffe LK, Doyle RJ, Streips UN. The energized membrane and cellular autolysis in Bacillus subtilis. Cell 1981; 25:753–763 [View Article] [PubMed]
     [Google Scholar]
  20. Tsukazaki T, Mori H, Echizen Y, Ishitani R, Fukai S et al. Structure and function of a membrane component SecDF that enhances protein export. Nature 2011; 474:235–238 [View Article] [PubMed]
     [Google Scholar]
  21. Gray D, Wang B, Gamba P, Strahl H, Hamoen L. Membrane depolarization kills dormant Bacillus subtilis cells by generating a lethal dose of ROS. In Review 2021 [View Article]
     [Google Scholar]
  22. Waggoner AS. Dye indicators of membrane potential. Annu Rev Biophys Bioeng 1979; 8:47–68 [View Article] [PubMed]
     [Google Scholar]
  23. Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H. Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Front Cell Dev Biol 2016; 4:29 [View Article]
     [Google Scholar]
  24. French S, Farha M, Ellis MJ, Sameer Z, Côté J-P et al. Potentiation of antibiotics against Gram-negative bacteria by Polymyxin B Analogue SPR741 from unique perturbation of the outer membrane. ACS Infect Dis 2020; 6:1405–1412 [View Article]
     [Google Scholar]
  25. Silvestro L, Weiser JN, Axelsen PH. Antibacterial and antimembrane activities of cecropin A in Escherichia coli. Antimicrob Agents Chemother 2000; 44:602–607 [View Article] [PubMed]
     [Google Scholar]
  26. Wu M, Maier E, Benz R, Hancock REW. Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 1999; 38:7235–7242 [View Article] [PubMed]
     [Google Scholar]
  27. Galperin My, Dibrov PA, Glagolev AN. delta mu H+ is required for flagellar growth in Escherichia coli. FEBS Lett 1982; 143:319–322 [View Article]
     [Google Scholar]
  28. Paul K, Erhardt M, Hirano T, Blair DF, Hughes KT. Energy source of flagellar type III secretion. Nature 2008; 451:489–492 [View Article] [PubMed]
     [Google Scholar]
  29. Santiveri M, Roa-Eguiara A, Kühne C, Wadhwa N, Hu H et al. Structure and function of stator units of the bacterial flagellar motor. Cell 2020; 183:244–257 [View Article]
     [Google Scholar]
  30. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article] [PubMed]
     [Google Scholar]
  31. de Jong IG, Beilharz K, Kuipers OP, Veening J-W. Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy. J Vis Exp 2011; 53:3145 [View Article] [PubMed]
     [Google Scholar]
  32. Xue C, Lin TY, Chang D, Guo Z. Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation. R Soc Open Sci 2017; 4:160696 [View Article] [PubMed]
     [Google Scholar]
  33. Renaud de la Faverie A, Guédin A, Bedrat A, Yatsunyk LA, Mergny J-L. Thioflavin T as a fluorescence light-up probe for G4 formation. Nucleic Acids Res 2014; 42:e65 [View Article]
     [Google Scholar]
  34. Sugimoto S, Arita-Morioka K, Mizunoe Y, Yamanaka K, Ogura T. Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels. Nucleic Acids Res 2015; 43:e92 [View Article] [PubMed]
     [Google Scholar]
  35. Prindle A, Liu J, Asally M, Ly S, Garcia-Ojalvo J et al. Ion channels enable electrical communication in bacterial communities. Nature 2015; 527:59–63 [View Article] [PubMed]
     [Google Scholar]
  36. Stratford JP, Edwards CLA, Ghanshyam MJ, Malyshev D, Delise MA et al. Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity. Proc Natl Acad Sci U S A 2019; 116:9552–9557 [View Article] [PubMed]
     [Google Scholar]
  37. Mancini L, Terradot G, Tian T, Pu Y, Li Y et al. A general workflow for characterization of nernstian dyes and their effects on bacterial physiology. Biophys J 2020; 118:4–14 [View Article] [PubMed]
     [Google Scholar]
  38. Vaara M. Agents that increase the permeability of the outer membrane. Microbiol Rev 1992; 56:395–411 [View Article] [PubMed]
     [Google Scholar]
  39. Wu M, Maier E, Benz R, Hancock REW. Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 1999; 38:7235–7242 [View Article] [PubMed]
     [Google Scholar]
  40. Morin N, Lanneluc I, Connil N, Cottenceau M, Pons AM et al. Mechanism of bactericidal activity of microcin L in Escherichia coli and Salmonella enterica. Antimicrob Agents Chemother 2011; 55:997–1007 [View Article] [PubMed]
     [Google Scholar]
  41. Clifton LA, Skoda MWA, Le Brun AP, Ciesielski F, Kuzmenko I et al. Effect of divalent cation removal on the structure of gram-negative bacterial outer membrane models. Langmuir 2015; 31:404–412 [View Article] [PubMed]
     [Google Scholar]
  42. Nikaido H, Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev 1985; 49:1–32 [View Article] [PubMed]
     [Google Scholar]
  43. Waggoner A. Optical probes of membrane potential. J Membr Biol 1976; 27:317–334 [View Article] [PubMed]
     [Google Scholar]
  44. Ehrenberg B, Montana V, Wei MD, Wuskell JP, Loew LM. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J 1988; 53:785–794 [View Article]
     [Google Scholar]
  45. Bashford CL. The measurement of membrane potential using optical indicators. Biosci Rep 1981; 1:183–196 [View Article] [PubMed]
     [Google Scholar]
  46. Daugelavicius R, Bakiene E, Bamford DH. Stages of polymyxin B interaction with the Escherichia coli cell envelope. Antimicrob Agents Chemother 2000; 44:2969–2978 [View Article] [PubMed]
     [Google Scholar]
  47. Bot CT, Prodan C. Quantifying the membrane potential during E. coli growth stages. Biophys Chem 2010; 146:133–137 [View Article] [PubMed]
     [Google Scholar]
  48. Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V et al. The complete genome sequence of Escherichia coli K-12. Science 1997; 277:1453–1462 [View Article] [PubMed]
     [Google Scholar]
  49. McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 2001; 413:852–856 [View Article] [PubMed]
     [Google Scholar]
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A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes
Microbiology 168, 001227 (2022); https://doi.org/10.1099/mic.0.001227
/content/journal/micro/10.1099/mic.0.001227
/content/journal/micro/10.1099/mic.0.001227
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