Advertisement

Analysis of Iron Requirements and Siderophore Production

  • Denise M. Burnside
  • Nicholas P. CianciottoEmail author
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)

Abstract

This chapter describes the methods for inducing, detecting, and purifying the Legionella pneumophila siderophore. The first protocol details the methods by which L. pneumophila is cultured to facilitate production of the siderophore, rhizoferrin. This chapter then describes how to purify siderophore from culture supernatants through sequential reversed-phase/weak-anion exchange chromatography and high-performance liquid chromatography. The next section describes assays which allow the detection of the iron-binding capability and the biological activity of the purified siderophore. Lastly, this chapter describes the growth of L. pneumophila in chemically defined liquid medium (CDM) containing various iron sources as a method to assess the iron requirements of L. pneumophila.

Key words

Legionella pneumophila CDM BYE BCYE Iron Siderophore HPLC Rhizoferrin Bioassay CAS assay Iron salts 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was supported by NIH grant AI034937 awarded to NPC.

References

  1. 1.
    Cianciotto NP (2007) Iron acquisition by Legionella pneumophila. Biometals 20:323–331CrossRefGoogle Scholar
  2. 2.
    Cianciotto NP (2015) An update on iron acquisition by Legionella pneumophila: new pathways for siderophore uptake and ferric iron reduction. Future Microbiol 10:841–851CrossRefGoogle Scholar
  3. 3.
    Robey M, Cianciotto NP (2002) Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection. Infect Immun 70:5659–5669CrossRefGoogle Scholar
  4. 4.
    Zheng H, Chatfield CH, Liles MR, Cianciotto NP (2013) Secreted pyomelanin of Legionella pneumophila promotes bacterial iron uptake and growth under iron-limiting conditions. Infect Immun 81:4182–4191CrossRefGoogle Scholar
  5. 5.
    Burnside DM, Wu Y, Shafaie S, Cianciotto NP (2015) The Legionella pneumophila siderophore legiobactin is a polycarboxylate that is identical in structure to rhizoferrin. Infect Immun 83:3937–3945CrossRefGoogle Scholar
  6. 6.
    Allard KA, Dao J, Sanjeevaiah P, McCoy-Simandle K, Chatfield CH, Crumrine DS, Castignetti D, Cianciotto NP (2009) Purification of legiobactin and importance of this siderophore in lung infection by Legionella pneumophila. Infect Immun 77:2887–2895CrossRefGoogle Scholar
  7. 7.
    Allard KA, Viswanathan VK, Cianciotto NP (2006) IbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin. J Bacteriol 188:1351–1363CrossRefGoogle Scholar
  8. 8.
    Chatfield CH, Mulhern BJ, Viswanathan VK, Cianciotto NP (2012) The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila. Microbiology 158:721–735CrossRefGoogle Scholar
  9. 9.
    Chatfield CH, Mulhern BJ, Burnside DM, Cianciotto NP (2011) Legionella pneumophila LbtU acts as a novel, TonB-independent receptor for the legiobactin siderophore. J Bacteriol 193:1563–1575CrossRefGoogle Scholar
  10. 10.
    Yip ES, Burnside DM, Cianciotto NP (2011) Cytochrome c4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c1 and c5 promote intracellular infection. Microbiology 157:868–878CrossRefGoogle Scholar
  11. 11.
    Edelstein PH, Edelstein MA (1993) Comparison of three buffers used in the formulation of buffered charcoal yeast extract medium. J Clin Microbiol 31:3329–3330PubMedPubMedCentralGoogle Scholar
  12. 12.
    Ristroph JD, Hedlund KW, Allen RG (1980) Liquid medium for growth of Legionella pneumophila. J Clin Microbiol 11:19–21PubMedPubMedCentralGoogle Scholar
  13. 13.
    Hoffman PS, Pine L, Bell S (1983) Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal. Appl Environ Microbiol 45:784–791PubMedPubMedCentralGoogle Scholar
  14. 14.
    Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56CrossRefGoogle Scholar
  15. 15.
    Sullivan JT, Jeffery EF, Shannon JD, Ramakrishnan G (2006) Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production. J Bacteriol 188:3785–3795CrossRefGoogle Scholar
  16. 16.
    Thieken A, Winkelmann G (1992) Rhizoferrin: a complexone type siderophore of the Mucorales and entomophthorales (Zygomycetes). FEMS Microbiol Lett 73:37–41CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Microbiology and ImmunologyNorthwestern University Medical SchoolChicagoUSA

Personalised recommendations

Cite protocol

Buy options