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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cianciotto NP (2007) Iron acquisition by Legionella pneumophila. Biometals 20:323–331
Cianciotto NP (2015) An update on iron acquisition by Legionella pneumophila: new pathways for siderophore uptake and ferric iron reduction. Future Microbiol 10:841–851
Robey M, Cianciotto NP (2002) Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection. Infect Immun 70:5659–5669
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 81:4182–4191
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 83:3937–3945
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 77:2887–2895
Allard KA, Viswanathan VK, Cianciotto NP (2006) IbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin. J Bacteriol 188:1351–1363
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 158:721–735
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 193:1563–1575
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 157:868–878
Edelstein PH, Edelstein MA (1993) Comparison of three buffers used in the formulation of buffered charcoal yeast extract medium. J Clin Microbiol 31:3329–3330
Ristroph JD, Hedlund KW, Allen RG (1980) Liquid medium for growth of Legionella pneumophila. J Clin Microbiol 11:19–21
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 45:784–791
Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56
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 188:3785–3795
Thieken A, Winkelmann G (1992) Rhizoferrin: a complexone type siderophore of the Mucorales and entomophthorales (Zygomycetes). FEMS Microbiol Lett 73:37–41
Acknowledgments
This work was supported by NIH grant AI034937 awarded to NPC.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Burnside, D.M., Cianciotto, N.P. (2019). Analysis of Iron Requirements and Siderophore Production. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 1921. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9048-1_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9048-1_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9047-4
Online ISBN: 978-1-4939-9048-1
eBook Packages: Springer Protocols