Abstract
More than 500 siderophores that bind ferric iron have been characterized and grouped by type based on their chemical structure. The chrome azurol S (CAS) assay is a universal colorimetric method that detects siderophores independent of their structure. In this assay, siderophores scavenge iron from an Fe-CAS-hexadecyltrimethylammonium bromide complex, and subsequent release of the CAS dye results in a color change from blue to orange. Solution-based experiments with CAS result in a quantitative measure of siderophore production, while an observable color change on CAS agar plates can be performed for qualitative detection of siderophores. Cross-feeding assays are another useful method to detect and characterize siderophores produced by bacteria. Under iron-limiting conditions, cross-feeding assays test the ability of an indicator strain to grow when supplied with a specific siderophore (from a test strain) to which it has a cognate receptor required for import into the cell. The cross-feeding assay can be tested with a variety of wild-type strains, siderophore biosynthesis mutants, and siderophore receptor mutants.
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Acknowledgments
We would like to thank Shelly Payne for helpful insight on the CAS agar protocol, Tyler Nusca for chelex-treated casamino acids and lyophilization of samples used in the CAS assay described in this protocol, and Alfredo Torres for generously providing the CFT073 entF::kan iucB::cam strain. We also thank Christopher Alteri for insight on designing the cross-feeding assay for P. mirabilis and Erin Garcia for construction of E. coli 536 mutants entF::kan and entF ybtS::kan. This work was supported in part by the Public Health Service Grants AI043360 and AI059722 from the National Institutes of Health.
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Himpsl, S.D., Mobley, H.L.T. (2019). Siderophore Detection Using Chrome Azurol S and Cross-Feeding Assays. In: Pearson, M. (eds) Proteus mirabilis. Methods in Molecular Biology, vol 2021. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9601-8_10
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DOI: https://doi.org/10.1007/978-1-4939-9601-8_10
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