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Chapter 60 : Genetic and Structural Examination of the Legiobactin Siderophore

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Genetic and Structural Examination of the Legiobactin Siderophore, Page 1 of 2

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Abstract:

Bacteria have evolved numerous ways to acquire iron from the environment, but a common mechanism is the production of siderophores. Siderophores are electronegative, iron-regulated, low-molecular-weight compounds produced by bacteria and fungi that bind ferric iron and facilitate its internalization via specific receptors. Siderophores are secondary metabolites mainly synthesized by nonribosomal peptide synthetases that are similar to those used for antibiotic synthesis. Iron-bound siderophores are usually recognized at the cell surface by specific receptors in both gram-positive and gram-negative bacteria that internalize the ferrisiderophore. Siderophores are extremely effective in binding the Fe ion because they contain the most effective iron binding ligands in nature, consisting of hydroxamate, catecholate, and α-hydroxycarboxylate ligands that form hexadentate Fe complexes, thus satisfying the six coordination sites on ferric ions. The authors showed that could produce a high-affinity iron-chelator. When grown at 37°C in a low-iron chemically defined medium (CDM), secretes a low-molecular-weight substance that is reactive in the Chrome Azurol S (CAS) assay. Indeed, legiobactin does not extract into common solvents that are used to extract catecholate and hydroxamate siderophores. The legiobactin peak is the portion of supernatants that promotes growth of iron-starved legionellae and is absent in the lbtA mutant supernatants. As was the case using C nuclear magnetic resonance (NMR) analysis, proton NMR analysis of purified legiobactin demonstrates that the siderophore contains only aliphatic residues. Currently, the authors are working toward determining the structure of legiobactin based on two-dimensional-NMR, elemental analysis, and Maldi experiments.

Citation: A. Allard K, Castignetti D, Crumrine D, Sanjeevaiah P, P. Cianciotto N. 2006. Genetic and Structural Examination of the Legiobactin Siderophore, p 242-245. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch60

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Figures

Image of FIGURE 1
FIGURE 1

Siderophore production by wildtype and an mutant. Wildtype 130b with pMMB2002 (⋄) or plbtA (♦) and mutant with pMM-B2002 (Δ) or plbtA (Δ) were grown in buffered yeast extract to an optical density at 660 nm (OD) of 1.0; inoculated into deferrated CDM to an OD of 0.3; and then incubated at 37°C. At various time points, the growth of the cultures was monitored spectrophotometrically (top), and the CAS reactivity of culture supernatants was examined (bottom). The values presented represent the means and standard deviations from duplicate cultures. The CAS reactivity of the mutant’s cultures was significantly different from that of the wildtype and complemented mutant cultures, at all times of incubation ( < 0.05; Student’s test). The results presented are representative of at least four independent experiments.

Citation: A. Allard K, Castignetti D, Crumrine D, Sanjeevaiah P, P. Cianciotto N. 2006. Genetic and Structural Examination of the Legiobactin Siderophore, p 242-245. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch60
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Image of FIGURE 2
FIGURE 2

Legiobactin H-NMR spectrum. Wild-type 130b was grown in buffered yeast extract to an OD of 1.0; inoculated into deferrated CDM to an OD of 0.3; and then incubated at 37°C. After 24 h, the CAS-reactive supernatants were harvested and filtered through 3-kDa cut-off ultrafiltration filters. Low-molecular-weight supernatants were then concentrated 16-fold by rotary evaporation and subjected to anion-exchange high-pressure liquid chromatography. Purified legiobactin was then concentrated and desalted by ultrafiltration using 500-kDa cut-off filters and dissolved ion D2O. 1H-NMR spectra were recorded on a Varian 300-Mhz NMR spectrometer.

Citation: A. Allard K, Castignetti D, Crumrine D, Sanjeevaiah P, P. Cianciotto N. 2006. Genetic and Structural Examination of the Legiobactin Siderophore, p 242-245. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch60
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References

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1. Byrd, T. F., and, M. A. Horwitz. 1989. Inter-feron gamma-activated human monocytes down-regulate transferrin receptors and inhibit the in-tracellular multiplication of Legionella pneumophila by limiting the availability of iron. J. Clin. Invest. 83:14571465.
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9. Wandersman, C., and, P. Delepelaire. 2004. Bacterial iron sources: from siderophores to he-mophores. Annu. Rev. Microbiol. 58:611647.
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