Extraction, Purification, and Identification of Yersiniabactin, the Siderophore of <i>Yersinia pestis</i>

Miller, Michael C.; DeMoll, Edward

doi:10.1002/9780471729259.mc05b03s23

Cited by 4 publications

(5 citation statements)

References 70 publications

(105 reference statements)

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“…7), this attribution was inconsistent with the electronic absorption spectrum, which showed no transitions in the lower visible/upper UV region characteristic of these species. [82][83][84][85] An assignment for SLC 3 that was more consistent with the electronic absorption spectrum is the Fe(III)hydroxamic acid complex 10 (m/z obs 496.16, m/z calc 496.14) with two coordinated hydroxamic acid groups (Fig. 6).…”

Section: Characterisation Of Siderophoresmentioning

confidence: 68%

The variable hydroxamic acid siderophore metabolome of the marine actinomycete Salinispora tropica CNB-440

Ejje¹,

Soe²,

Gu³

et al. 2013

Metallomics

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The recently sequenced genome of the marine actinomycete Salinispora tropica CNB-440 revealed a high frequency of gene clusters which code for the biosynthesis of known and novel secondary metabolites. Of these metabolites, bioinformatics analysis predicted that S. tropica CNB-440 could potentially biosynthesize, as high affinity Fe(iii) ligands, siderophores from the hydroxamic acid desferrioxamine class (sid1 gene cluster) and the phenolate-thia(oxa)zoli(di)ne class (sid2 and sid4 gene clusters). In this work, we have used Ni(ii)-based immobilized metal ion affinity chromatography (IMAC) to pre-fractionate the hydroxamic acid siderophore metabolome of S. tropica CNB-440 from the secondary metabolome, to reveal low abundance siderophores. LC-MS measurements and electronic absorption spectra from purified extracts incubated with exogenous Fe(iii) revealed eight siderophores from the desferrioxamine class (DFOA2, DFOA1a, DFOA1b, DFOB, DFON, DFOD2, DFOE, DFOD1), which included two constitutional isomers (DFOA1a, DFOA1b), and one new siderophore (DFON), the latter which would require assembly from a combination of 1,5-diaminopentane and 1,6-diaminohexane as diamine substrates. Three additional species (m/zobs 496.14, 792.34 and 804.34) with electronic absorption spectra characteristic of complexes formed between Fe(iii) and hydroxamic acid-type siderophores were evident under some conditions. The signal at m/zobs 792.34 eluted in the hydrophobic region of the reverse-phase LC and correlated with a DFOD1 analogue with a C-terminal branched chain fatty acid ([M + K(+)](+)m/zcalc 792.35), which has been previously identified from marine sediment dwelling Micrococcus luteus KLE1011. The S. tropica CNB-440 hydroxamic acid siderophore metabolome was modulated by culture conditions (pH 7, 22 °C; pH 7, 28 °C; pH 9, 28 °C) designed to simulate the variable marine environment. An increase in temperature at constant pH value showed increased levels of DFOA2 and DFOA1, and decreased levels of DFOB and DFOE. An increase in pH value at constant temperature showed decreased levels of DFOA2 and DFOA1, and increased levels of DFOB, DFON and DFOE. These results indicate that the marine adaptation of S. tropica CNB-440 could involve its ability to select a suite of siderophores from a large number of candidates, which are optimized for the iron microenvironment.

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Section: Characterisation Of Siderophoresmentioning

confidence: 68%

The variable hydroxamic acid siderophore metabolome of the marine actinomycete Salinispora tropica CNB-440

Ejje¹,

Soe²,

Gu³

et al. 2013

Metallomics

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“…62, 63 No iron was added to the spent medium before extraction. Eight liters of spent medium were extracted three times in 500 ml aliquots with 200 ml portions of ethyl acetate.…”

Section: Methodsmentioning

confidence: 99%

Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice

et al. 2017

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A number of bacterial pathogens require the ZnuABC Zinc (Zn2+) transporter and/or a second Zn2+ transport system to overcome Zn2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn2+ in vitro under the conditions tested. However, we detect a significant increase in Zn2+-binding ability of filtered supernatants from a Ybt+ strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.

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“…Apo-Ybt was purified in a multi-step procedure modified from previous methods (Miller et al, 2010;Miller and DeMoll, 2011). No iron was added to the spent media before extraction.…”

Section: Purification Of Apo-ybtmentioning

confidence: 99%

The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice

Bobrov

Kirillina

Fetherston

et al. 2014

Molecular Microbiology

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130

157

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Summary Bacterial pathogens must overcome host sequestration of zinc (Zn2+), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn2+ by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn2+-deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn2+ acquisition. Studies with the Zn2+-dependent transcriptional reporter znuA∷lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn2+. However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, that are required for Fe3+ acquisition by Ybt, are not needed for Ybt-dependent Zn2+ uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn2+ uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicemic plague mouse model.

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Extraction, Purification, and Identification of Yersiniabactin, the Siderophore of Yersinia pestis

Cited by 4 publications

References 70 publications

The variable hydroxamic acid siderophore metabolome of the marine actinomycete Salinispora tropica CNB-440

The variable hydroxamic acid siderophore metabolome of the marine actinomycete Salinispora tropica CNB-440

Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice

The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice

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