Monitoring Iron Uptake by Siderophores

Hoegy, Françoise; Schalk, Isabelle J.

doi:10.1007/978-1-4939-0473-0_28

Cited by 14 publications

(15 citation statements)

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“…The bacteria were then washed with 50 mM Tris‐HCl pH 8.0, to eliminate the siderophores used to induce PfeA expression, and diluted to an OD 600nm of 1. Bacteria were then incubated in the presence of 200 nM chelator‐ 55 Fe, and the incorporation of radioactivity into the bacteria over time was monitored by filtration (Schalk et al ., ; Hoegy and Schalk, ). The experiments were repeated with cells pretreated with 200 μM CCCP.…”

Section: Methodsmentioning

confidence: 99%

“…Siderophore– 55 Fe complexes were prepared at 55 Fe concentrations of 20 μM, with a siderophore : iron (mol : mol) ratio of 20:1. Iron uptake assays were carried out as previously described for PVD–Fe transport (Schalk et al ., ; Hoegy and Schalk, ), except that bacteria were grown in CAA medium in the presence or absence of 10 μM enterobactin or catechol chelator, to induce PfeA expression. The bacteria were then washed with 50 mM Tris‐HCl pH 8.0, to eliminate the siderophores used to induce PfeA expression, and diluted to an OD 600nm of 1.…”

Section: Methodsmentioning

confidence: 99%

“…For the data presented in Table , the radioactivity incorporated into the bacteria was monitored after 15 min of incubation with the chelator– 55 Fe complexes. Iron‐55 uptake assays in the presence of PCH‐ 55 Fe (200 nM) were carried out as for enterobactin except that the bacteria were harvest by centrifugation, as previously described (Hoegy et al ., ; Hoegy and Schalk, ), and not by filtration to avoid an adsorption of PCH‐ 55 Fe on the GFB filters (Whatman).…”

Section: Methodsmentioning

confidence: 99%

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Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore–antibiotic conjugates development

Gasser

Baco

Cunrath

et al. 2016

Environmental Microbiology

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Previous studies have suggested that antibiotic vectorization by siderophores (iron chelators produced by bacteria) considerably increases the efficacy of such drugs. The siderophore serves as a vector: when the pathogen tries to take up iron via the siderophore, it also takes up the antibiotic. Catecholates are among the most common iron-chelating compounds used in synthetic siderophore-antibiotic conjugates. Using reverse transcription polymerase chain reaction and proteomic approaches, we showed that the presence of catecholate compounds in the medium of Pseudomonas aeruginosa led to strong activation of the transcription and expression of the outer membrane transporter PfeA, the ferri-enterobactin importer. Iron-55 uptake assays on bacteria with and without PfeA expression confirmed that catechol compounds imported iron into P. aeruginosa cells via PfeA. Uptake rates were between 0.3 × 10(3) and 2 × 10(3) Fe atoms/bacterium/min according to the used catechol siderophore in iron-restricted medium, and remained as high as 0.8 × 10(3) Fe atoms/bacterium/min for enterobactin, even in iron-rich medium. Reverse transcription polymerase chain reaction and proteomic approaches showed that in parallel to this switching on of PfeA expression, a repression of the expression of pyochelin (PCH) pathway genes (PCH being one of the two siderophores produced by P. aeruginosa for iron acquisition) was observed.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore–antibiotic conjugates development

Gasser

Baco

Cunrath

et al. 2016

Environmental Microbiology

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“…Siderophore-mediated iron uptake was performed as described by Fuchs et al (2001) and adapted by Hoegy and Schalk (2014). Briefly, labeled ferrisiderophores were prepared by mixing a 4μl 55 FeCl 3 250 μM (Amersham) with 4 μl of a 1 mM pyoverdine solution obtained from the cultures of the different strains.…”

Section: Methodsmentioning

confidence: 99%

Iron Uptake Analysis in a Set of Clinical Isolates of Pseudomonas putida

et al. 2016

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Pseudomonas putida strains are frequent inhabitants of soil and aquatic niches and they are occasionally isolated from hospital environments. As the available iron sources in human tissues, edaphic, and aquatic niches are different, we have analyzed iron-uptake related genes in different P. putida strains that were isolated from all these environments. We found that these isolates can be grouped into different clades according to the genetics of siderophore biosynthesis and recycling. The pyoverdine locus of the six P. putida clinical isolates that have so far been completely sequenced, are not closely related; three strains (P. putida HB13667, HB3267, and NBRC14164T) are grouped in Clade I and the other three in Clade II, suggesting possible different origins and evolution. In one clinical strain, P. putida HB4184, the production of siderophores is induced under high osmolarity conditions. The pyoverdine locus in this strain is closely related to that of strain P. putida HB001 which was isolated from sandy shore soil of the Yellow Sea in Korean marine sand, suggesting their possible origin, and evolution. The acquisition of two unique TonB-dependent transporters for xenosiderophore acquisition, similar to those existing in the opportunistic pathogen P. aeruginosa PAO, is an interesting adaptation trait of the clinical strain P. putida H8234 that may confer adaptive advantages under low iron availability conditions.

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“…Because of the low solubility of iron under natural conditions, the availability of free iron in solution is extremely limited (10 Ϫ8 to 10 Ϫ9 M) and far below the level required for microbial growth (ϳ10 Ϫ6 M) (6). Siderophores are ferric iron-chelating molecules with a high affinity for iron (binding constants from 10 20 to 10 50 M Ϫ1 ) and a low molecular mass, 200 to 2,000 Da (7,8), and are synthesized and secreted by both Gram-positive and Gram-negative bacteria in response to iron deprivation.…”

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confidence: 99%

A Complex Mechanism Involving LysR and TetR/AcrR That Regulates Iron Scavenger Biosynthesis in Pseudomonas donghuensis HYS

2018

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7-Hydroxytropolone (7-HT) is a symmetrical seven-membered heteroatomic ring with a carboxyl group and two hydroxyl groups and was recently reported to be an iron scavenger of HYS. Cluster 1 includes 12 genes related to the synthesis of 7-HT; among these genes, those for two regulators, Orf1 and Orf12, were predicted to regulate 7-HT biosynthesis and to be LysR-type transcriptional regulators (LTTRs) and TetR/AcrR family transcriptional regulators, respectively. Data from real-time quantitative PCR and β-galactosidase and classical siderophore assays indicated that the transcription levels of and , as well as those of crucial genes to , were repressed under high-iron conditions. The deletion of and led to an absence of 7-HT and a decrease in expression. Orf1 and Orf12 were essential for the production of 7-HT through These two regulators are regulated by the Gac/Rsm system; Orf1 facilitates the expression of Orf12, and Orf12 concomitantly stimulates the expression of to synthesize 7-HT. The overexpression of Orf12 decreased 7-HT yields, possibly through decreased expression. This work thus outlines a complex mechanism regulating the biosynthesis of the iron scavenger 7-HT in HYS. The synergy between Orf1 and Orf12 ensures that 7-HT acts as an iron chelator despite being toxic to bacteria and provides new ideas for the novel regulation of dual-functional secondary metabolism and research on 7-HT and its derivates in other bacteria. A complex regulation mechanism including two regulators, LysR and TetR/AcrR, in the biosynthesis of the novel iron scavenger 7-hydroxytropolone (7-HT) was verified in HYS. The coaction of LysR Orf1 and TetR/AcrR Orf12 may balance the toxicity and iron chelation of 7-HT in HYS to overcome iron deficiency, as well as improve the bacterial competitiveness under iron-scarce conditions because of the toxicity of 7-HT toward other bacteria, making the accurate regulation of 7-HT biosynthesis indispensable. This regulation mechanism may be ubiquitous in the group but may better explain the group's strong adaptability.

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Monitoring Iron Uptake by Siderophores

Cited by 14 publications

References 16 publications

Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore–antibiotic conjugates development

Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore–antibiotic conjugates development

Iron Uptake Analysis in a Set of Clinical Isolates of Pseudomonas putida

A Complex Mechanism Involving LysR and TetR/AcrR That Regulates Iron Scavenger Biosynthesis in Pseudomonas donghuensis HYS

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