GNPS - Iron titration into siderophore standard mixture and cultures
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Commercial Standards E. Coli Nissle 1917 Arthrobacter Sp. (ncbitaxon:1667)
SUBMITTER: 
Pieter C. Dorrestein
PROVIDER: MSV000085103 | GNPS | Sat Mar 14 18:13:00 GMT 2020
REPOSITORIES: GNPS
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Project description:Iron was titrated into 5 commercial siderophore standards to calculate dissociation constants.
2020-03-14 | MSV000085102 | GNPS
Project description:Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. In two experiments we treated A549 (lung cancer cell line) cells with Lcn2, Ent, and iron, alone and in combination. In experiment 1, biological duplicates of 4 conditions were used: PBS control, Lcn2, Lcn2+Ent, and Lcn2+Ent+iron. In experiment 2, 4 biological replicates of 4 conditions were used: PBS control, Ent, iron, and Ent+iron. Targets made from the samples were hybridized to Affymetrix Human Gene 1.0 ST arrays to measure transcript abundances. The RMA algorithm was used to estimate transcript levels. Replicate samples were exchangeable, so we fit one-way ANOVA models to log2-transformed data separately to each experiment, and tested for pairwise differences between groups in each experiment, as well as asking if the Ent vs. PBS differences were larger or smaller than the Ent+iron vs. iron differences (Ent by iron interactions). We report results for 29096 probe-sets that were not annotated as positive or negative controls on the array. A supplementary Excel workbook is provided that contains the estimated expression level, some probe-set annotation, and simple statistical analysis for each probe-set. It may be convenient for some users, however obtaining newer probe-set annotation may be advisable.
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Project description:Iron (Fe) plays a pivotal role in several metabolic and biosynthetic pathways essential for plant growth. Fe deficiency in plants severely affects the overall crop yield. Despite several studies on iron deficiency responses in different plant species, these mechanisms remain unclear in the allohexaploid wheat, which is the most widely cultivated commercial crop. In order to gain a comprehensive insight into molecular responses of bread wheat when exposed to iron deficiency, we studied transcriptomic changes in the roots and flag leaves of wheat plants subjected to iron-deficient and iron-sufficient conditions during early grain filling.
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Project description:Iron titration was performed (for replicates) into mixture of commercial standards (DFE, DFB, enterobactin, rhodotorulic acid, and ferrichrome), into cheese culture (JB 182), and E coli Nissle culture. Then iron concentration was kept constant (at 5 different concentrations) while the concentration of standard mixture was increased. This will be another way to determine Kd of each compound.
2020-03-31 | MSV000085206 | MassIVE
Project description:Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. In two experiments we treated A549 (lung cancer cell line) cells with Lcn2, Ent, and iron, alone and in combination. In experiment 1, biological duplicates of 4 conditions were used: PBS control, Lcn2, Lcn2+Ent, and Lcn2+Ent+iron. In experiment 2, 4 biological replicates of 4 conditions were used: PBS control, Ent, iron, and Ent+iron. Targets made from the samples were hybridized to Affymetrix Human Gene 1.0 ST arrays to measure transcript abundances. The RMA algorithm was used to estimate transcript levels. Replicate samples were exchangeable, so we fit one-way ANOVA models to log2-transformed data separately to each experiment, and tested for pairwise differences between groups in each experiment, as well as asking if the Ent vs. PBS differences were larger or smaller than the Ent+iron vs. iron differences (Ent by iron interactions). We report results for 29096 probe-sets that were not annotated as positive or negative controls on the array. A supplementary Excel workbook is provided that contains the estimated expression level, some probe-set annotation, and simple statistical analysis for each probe-set. It may be convenient for some users, however obtaining newer probe-set annotation may be advisable. A549 (ATCC CCL-185) cells, a human type II pneumocyte cell line, were cultured in F12K (Invitrogen, Carlsbad, CA) media supplemented with 10% fetal bovine serum (Invitrogen) and 1:100 penicillin streptomycin (Invitrogen). 24-well plates were seeded with A549 cells at a concentration of 35000 cells/well. After two days, cells were weaned from serum and antibiotics overnight. Cells were then stimulated overnight with the indicated combinations of 50 uM Ferric ammonium citrate (FAC) (Sigma, St. Louis, MO), 50 um Ent (Sigma or EMC Microcollections, Tubingen, Germany), and/or 25 uM lipocalin 2 (Lcn2) in F12K media lacking serum or antibiotics. Prior to incubation with cells, siderophore-Lcn2 complexes were prepared by sequential incubation at room temperature of FAC and Ent for 30 minutes followed by addition of Lcn2 and incubation for an additional 30 minutes. In experiment 1, 2 biological replicates of each of these 4 conditions were grown: PBS, 25 uM lipocalin 2 (Lcn2), a combination of 25 uM Lcn2 with 50 uM Enterobactin (Ent), and a combination of Lcn2, Ent and 50 uM Ferric Ammonium Citrate (FAC). In experiment 2, 4 biological replicates of each of these 4 conditions were grown: PBS, 50 uM Ent, 50 uM FAC, and a combination of 50 uM Ent and 50 uM FAC. Cells were harvested and RNA was extracted using an miRNeasy Mini Kit (Qiagen). Biotinylated cDNA targets were prepared according to the Ambion WT expression kit protocol from 150 ng total RNA. There are no batches within each experiment - the samples in each group are exchangeable. Targets were hybridized to Affymetrix Human Gene 1.0 ST arrays. Arrays were scanned and quantified by usual procedures, and transcript abundances for 29096 non-control probe-sets estimated by an RMA algorithm.
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Project description:Iron is the fourth most abundant element in the Earth’s crust. However, the poor solubility of iron due to oxidation of ferrous iron to the almost insoluble ferric iron under aerobic conditions constitutes a considerable challenge for living organisms to obtain sufficient amounts of the iron available. In the present study, we set out to characterize the global gene expression of C. glutamicum under iron limitation in comparison to iron-replete conditions.
2020-02-24 | GSE92359 | GEO
Project description:Iron is the fourth most abundant element in the Earth’s crust. However, the poor solubility of iron due to oxidation of ferrous iron to the almost insoluble ferric iron under aerobic conditions constitutes a considerable challenge for living organisms to obtain sufficient amounts of the iron available. In the present study, we set out to characterize the global gene expression of C. glutamicum under iron limitation in comparison to iron-replete conditions.
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Project description:Glutamicibacter arilaitensis JB182 cheese culture extracts were analyzed with a post-LC iron infusion.
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Project description:Soilborne fungal pathogens cause devastating yield losses, are highly persistent and difficult to control. To culminate infection, these organisms must cope with limited availability of iron. Here we show that the bZIP protein HapX functions as a key regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. Deletion of hapX does not affect iron uptake, but causes derepression of genes involved in iron-consuming pathways, leading to impaired growth under iron-depleted conditions. F. oxysporum strains lacking HapX are reduced in their capacity to invade and kill tomato plants and immunodepressed mice. The virulence defect of ΔhapX on tomato plants is exacerbated by coinoculation of roots with a biocontrol strain of Pseudomonas putida, but not with a siderophore-deficient mutant, indicating that HapX contributes to iron competition of F. oxysporum in the tomato rhizosphere. These results establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.
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