FUR- mutant vs. WT
Ontology highlight
ABSTRACT:
ORGANISM(S): Salmonella Salmonella enterica subsp. enterica serovar Typhimurium
PROVIDER: GSE18441 | GEO | 2011/10/14
SECONDARY ACCESSION(S): PRJNA118243
REPOSITORIES: GEO
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Project description:Ferric uptake regulator (Fur) is the major regulator of iron acquisition in Escherichia coli and other bacteria. In the present study, the role of Fur in anaerobic S. enterica serovar Typhimurium was determined by transcriptome analysis, reporter assays, and enzymatic assays. In anaerobic ?fur, 298 genes were differentially expressed. In general, Fur repressed genes required for iron acquisition/storage, metabolism, electron transport, oxidative/nitrosative stress, and modulators of virulence. There were 73 genes whose expression required Fur, eleven of which contain a putative Fur box 5? of the gene. Transcription of sodA was >9-fold higher in ?fur but there was no corresponding increase in the activity of SodA. Anaerobic cultures of the fur mutant and the WT were used to inoculate two sets of three independent flasks each containing 150 ml of anoxic LB-MOPS-X. The three independent cultures were grown to an optical density at 600 nm (OD600) of 0.25 to 0.35, pooled, and treated with RNAlater (QIAGEN, Valencia, CA) to fix the cells and preserve the quality of the RNA. Total RNA was extracted with the RNeasy RNA extraction kit (QIAGEN), and the samples were treated with RNase-free DNase (Invitrogen, Carlsbad, CA). The absence of contaminating DNA and the quality of the RNA was confirmed by PCR amplification of known genes and by using agarose gel electrophoresis. Aliquots of the RNA samples were kept at –80°C for use in the microarray and quantitative real-time reverse transcription-PCR (qRT-PCR) studies. The SuperScript Indirect cDNA labeling system (Invitrogen) was used to synthesize the cDNA for the hybridizations. Each experiment consisted of two hybridizations, on two slides, and was carried out in Corning Hybridization Chambers at 42°C overnight. Dye swapping was performed to avoid dye-associated effects on cDNA synthesis. The slides were washed at increasing stringencies (2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% sodium dodecyl sulfate [SDS], 42°C; 0.1% SSC, 0.1% SDS, room temperature; 0.1% SSC, room temperature). Following hybridization, the microarrays were scanned for the Cy3 and Cy5 fluorescent signals with a ScanArray 4000 microarray scanner from GSI Lumonics (Watertown, MA). The intensity of every spot was codified as the sum of the intensities of all the pixels within a circle positioned over the spot itself and the background as the sum of the intensities of an identical number of pixels in the immediate surroundings of the circled spot. Cy3 and Cy5 values for each spot were normalized over the total intensity for each dye to account for differences in total intensity between the two scanned images. The consistency of the data obtained from the microarray analysis was evaluated by a pair-wise comparison, calculated with a two-tailed Student's t test and analyzed by the MEAN and TTEST procedures of SAS-STAT statistical software (SAS Institute, Cary, NC) . The signal intensity at each spot from the fur mutant and the WT were background subtracted, normalized, and used to calculate the ratio of gene expression between the two strains. All replicas were combined, and the median expression ratios and standard deviations were calculated for open reading frames (ORFs) showing 2.5-fold change.
2011-10-13 | E-GEOD-18441 | biostudies-arrayexpress
Project description:Clostridium acetobutylicum is a Gram-positive, endospore-forming bacterium that is considered as a strict anaerobe. It ferments sugars to the organic acids acetate and butyrate or shifts to formation of the solvents - ethanol, butanol and acetone. In most bacteria the major regulator of iron homeostasis is Fur (ferric uptake regulator). Analysis of the genome of Clostridium acetobutylicum has revealed three genes encoding Fur-like proteins. The amino acid sequece of one of them showed 70% similarity to the Fur protein of the closely related Bacillus subtilis.<br>Thus, to gain insight into the role of Fur and the mechanisms for maintenance of iron homeostasis in this strict anaerobic organism, we determined its transcriptional profile in response to iron limitation and inactivation of fur.
2012-03-30 | E-MEXP-3401 | biostudies-arrayexpress
Project description:In Neisseria gonorrhoeae, Fur (ferric uptake regulator) protein regulates iron homeostasis gene expression through binding to conserved sequences in promoters of iron-responsive genes. We have expanded the gonococcal Fur regulon using a custom microarray to monitor iron-responsive gene expression throughout the growth curve combined with a genome-wide in silico analysis to predict Fur boxes (FB), and in vivo FuRTA assays to detect genes able to bind Fur. Keywords: time course: (1hr ,2hr, 3hr, 4hr)
2010-01-01 | GSE16352 | GEO
Project description:In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identify 46 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB dependent receptor gene clusters, feoAB), riboflavin biosynthesis and some genes encoding hypothetical proteins. Most of these genes are associated with Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 50 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, are also included in this group many genes encoding TonB dependent receptors and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses performed in the promoters of these genes suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of more 103 genes, including upregulation of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as downregulation of genes implicated in amino acid metabolism, chemotaxis and motility. Altogether, our results showed that adaptation of C. crescentus to iron limitation involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins as a general strategy Two experimental procedures, each of them performed in two replicates. A total of four independent biological samples were used
2013-08-15 | E-GEOD-45653 | biostudies-arrayexpress
Project description:Global tranascriptional profiling of Bacillus subtilis cells comparing fur mutant to mutants of the iron-sparing response: fur fsrA double mutant, fur fbpAB triple mutant, fur fbpC double mutant, and fur fbpABC quadruple mutant Abstract of associated manuscript: The Bacillus subtilis ferric uptake regulator (Fur) protein is the major sensor of cellular iron status. When iron is limiting for growth, derepression of the Fur regulon increases the cellular capacity for iron uptake and mobilizes an iron-sparing response mediated, in large part, by a small non-coding RNA named FsrA. FsrA functions together with three small, basic proteins (FbpABC) to repress many "low-priority" iron-containing enzymes. We have used transcriptome analyses to define the scope of the iron-sparing response and to define subsets of genes dependent on either FbpAB or FbpC for their repression. Enzymes of the tricarboxylic acid cycle, including both aconitase and succinate dehydrogenase, are major targets of FsrA-mediated repression and, as a consequence, flux through this pathway is significantly decreased under iron limitation. FsrA also mediates a physiologically significant repression of iron-sulfur containing enzymes required for ammonium assimilation (the GltAB glutamate synthase) and catabolism of lactic acid (LutABC). Repression of the lutABC operon requires both FsrA and FbpB which supports a model in which the Fbp proteins function to enable repression of subsets of the FsrA regulon.
2011-02-19 | GSE27416 | GEO
Project description:In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identify 46 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB dependent receptor gene clusters, feoAB), riboflavin biosynthesis and some genes encoding hypothetical proteins. Most of these genes are associated with Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 50 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, are also included in this group many genes encoding TonB dependent receptors and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses performed in the promoters of these genes suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of more 103 genes, including upregulation of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as downregulation of genes implicated in amino acid metabolism, chemotaxis and motility. Altogether, our results showed that adaptation of C. crescentus to iron limitation involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins as a general strategy
2013-08-15 | GSE45653 | GEO
Project description:Purpose: The goal is to study the regulatory role of ferric uptake regulator (Fur) during anaerobic respiration in Shewanella piezotolerans WP3
2016-06-10 | GSE47773 | GEO
Project description:Bacterial cells often modulate their transcriptional profiles in response to the changes in iron availability. Ferric uptake regulator (Fur), as a global iron biosensor, plays a central role in maintaining iron homeostasis in Bacillus subtilis. Here we utilized a high affinity Fe2+ efflux transporter, Listeria monocytogenes FrvA, as an inducible genetic tool to deplete intracellular iron. We then characterized the responses of the Fur, FsrA, and PerR regulons as cells transition from iron sufficiency to deficiency. Our results indicate that the Fur regulon is derepressed in three distinct waves. First, elemental iron uptake (ywbLMN), ferric citrate uptake (ymfCDEF-yhfQ), and petrobactin uptake (yclNOPQ) systems are induced to prevent iron deficiency. Second, B. subtilis synthesizes its own siderophore bacillibactin (dhbACEBF) and turns on bacillibactin uptake (feuABC-yusV) along with flavodoxin (ykuNOP) and hydroxamate siderophore uptake (fhuBCGD-yxeB) to scavenge iron from the environment. Third, as iron levels decline further, an iron sparing response (fsrA, fbpAB, and fbpC) is induced to block the translation of nonessential iron-using proteins and permit only essential iron-dependent enzymes to utilize the limited iron. ChIP experiments demonstrate that in vivo occupancy of Fur correlates with derepression of each operon, and the graded response observed here results, at least in part, from higher affinity binding of Fur to the late induced genes. These results provide insights into the distinct roles of Fur-regulated target genes as intracellular iron levels decline.
2017-06-30 | GSE100668 | GEO
Project description:Nontypeable Haemophilus influenzae (NTHi) is a commensal microorganism of the normal human nasopharyngeal flora, yet also an opportunistic pathogen of the upper and lower respiratory tracts. Changes in gene expression patterns in response to host microenvironments are likely critical for persistence. One such system of gene regulation is the ability to carefully regulate iron uptake. A central regulatory system that controls iron uptake, mediated by the ferric uptake regulator Fur, is present in multiple bacteria, including NTHi. To understand the regulation of iron homeostasis in NTHi, fur was deleted in the prototypic NTHi clinical isolate, 86-028NP. Using an NTHi-specific microarray, we identified genes whose expression was repressed or activated by Fur.
2013-02-05 | GSE39874 | GEO
Project description:Purpose: The goal is to study the regulatory role of ferric uptake regulator (Fur) during anaerobic respiration in Shewanella piezotolerans WP3 mRNA profiles of WP3 wild type (WP3) and the fur mutant (Fur) were generated by deep sequencing using Illumina HiSeq 2000.
2016-06-10 | E-GEOD-47773 | biostudies-arrayexpress