Expression Of Fur Research Articles

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids.

SUMMARYLipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Fur Protein Regulates the Motility of Avian Pathogenic Escherichia coli AE17 Through Promoter Regions of the Flagella Key Genes flhD.

Avian pathogenic Escherichia coli (APEC) is an important pathogen causing several diseases in birds. It is responsible for local and systemic infections in poultry, seriously impeding the development of the poultry industry, and poses a potential risk to public health. The iron absorption regulatory protein Fur and the noncoding RNA, RyhB, that it negatively regulates are important factors in bacterial iron uptake, but the regulation of bacterial virulence genes varies greatly among different bacteria. We found that Fur is very important for the mobility of APEC. The expression of fur and RyhB is extensively regulated in APEC, and RyhB expression is also negatively regulated by Fur. A transcriptomic analysis showed that the genes significantly differentially regulated by Fur are related to cell movement, including pilus- or flagellum-dependent cell motility. To verify these results, we examined the effects of fur knockdown on cell movement by measuring the diameter of the bacteria colonies. Consistent with the RNA sequencing results, the mobility of AE17Δfur was significantly reduced compared with that of the wild type, and it had almost lost its ability to move. Using an electrophoretic mobility assay, we confirmed that the Fur protein directly binds to the promoter region of the key flagellum-related gene flhD, thereby affecting the assembly and synthesis of the APEC flagellum. This study extends our understanding of gene regulation in APEC.

Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation

ObjectiveTo investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis (Y. pestis), as well as the roles of RyhBs in biofilm formation. MethodsRegulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting. ResultsFur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription, while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level. In addition, both RyhB1 and RyhB2 positively regulated Y. pestis biofilm exopolysaccharide (EPS) production and the expression of hmsHFRS and hmsT. ConclusionFur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in Y. pestis.

The inability of Bacillus licheniformis perR mutant to grow is mainly due to the lack of PerR-mediated fur repression.

PerR, a member of Fur family protein, is a metal-dependent H2O2 sensing transcription factor that regulates genes involved in peroxide stress response. Industrially important bacterium Bacillus licheniformis contains three PerR-like proteins (PerRBL, PerR2, and PerR3) compared to its close relative Bacillus subtilis. Interestingly, unlike other bacteria including B. subtilis, no authentic perR BL null mutant could be established for B. licheniformis. Thus, we constructed a conditional perR BL mutant using a xylose-inducible promoter, and investigated the genes under the control of PerRBL. PerRBL regulon genes include katA, mrgA, ahpC, pfeT, hemA, fur, and perR as observed for PerRBS. However, there is some variation in the expression levels of fur and hemA genes between B. subtilis and B. licheniformis in the derepressed state. Furthermore, katA, mrgA, and ahpC are strongly induced, whereas the others are only weakly or not induced by H2O2 treatment. In contrast to the B. subtilis perR null mutant which frequently gives rise to large colony phenotype mainly due to the loss of katA, the suppressors of B. licheniformis perR mutant, which can form colonies on LB agar, were all catalase-positive. Instead, many of the suppressors showed increased levels of siderophore production, suggesting that the suppressor mutation is linked to the fur gene. Consistent with this, perR fur double mutant could grow on LB agar without Fe supplementation, whereas perR katA double mutant could only grow on LB agar with Fe supplementation. Taken together, our data suggest that in B. licheniformis, despite the similarity in PerRBL and PerRBS regulon genes, perR is an essential gene required for growth and that the inability of perR null mutant to grow is mainly due to elevated expression of Fur.

Phased Expression Technique for Realistic Embodiment of Tiger Fur

In recent Hollywood movies such as , etc, animal-shaped CG creatures are being realistically presented. Materialization of fur serves as an important factor to give each character distinctive features as well as particularly describes CG creatures more realistically. In case of major studios, the fur effect is materialized by using in-house tool or through their own R&D. In most studios, however, the effect incurs technical and financial difficulties. This study, therefore, is to suggest an effective fur making technique using five plugins through experimental research. For this research purpose, this study first draws existing technical limitations and problems through literature review on fur making using CG and case studies of movies. Second, this study establishes optimized work process based on the limitations and problems by utilizing features of general plugins on three steps of ①Fur shape modeling, ②Fur dynamics or animation and ③Fur rendering as a technical method for realistic expression of fur and analyzes its technical utility. Through this procedure, it is to present an effective way of working and it is considered as a useful research that can be applied to projects of studios in general.

Expression of fur and its antisense α-fur from Microcystis aeruginosa PCC7806 as response to light and oxidative stress

Ferric uptake regulation (Fur) proteins are prokaryotic transcriptional regulators that integrate signaling of iron metabolism and oxidative stress responses with several environmental stresses. In photosynthetic organisms, Fur proteins regulate many genes involved in photosynthesis, nitrogen metabolism and other key processes. Also, Fur triggers the expression of virulence factors in many bacterial pathogens, and Fur from Microcystis aeruginosa has been shown to bind promoter regions of the microcystin synthesis gene cluster. In this work, we studied transcriptional responses of fur genes under different light intensities and oxidative stress. An antisense of fur, the α-fur RNA, plays an important role in regulating fur expression under oxidative stress, affecting levels of Fur protein in cells. Importantly, an active photosynthetic electron chain is required for the expression of the fur gene.

Ferric iron uptake genes are differentially expressed in the presence of copper sulfides in Acidithiobacillus ferrooxidans strain LR

Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide ores. This work aimed to investigate the relative expression of genes related to the iron uptake system when A. ferrooxidans LR was maintained in contact with chalcopyrite or bornite as the sole energy source. Real-time quantitative PCR analysis revealed that the presence of bornite had no effect on the expression of seven genes related to the siderophore-mediated Fe(III) uptake system, while in the presence of chalcopyrite the expression of the genes was up-regulated. Bioinformatic analysis of the genomic region where these genes were found revealed the existence of three new putative DNA-binding sequences for the ferric iron uptake transcriptional regulator (Fur). Electrophoretic mobility shift assays demonstrated that a purified A. ferrooxidans His-tagged Fur protein was able to bind in vitro to each of these putative Fur boxes, suggesting that Fur regulated the expression of these genes. The expression of fur and two known Fur-regulated genes, mntH and dsrK, was also investigated in the presence of chalcopyrite. While the expression of fur and mntH was up-regulated, the expression of dsrK was down-regulated. The low amount of ferrous iron in the medium was probably responsible for the up-regulation of fur and the genes related to the siderophore-mediated Fe(III) uptake system when A. ferrooxidans LR was kept in the presence of chalcopyrite. A homology model of the A. ferrooxidans Fur was constructed and revealed that the putative DNA-binding surface presents conserved positively charged residues, supporting a previously suggested mode of interaction with DNA. The up-regulation of fur and the siderophore-mediated Fe(III) uptake genes, and the down-regulation of dsrK suggest that in the presence of chalcopyrite Fur acts as a transcription inducer and repressor.

Transcriptomic Response ofListeria monocytogenesto Iron Limitation andfurMutation

Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur- and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.

Staphylococcus and the Healing Power of Pus

Staphylococcus aureus infections induce formation of neutrophil-rich abscesses filled with debris from dead phagocytes. Corbin and colleagues report that this pus has antimicrobial powers through the activity of calprotectin. Calprotectin, a member of the S100 family of proinflammatory proteins, acts through chelation of manganese. As manganese is an essential cofactor for several enzymes in S. aureus, this impacts bacterial growth and the bacterium's ability to withstand oxidative stress.

Identification of a Ferric uptake regulator fromMicrocystis aeruginosaPCC7806

Ferric uptake regulator (Fur) proteins are widely recognized as repressors that in many prokaryotes regulate a large number of genes involved in iron homeostasis and oxidative stress response. In our study, we were able to identify the complete sequence of the fur gene from Microcystis aeruginosa using inverse-polymerase chain reaction. DNA sequence analysis confirmed the presence of a 183 amino-acid open reading frame that showed high identity with Fur proteins reported for cyanobacteria. The recombinant Fur protein has been purified and electrophoretical mobility shift assays shown to be active. Mn2+ and dithiothreitol enable Fur to bind to its promoter, with dithiothreitol being more potent. The expression of Fur in Microcystis was induced about twofold in iron-deficient conditions.

OxyR and SoxRS regulation of fur.

The cytotoxic effects of reactive oxygen species are largely mediated by iron. Hydrogen peroxide reacts with iron to form the extremely reactive and damaging hydroxyl radical via the Fenton reaction. Superoxide anion accelerates this reaction because the dismutation of superoxide leads to increased levels of hydrogen peroxide and because superoxide elevates the intracellular concentration of iron by attacking iron-sulfur proteins. We found that regulators of the Escherichia coli responses to oxidative stress, OxyR and SoxRS, activate the expression of Fur, the global repressor of ferric ion uptake. A transcript encoding Fur was induced by hydrogen peroxide in a wild-type strain but not in a DeltaoxyR strain, and DNase I footprinting assays showed that OxyR binds to the fur promoter. In cells treated with the superoxide-generating compound paraquat, we observed the induction of a longer transcript encompassing both fur and its immediate upstream gene fldA, which encodes a flavodoxin. This polycistronic mRNA is induced by paraquat in a wild-type strain but not in a DeltasoxRS strain, and SoxS was shown to bind to the fldA promoter. These results demonstrate that iron metabolism is coordinately regulated with the oxidative stress defenses.

Purification of Vibrio cholerae fur and estimation of its intracellular abundance by antibody sandwich enzyme-linked immunosorbent assay.

The Vibrio cholerae fur gene was previously cloned and sequenced. A putative Fur box was identified in the divergent promoters of irgA, a virulence factor of V. cholerae, and irgB, a transcriptional activator of irgA. In this work, V. cholerae Fur was overexpressed in Escherichia coli and purified to approximately 95% homogeneity. The purified protein bound a DNA fragment containing the irgA-irgB promoter in a gel shift assay. The purified protein was used to raise monoclonal and polyclonal antibodies to V. cholerae Fur, and a Fur sandwich enzyme-linked immunosorbent assay was developed to estimate the intracellular abundance of Fur under a variety of growth conditions. The number of Fur molecules per cell during exponential growth was approximately 2,500, which is higher than most measurements for other bacterial repressors but comparable to the intracellular concentration of the leucine-responsive regulatory protein. The number of Fur molecules per cell increased in the late logarithmic and stationary phases. Growth of V. cholerae in low-iron medium did not alter the intracellular abundance of Fur significantly. Growth under microaerophilic conditions resulted in a significant, approximately twofold decrease in the intracellular levels of Fur. The measurements of intracellular Fur abundance indicate that a large amount of this repressor is produced constitutively and that the concentration of Fur in the cell varies by less than a factor of 2 under the conditions studied. We hypothesize that the high constitutive expression of Fur is necessary for its role as an iron-responsive regulator.

Iron content and FNR-dependent gene regulation in Escherichia coli

The significance of intracellular iron levels of Escherichia coli on the expression of the fumarate reductase operon (frd), which is regulated by the transcriptional activator FNR, was studied in vivo. The iron contents of aerobically and anaerobically grown E. coli were determined and related to the expression of frd and of genes (fiu, fepA, fhuF) which are regulated by the iron uptake regulatory protein Fur. The iron contents varied from 1.6 to 6.9 mumol Fe/g protein with no significant difference in aerobic and anaerobic bacteria. Expression of frd was not related to the different iron levels, but to oxygen supply. Only severe iron limitation in iron-depleted medium, which caused lower iron contents (0.8 to 1.6 mumol/g), reduced the expression of frd under anaerobic conditions. On the other hand, expression of fiu, fepA and fhuF clearly responded to iron supply and cellular content, but only slightly to changed O2 supply. Generally, expression of frd responded only to much stricter iron limitation, than expression of Fur regulated genes. It is concluded that the functional state of FNR during aerobic/anaerobic switch is not regulated by iron content and reversible binding of Fe2+ under physiological conditions. Therefore FNR does not communicate with the iron pool regulating the Fur protein.