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Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus.

da Silva Neto JF, Braz VS, Italiani VC, Marques MV - Nucleic Acids Res. (2009)

Bottom Line: Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis.Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency.In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-000 São Paulo, SP, Brazil.

ABSTRACT
In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

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Analysis of expression of selected genes in response to iron and Fur. (A) Expression was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of either 100 μM FeSO4 (Fe) or 100 μM 2′-dipyridyl (DDP). The last panel shows as a control the expression of CC2194 in NA1000, the fur mutant (fur) and fur complemented with the gene in trans (fur+), indicating that the presence of Fur restores the wild-type expression. (B) Expression driven by the nuoA and sdhC promoters was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of 100 μM FeSO4. The assays were carried out using either the wild-type promoters (P) or the mutagenized promoters (P*) as described in Figure 3, introduced into the NA1000 or SP0057 strains. β-Galactosidase activity is expressed in Miller units (38) and is the average of at least three independent assays.
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Figure 5: Analysis of expression of selected genes in response to iron and Fur. (A) Expression was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of either 100 μM FeSO4 (Fe) or 100 μM 2′-dipyridyl (DDP). The last panel shows as a control the expression of CC2194 in NA1000, the fur mutant (fur) and fur complemented with the gene in trans (fur+), indicating that the presence of Fur restores the wild-type expression. (B) Expression driven by the nuoA and sdhC promoters was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of 100 μM FeSO4. The assays were carried out using either the wild-type promoters (P) or the mutagenized promoters (P*) as described in Figure 3, introduced into the NA1000 or SP0057 strains. β-Galactosidase activity is expressed in Miller units (38) and is the average of at least three independent assays.

Mentions: To determine the effect of Fur in gene expression in vivo, some of the promoter regions of genes with validated Fur-binding sites were cloned in front of a lacZ reporter gene and plasmids containing each transcriptional fusion were introduced into the wild-type NA1000 strain and into the fur mutant strain. Expression driven by each promoter was assessed by measuring β-galactosidase activity in the presence of either 100 μM FeSO4 or 100 μM of the iron chelator 2,2-dipyridyl (Figure 5A). The results showed that the expression driven by the sdhC, acnA and nuoA promoters was diminished in the presence of dipyridyl and in the fur mutant strain, indicating that these genes are activated by Fur in the presence of iron. The TonB-dependent receptors-encoding genes CC0139, CC0028, CC2928 and CC2194, as well as the feoAB operon, were induced in the presence of dipyridyl in the wild-type strain and constitutively derepressed in the fur mutant strain independently of iron, indicating that Fur represses these genes in response to high iron level. As a control, expression of CC2194 was also determined in the Fur complemented strain. The iron-dependent repression of the CC2194 gene was completely restored in this strain.Figure 5.


Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus.

da Silva Neto JF, Braz VS, Italiani VC, Marques MV - Nucleic Acids Res. (2009)

Analysis of expression of selected genes in response to iron and Fur. (A) Expression was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of either 100 μM FeSO4 (Fe) or 100 μM 2′-dipyridyl (DDP). The last panel shows as a control the expression of CC2194 in NA1000, the fur mutant (fur) and fur complemented with the gene in trans (fur+), indicating that the presence of Fur restores the wild-type expression. (B) Expression driven by the nuoA and sdhC promoters was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of 100 μM FeSO4. The assays were carried out using either the wild-type promoters (P) or the mutagenized promoters (P*) as described in Figure 3, introduced into the NA1000 or SP0057 strains. β-Galactosidase activity is expressed in Miller units (38) and is the average of at least three independent assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724300&req=5

Figure 5: Analysis of expression of selected genes in response to iron and Fur. (A) Expression was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of either 100 μM FeSO4 (Fe) or 100 μM 2′-dipyridyl (DDP). The last panel shows as a control the expression of CC2194 in NA1000, the fur mutant (fur) and fur complemented with the gene in trans (fur+), indicating that the presence of Fur restores the wild-type expression. (B) Expression driven by the nuoA and sdhC promoters was determined from cells harboring the respective promoter fusions to a lacZ reporter gene after incubation at 30°C for 2 h in presence of 100 μM FeSO4. The assays were carried out using either the wild-type promoters (P) or the mutagenized promoters (P*) as described in Figure 3, introduced into the NA1000 or SP0057 strains. β-Galactosidase activity is expressed in Miller units (38) and is the average of at least three independent assays.
Mentions: To determine the effect of Fur in gene expression in vivo, some of the promoter regions of genes with validated Fur-binding sites were cloned in front of a lacZ reporter gene and plasmids containing each transcriptional fusion were introduced into the wild-type NA1000 strain and into the fur mutant strain. Expression driven by each promoter was assessed by measuring β-galactosidase activity in the presence of either 100 μM FeSO4 or 100 μM of the iron chelator 2,2-dipyridyl (Figure 5A). The results showed that the expression driven by the sdhC, acnA and nuoA promoters was diminished in the presence of dipyridyl and in the fur mutant strain, indicating that these genes are activated by Fur in the presence of iron. The TonB-dependent receptors-encoding genes CC0139, CC0028, CC2928 and CC2194, as well as the feoAB operon, were induced in the presence of dipyridyl in the wild-type strain and constitutively derepressed in the fur mutant strain independently of iron, indicating that Fur represses these genes in response to high iron level. As a control, expression of CC2194 was also determined in the Fur complemented strain. The iron-dependent repression of the CC2194 gene was completely restored in this strain.Figure 5.

Bottom Line: Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis.Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency.In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-000 São Paulo, SP, Brazil.

ABSTRACT
In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

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