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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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Promoter sequences of selected genes, indicating the Fur-binding sites (shaded) predicted in silico or experimentally demonstrated (sdhC and CC2194). Boxes indicate conserved –35 and –10 sequences of Caulobacter σ70 promoters (TTGAC-16 bp-G/CCTANA) and previously identified transcription start sites (39) are indicated in bold. Annotated start codons are also indicated in bold letters, except for the putative start codon of CC0028, which was proposed in this work.
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Figure 6: Promoter sequences of selected genes, indicating the Fur-binding sites (shaded) predicted in silico or experimentally demonstrated (sdhC and CC2194). Boxes indicate conserved –35 and –10 sequences of Caulobacter σ70 promoters (TTGAC-16 bp-G/CCTANA) and previously identified transcription start sites (39) are indicated in bold. Annotated start codons are also indicated in bold letters, except for the putative start codon of CC0028, which was proposed in this work.

Mentions: Transcriptional start sites previously determined (39) were used to analyze the position of some verified Fur-binding sites in relation to the promoter region of the respective genes (Figure 6). The Fur-binding sites of genes nuoA, sdhC and acnA are found upstream of the deduced –35 promoter regions, consistent with them being activated by Fur, as verified by gene expression assays (Figure 5). On the other hand, the Fur-binding sites of genes CC0139, CC0028, CC2928, C2194 and feoA are located overlapping or downstream of the RNA polymerase binding site, suggesting transcriptional repression by steric hindrance. In fact, all these genes were repressed by Fur in response to high iron level (Figure 5A). Thus, transcription start sites correlated well with –35 and –10 promoter elements and with predicted Fur-binding sites, except for sdhC. In this gene, the proposed transcriptional start site was found between the –35 and –10 elements of an excellent C. crescentus σ70 consensus (TTGAC-16-CCTANA) and the role of Fur as activator indicates that the Fur-binding site is correctly positioned with regard to this consensus, indicating that the transcriptional start site is probably a few base pairs downstream.Figure 6.


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)

Promoter sequences of selected genes, indicating the Fur-binding sites (shaded) predicted in silico or experimentally demonstrated (sdhC and CC2194). Boxes indicate conserved –35 and –10 sequences of Caulobacter σ70 promoters (TTGAC-16 bp-G/CCTANA) and previously identified transcription start sites (39) are indicated in bold. Annotated start codons are also indicated in bold letters, except for the putative start codon of CC0028, which was proposed in this work.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Promoter sequences of selected genes, indicating the Fur-binding sites (shaded) predicted in silico or experimentally demonstrated (sdhC and CC2194). Boxes indicate conserved –35 and –10 sequences of Caulobacter σ70 promoters (TTGAC-16 bp-G/CCTANA) and previously identified transcription start sites (39) are indicated in bold. Annotated start codons are also indicated in bold letters, except for the putative start codon of CC0028, which was proposed in this work.
Mentions: Transcriptional start sites previously determined (39) were used to analyze the position of some verified Fur-binding sites in relation to the promoter region of the respective genes (Figure 6). The Fur-binding sites of genes nuoA, sdhC and acnA are found upstream of the deduced –35 promoter regions, consistent with them being activated by Fur, as verified by gene expression assays (Figure 5). On the other hand, the Fur-binding sites of genes CC0139, CC0028, CC2928, C2194 and feoA are located overlapping or downstream of the RNA polymerase binding site, suggesting transcriptional repression by steric hindrance. In fact, all these genes were repressed by Fur in response to high iron level (Figure 5A). Thus, transcription start sites correlated well with –35 and –10 promoter elements and with predicted Fur-binding sites, except for sdhC. In this gene, the proposed transcriptional start site was found between the –35 and –10 elements of an excellent C. crescentus σ70 consensus (TTGAC-16-CCTANA) and the role of Fur as activator indicates that the Fur-binding site is correctly positioned with regard to this consensus, indicating that the transcriptional start site is probably a few base pairs downstream.Figure 6.

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.

Show MeSH