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Role of a Fur homolog in iron metabolism in Nitrosomonas europaea.

Vajrala N, Sayavedra-Soto LA, Bottomley PJ, Arp DJ - BMC Microbiol. (2011)

Bottom Line: Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.Our studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene.Additional studies are required to fully delineate role of this fur homolog.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Botany and Plant Pathology, 2082 Cordley, Oregon State University, Corvallis, OR 97331, USA.

ABSTRACT

Background: In response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of Nitrosomonas europaea, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family.

Results: Of the three N. europaea fur homologs, only the Fur homolog encoded by gene NE0616 complemented the Escherichia coli H1780 fur mutant. A N. europaea fur:kanP mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 fur homolog. The total cellular iron contents of the fur:kanP mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the fur:kanP mutant exhibited increased sensitivity to iron at or above 500 μM concentrations. Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.

Conclusions: Our studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene. Additional studies are required to fully delineate role of this fur homolog.

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Related in: MedlinePlus

In vitro transposon mutagenesis scheme and mutant confirmation. (A) The physical structure of a 5,810-bp fragment of the N. europaea chromosome is shown in the center (heavy black line), with positions of NE0616 (fur) gene shown as grey arrow, the fur box (fb) located in NE0616 promoter region shown as white rectangle. The regions covered by the plasmids pFur616, pFur616-kanP, pFur616-kanC whose DNA sequences were determined are shown as thin black lines with the names of the respective plasmids shown below each line. The position and relative orientation of each in vitro-constructed Tn5-Kan2 cassette insertion mutation are indicated by a flag on the lines. The restriction endonuclease sites P (Pst1) and E (EcoR1) used for Southern blot confirmation are indicated. (B) Verification of mutagenesis of fur:kanP in N. europaea by Southern hybridization. Genomic DNA from the wild type (WT), fur:kanP mutant (MT) were digested with E (EcoRI) and P (Pst1), and probed with (left) fur ORF sequence and (right) kan sequence.
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Figure 4: In vitro transposon mutagenesis scheme and mutant confirmation. (A) The physical structure of a 5,810-bp fragment of the N. europaea chromosome is shown in the center (heavy black line), with positions of NE0616 (fur) gene shown as grey arrow, the fur box (fb) located in NE0616 promoter region shown as white rectangle. The regions covered by the plasmids pFur616, pFur616-kanP, pFur616-kanC whose DNA sequences were determined are shown as thin black lines with the names of the respective plasmids shown below each line. The position and relative orientation of each in vitro-constructed Tn5-Kan2 cassette insertion mutation are indicated by a flag on the lines. The restriction endonuclease sites P (Pst1) and E (EcoR1) used for Southern blot confirmation are indicated. (B) Verification of mutagenesis of fur:kanP in N. europaea by Southern hybridization. Genomic DNA from the wild type (WT), fur:kanP mutant (MT) were digested with E (EcoRI) and P (Pst1), and probed with (left) fur ORF sequence and (right) kan sequence.

Mentions: To address the physiological role fur plays in N. europaea, we attempted to generate an N. europaea fur mutant but were unsuccessful. However, we were successful in isolating an N. europaea fur:kanP mutant strain with Kmr inserted in the Fur box located in the promoter region of NE0616 gene (Figure 4A). The pFur616-kanP plasmid was electroporated into N. europaea wild-type cells. The fur:kanP mutant was obtained through homologous recombination and confirmed by PCR (data not shown) and Southern hybridization (Figure 4B). The fur probe detected a 3.96 Kb EcoR1 fragment and a 4.85 Kb Pst1 fragment in wild type and a ~ 5 Kb EcoR1 fragment and a ~ 4.3 Kb Pst1 fragment (calculated size based on the DNA sequences) in fur:kanP mutant strain. The kanamycin-cassette probe detected the same ~ 5 Kb EcoR1 fragment and the ~ 4.3 Kb Pst1 fragment in fur:kanP mutant but not in the wild type. These results confirm that a single copy of Kmr was correctly inserted in the Fur box located in the promoter region of NE0616 gene of the N. europaea genome (Figure 4A). A fur transcript was not detected in the fur:kanP mutant by either RT-PCR or qRT-PCR analysis (up to 28 cycles) indicating the inactivation of fur gene due to Kmr insertion in its promoter region. Transcripts of ammonia monooxygenase C (amoC) component used as positive control both for the efficiency of the RT-PCR procedure and for RNA and cDNA recovery showed no significant difference in expression in wild type and the fur:kanP mutant (data not shown).


Role of a Fur homolog in iron metabolism in Nitrosomonas europaea.

Vajrala N, Sayavedra-Soto LA, Bottomley PJ, Arp DJ - BMC Microbiol. (2011)

In vitro transposon mutagenesis scheme and mutant confirmation. (A) The physical structure of a 5,810-bp fragment of the N. europaea chromosome is shown in the center (heavy black line), with positions of NE0616 (fur) gene shown as grey arrow, the fur box (fb) located in NE0616 promoter region shown as white rectangle. The regions covered by the plasmids pFur616, pFur616-kanP, pFur616-kanC whose DNA sequences were determined are shown as thin black lines with the names of the respective plasmids shown below each line. The position and relative orientation of each in vitro-constructed Tn5-Kan2 cassette insertion mutation are indicated by a flag on the lines. The restriction endonuclease sites P (Pst1) and E (EcoR1) used for Southern blot confirmation are indicated. (B) Verification of mutagenesis of fur:kanP in N. europaea by Southern hybridization. Genomic DNA from the wild type (WT), fur:kanP mutant (MT) were digested with E (EcoRI) and P (Pst1), and probed with (left) fur ORF sequence and (right) kan sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: In vitro transposon mutagenesis scheme and mutant confirmation. (A) The physical structure of a 5,810-bp fragment of the N. europaea chromosome is shown in the center (heavy black line), with positions of NE0616 (fur) gene shown as grey arrow, the fur box (fb) located in NE0616 promoter region shown as white rectangle. The regions covered by the plasmids pFur616, pFur616-kanP, pFur616-kanC whose DNA sequences were determined are shown as thin black lines with the names of the respective plasmids shown below each line. The position and relative orientation of each in vitro-constructed Tn5-Kan2 cassette insertion mutation are indicated by a flag on the lines. The restriction endonuclease sites P (Pst1) and E (EcoR1) used for Southern blot confirmation are indicated. (B) Verification of mutagenesis of fur:kanP in N. europaea by Southern hybridization. Genomic DNA from the wild type (WT), fur:kanP mutant (MT) were digested with E (EcoRI) and P (Pst1), and probed with (left) fur ORF sequence and (right) kan sequence.
Mentions: To address the physiological role fur plays in N. europaea, we attempted to generate an N. europaea fur mutant but were unsuccessful. However, we were successful in isolating an N. europaea fur:kanP mutant strain with Kmr inserted in the Fur box located in the promoter region of NE0616 gene (Figure 4A). The pFur616-kanP plasmid was electroporated into N. europaea wild-type cells. The fur:kanP mutant was obtained through homologous recombination and confirmed by PCR (data not shown) and Southern hybridization (Figure 4B). The fur probe detected a 3.96 Kb EcoR1 fragment and a 4.85 Kb Pst1 fragment in wild type and a ~ 5 Kb EcoR1 fragment and a ~ 4.3 Kb Pst1 fragment (calculated size based on the DNA sequences) in fur:kanP mutant strain. The kanamycin-cassette probe detected the same ~ 5 Kb EcoR1 fragment and the ~ 4.3 Kb Pst1 fragment in fur:kanP mutant but not in the wild type. These results confirm that a single copy of Kmr was correctly inserted in the Fur box located in the promoter region of NE0616 gene of the N. europaea genome (Figure 4A). A fur transcript was not detected in the fur:kanP mutant by either RT-PCR or qRT-PCR analysis (up to 28 cycles) indicating the inactivation of fur gene due to Kmr insertion in its promoter region. Transcripts of ammonia monooxygenase C (amoC) component used as positive control both for the efficiency of the RT-PCR procedure and for RNA and cDNA recovery showed no significant difference in expression in wild type and the fur:kanP mutant (data not shown).

Bottom Line: Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.Our studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene.Additional studies are required to fully delineate role of this fur homolog.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Botany and Plant Pathology, 2082 Cordley, Oregon State University, Corvallis, OR 97331, USA.

ABSTRACT

Background: In response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of Nitrosomonas europaea, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family.

Results: Of the three N. europaea fur homologs, only the Fur homolog encoded by gene NE0616 complemented the Escherichia coli H1780 fur mutant. A N. europaea fur:kanP mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 fur homolog. The total cellular iron contents of the fur:kanP mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the fur:kanP mutant exhibited increased sensitivity to iron at or above 500 μM concentrations. Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.

Conclusions: Our studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene. Additional studies are required to fully delineate role of this fur homolog.

Show MeSH
Related in: MedlinePlus