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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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Fur Titration Assays (FURTA). (A) Complementation of an E. coli fur mutant H1780 by N. europaea Fur homologs.E. coli H1780 (pFur616)-upper left quadrant; H1780 (pFur616-kanC)-upper right quadrant; H1780 (pFur730)-lower left quadrant; H1780 (pFur1722)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs. (B) E. coli H1717 plated on McConkey medium with 30 μM Fe supplement-upper left quadrant, no Fe supplement-upper right quadrant; H1717 (pFur616)-lower left quadrant; H1717 (pFur616-kanP)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs.
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Figure 3: Fur Titration Assays (FURTA). (A) Complementation of an E. coli fur mutant H1780 by N. europaea Fur homologs.E. coli H1780 (pFur616)-upper left quadrant; H1780 (pFur616-kanC)-upper right quadrant; H1780 (pFur730)-lower left quadrant; H1780 (pFur1722)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs. (B) E. coli H1717 plated on McConkey medium with 30 μM Fe supplement-upper left quadrant, no Fe supplement-upper right quadrant; H1717 (pFur616)-lower left quadrant; H1717 (pFur616-kanP)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs.

Mentions: All strains evaluated for Lac phenotype were grown on McConkey Lactose plates with 30 μM iron supplement, since iron is required to ensure that Fur is functional as a repressor [6]. In these studies, E. coli H1780, H1780 (pFur616), H1780 (pFur616-kanC), H1780 (pFur730) and H1780 (pFur1722) strains were compared. Lac+ phenotype was observed for E. coli H1780 whether grown in the presence or absence of added Fe supplement as predicted since it is deficient in Fur protein (data not shown). Complementation of E. coli H1780 with pFur616 rescued the Fur defect of this strain and resulted in the repression of transcription of the fiu-lacZ reporter gene, as shown by the Lac- phenotype (Figure 3A; upper left quadrant). When pFur616-kanC plasmid containing the disrupted NE0616 gene, was transformed into the E. coli H1780 mutant, Lac+ phenotype was maintained (Figure 3A; upper right quadrant). When pFur730 and pFur1722 plasmids containing the N. europaea fur homologs NE0730 and NE1722 were transformed separately into E. coli H1780 strain, Lac+ phenotype was observed (Figure 3A; lower left and right quadrants). These results clearly demonstrate that the N. europaea NE0616 fur homolog is expressed in E. coli in a functional form and is capable of regulating the Fur-dependent fiu promoter in H1780. The other N. europaea fur homologs (NE0730 and NE1722) were not capable of regulating the fiu promoter in H1780. NE0616 is here after referred to as N. europaea fur.


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

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

Fur Titration Assays (FURTA). (A) Complementation of an E. coli fur mutant H1780 by N. europaea Fur homologs.E. coli H1780 (pFur616)-upper left quadrant; H1780 (pFur616-kanC)-upper right quadrant; H1780 (pFur730)-lower left quadrant; H1780 (pFur1722)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs. (B) E. coli H1717 plated on McConkey medium with 30 μM Fe supplement-upper left quadrant, no Fe supplement-upper right quadrant; H1717 (pFur616)-lower left quadrant; H1717 (pFur616-kanP)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050691&req=5

Figure 3: Fur Titration Assays (FURTA). (A) Complementation of an E. coli fur mutant H1780 by N. europaea Fur homologs.E. coli H1780 (pFur616)-upper left quadrant; H1780 (pFur616-kanC)-upper right quadrant; H1780 (pFur730)-lower left quadrant; H1780 (pFur1722)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs. (B) E. coli H1717 plated on McConkey medium with 30 μM Fe supplement-upper left quadrant, no Fe supplement-upper right quadrant; H1717 (pFur616)-lower left quadrant; H1717 (pFur616-kanP)-lower right quadrant plated on McConkey medium with 30 μM Fe supplement and grown at 37°C for 24 hrs.
Mentions: All strains evaluated for Lac phenotype were grown on McConkey Lactose plates with 30 μM iron supplement, since iron is required to ensure that Fur is functional as a repressor [6]. In these studies, E. coli H1780, H1780 (pFur616), H1780 (pFur616-kanC), H1780 (pFur730) and H1780 (pFur1722) strains were compared. Lac+ phenotype was observed for E. coli H1780 whether grown in the presence or absence of added Fe supplement as predicted since it is deficient in Fur protein (data not shown). Complementation of E. coli H1780 with pFur616 rescued the Fur defect of this strain and resulted in the repression of transcription of the fiu-lacZ reporter gene, as shown by the Lac- phenotype (Figure 3A; upper left quadrant). When pFur616-kanC plasmid containing the disrupted NE0616 gene, was transformed into the E. coli H1780 mutant, Lac+ phenotype was maintained (Figure 3A; upper right quadrant). When pFur730 and pFur1722 plasmids containing the N. europaea fur homologs NE0730 and NE1722 were transformed separately into E. coli H1780 strain, Lac+ phenotype was observed (Figure 3A; lower left and right quadrants). These results clearly demonstrate that the N. europaea NE0616 fur homolog is expressed in E. coli in a functional form and is capable of regulating the Fur-dependent fiu promoter in H1780. The other N. europaea fur homologs (NE0730 and NE1722) were not capable of regulating the fiu promoter in H1780. NE0616 is here after referred to as N. europaea fur.

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