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The Porphyromonas gingivalis ferric uptake regulator orthologue binds hemin and regulates hemin-responsive biofilm development.

Butler CA, Dashper SG, Zhang L, Seers CA, Mitchell HL, Catmull DV, Glew MD, Heath JE, Tan Y, Khan HS, Reynolds EC - PLoS ONE (2014)

Bottom Line: Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation.This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability.ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability.

View Article: PubMed Central - PubMed

Affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Victoria, Australia.

ABSTRACT
Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.

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Dimerization of Zn(II)Har, Zn(II)C97A and Zn(II)Har150.Representative elution profiles of Zn(II)Har/Zn(II)C97A (A) and Zn(II)Har150 (B) from a Superdex 75 analytical gel filtration column at 4°C in an AKTA FPLC Chromatographic System (GE Healthcare). Proteins (100 µg) were applied to the column pre-equilibrated with 500 mM NaCl containing buffers (20 mM) at pH 6.0 (MES), pH 7.0 (HEPES), 7.4 (KPi) and 8.5 (borate). The molar masses were calculated against a calibration curve of retention volumes (Ve) of the protein standards, which are indicated at the top of the chart. The elution profiles at pH 7.0 are presented.
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pone-0111168-g003: Dimerization of Zn(II)Har, Zn(II)C97A and Zn(II)Har150.Representative elution profiles of Zn(II)Har/Zn(II)C97A (A) and Zn(II)Har150 (B) from a Superdex 75 analytical gel filtration column at 4°C in an AKTA FPLC Chromatographic System (GE Healthcare). Proteins (100 µg) were applied to the column pre-equilibrated with 500 mM NaCl containing buffers (20 mM) at pH 6.0 (MES), pH 7.0 (HEPES), 7.4 (KPi) and 8.5 (borate). The molar masses were calculated against a calibration curve of retention volumes (Ve) of the protein standards, which are indicated at the top of the chart. The elution profiles at pH 7.0 are presented.

Mentions: Size exclusion chromatography of Zn(II)Har, Zn(II)C97A and Zn(II)Har50 in reducing or non-reducing environments showed a single major peak in each elution profile with an apparent molar mass of 41.0 kDa which corresponded to dimeric Har or C97A with two zinc ions, or 35.0 kDa which corresponded to dimeric Zn(II)Har150 (Fig. 3). The Zn(II)Har homodimer was stable over the pH range 6.0–8.5 and no variation in the elution profile of the dimer was found at 4°C and ambient temperature (22°C) suggesting a stable dimeric form of Zn(II)Har (data not shown). Since all the seven thiol groups are unpaired, such dimerizaton was not caused by intermolecular disulfide formation.


The Porphyromonas gingivalis ferric uptake regulator orthologue binds hemin and regulates hemin-responsive biofilm development.

Butler CA, Dashper SG, Zhang L, Seers CA, Mitchell HL, Catmull DV, Glew MD, Heath JE, Tan Y, Khan HS, Reynolds EC - PLoS ONE (2014)

Dimerization of Zn(II)Har, Zn(II)C97A and Zn(II)Har150.Representative elution profiles of Zn(II)Har/Zn(II)C97A (A) and Zn(II)Har150 (B) from a Superdex 75 analytical gel filtration column at 4°C in an AKTA FPLC Chromatographic System (GE Healthcare). Proteins (100 µg) were applied to the column pre-equilibrated with 500 mM NaCl containing buffers (20 mM) at pH 6.0 (MES), pH 7.0 (HEPES), 7.4 (KPi) and 8.5 (borate). The molar masses were calculated against a calibration curve of retention volumes (Ve) of the protein standards, which are indicated at the top of the chart. The elution profiles at pH 7.0 are presented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222909&req=5

pone-0111168-g003: Dimerization of Zn(II)Har, Zn(II)C97A and Zn(II)Har150.Representative elution profiles of Zn(II)Har/Zn(II)C97A (A) and Zn(II)Har150 (B) from a Superdex 75 analytical gel filtration column at 4°C in an AKTA FPLC Chromatographic System (GE Healthcare). Proteins (100 µg) were applied to the column pre-equilibrated with 500 mM NaCl containing buffers (20 mM) at pH 6.0 (MES), pH 7.0 (HEPES), 7.4 (KPi) and 8.5 (borate). The molar masses were calculated against a calibration curve of retention volumes (Ve) of the protein standards, which are indicated at the top of the chart. The elution profiles at pH 7.0 are presented.
Mentions: Size exclusion chromatography of Zn(II)Har, Zn(II)C97A and Zn(II)Har50 in reducing or non-reducing environments showed a single major peak in each elution profile with an apparent molar mass of 41.0 kDa which corresponded to dimeric Har or C97A with two zinc ions, or 35.0 kDa which corresponded to dimeric Zn(II)Har150 (Fig. 3). The Zn(II)Har homodimer was stable over the pH range 6.0–8.5 and no variation in the elution profile of the dimer was found at 4°C and ambient temperature (22°C) suggesting a stable dimeric form of Zn(II)Har (data not shown). Since all the seven thiol groups are unpaired, such dimerizaton was not caused by intermolecular disulfide formation.

Bottom Line: Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation.This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability.ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability.

View Article: PubMed Central - PubMed

Affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Victoria, Australia.

ABSTRACT
Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.

Show MeSH
Related in: MedlinePlus