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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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Spectrometric determination of hemin binding by Zn(II)Har and Zn(II)C97A.Hemin (Hm, 1–2 µM) in TBS was incubated with Zn(II)Har and Zn(II)C97A at protein to hemin molar ratios of 0∶1 to 4∶1 for 1 h and solution spectra collected on a Cary 50 UV-visible spectrometer (Varian). Lysozyme (Lys) was used as a negative control (green lines). (A) Absorption spectra of 2 µM free hemin, 8 µM Zn(II)Har or Zn(II)C97A, and hemin plus four equivalents of Zn(II)Har or Zn(II)C97A. Based on the hemin binding affinities of Zn(II)Har and Zn(II)C97A estimated in (B), at the starting protein:hemin molar ratio of 4∶1, free hemin in the equilibrium solution was 3.8% and 15.3% of the total hemin after reaction with Zn(II)Har and Zn(II)C97A, respectively. (B) Spectra of 1∶1 protein to hemin (2 µM) molar ratio are presented as a subtraction from the spectrum of hemin only (red line for Zn(II)Har, brown line for Zn(II)C97A, green line for lysozyme). The hemin binding affinity of the protein was estimated by fitting the absorbance changes at 419 nm for Zn(II)Har and Zn(II)C97A against protein concentrations (inset) using the biochemical analysis program Dynafit [31]. Inset: Fitted titration curves, apparent dissociation constants (Kd) and the titration data point sets of the normalised absorbance at 419 nm for Zn(II)Har and Zn(II)C97A. Estimation of binding stoichiometry is shown in blue. P: protein.
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pone-0111168-g004: Spectrometric determination of hemin binding by Zn(II)Har and Zn(II)C97A.Hemin (Hm, 1–2 µM) in TBS was incubated with Zn(II)Har and Zn(II)C97A at protein to hemin molar ratios of 0∶1 to 4∶1 for 1 h and solution spectra collected on a Cary 50 UV-visible spectrometer (Varian). Lysozyme (Lys) was used as a negative control (green lines). (A) Absorption spectra of 2 µM free hemin, 8 µM Zn(II)Har or Zn(II)C97A, and hemin plus four equivalents of Zn(II)Har or Zn(II)C97A. Based on the hemin binding affinities of Zn(II)Har and Zn(II)C97A estimated in (B), at the starting protein:hemin molar ratio of 4∶1, free hemin in the equilibrium solution was 3.8% and 15.3% of the total hemin after reaction with Zn(II)Har and Zn(II)C97A, respectively. (B) Spectra of 1∶1 protein to hemin (2 µM) molar ratio are presented as a subtraction from the spectrum of hemin only (red line for Zn(II)Har, brown line for Zn(II)C97A, green line for lysozyme). The hemin binding affinity of the protein was estimated by fitting the absorbance changes at 419 nm for Zn(II)Har and Zn(II)C97A against protein concentrations (inset) using the biochemical analysis program Dynafit [31]. Inset: Fitted titration curves, apparent dissociation constants (Kd) and the titration data point sets of the normalised absorbance at 419 nm for Zn(II)Har and Zn(II)C97A. Estimation of binding stoichiometry is shown in blue. P: protein.

Mentions: Addition of Zn(II)Har or Zn(II)C97A to hemin induced a solution spectrum change that indicated the formation of a complex between hemin and each protein. UV-visible spectra of the Zn(II)Har-hemin and Zn(II)C97A-hemin complexes showed a blue shift of the typical hemin absorption peak at 388 nm in the near UV range by ∼16 nm (372 nm; Fig. 4A) consistent with hemin binding to HRMs [39] and indicating both proteins had affinity for hemin. Difference absorption spectra of the Zn(II)Har-hemin and Zn(II)C97A-hemin complexes showed a second absorption maxima at ∼420 nm (Fig. 4B). The intensities of the Soret maxima for Zn(II)C97A were reduced with respect to that of the wild-type spectrum. Addition of lysozyme to hemin resulted in no obvious change to the hemin spectrum. Titration of a hemin solution with Zn(II)Har or Zn(II)C97A showed that Zn(II)Har bound one hemin molecule per monomer with high affinity (Kd of 0.23±0.12 µM), and Zn(II)C97A had a four-fold lower affinity for hemin with a Kd of 1.00±0.37 µM (Fig. 4B, inset).


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)

Spectrometric determination of hemin binding by Zn(II)Har and Zn(II)C97A.Hemin (Hm, 1–2 µM) in TBS was incubated with Zn(II)Har and Zn(II)C97A at protein to hemin molar ratios of 0∶1 to 4∶1 for 1 h and solution spectra collected on a Cary 50 UV-visible spectrometer (Varian). Lysozyme (Lys) was used as a negative control (green lines). (A) Absorption spectra of 2 µM free hemin, 8 µM Zn(II)Har or Zn(II)C97A, and hemin plus four equivalents of Zn(II)Har or Zn(II)C97A. Based on the hemin binding affinities of Zn(II)Har and Zn(II)C97A estimated in (B), at the starting protein:hemin molar ratio of 4∶1, free hemin in the equilibrium solution was 3.8% and 15.3% of the total hemin after reaction with Zn(II)Har and Zn(II)C97A, respectively. (B) Spectra of 1∶1 protein to hemin (2 µM) molar ratio are presented as a subtraction from the spectrum of hemin only (red line for Zn(II)Har, brown line for Zn(II)C97A, green line for lysozyme). The hemin binding affinity of the protein was estimated by fitting the absorbance changes at 419 nm for Zn(II)Har and Zn(II)C97A against protein concentrations (inset) using the biochemical analysis program Dynafit [31]. Inset: Fitted titration curves, apparent dissociation constants (Kd) and the titration data point sets of the normalised absorbance at 419 nm for Zn(II)Har and Zn(II)C97A. Estimation of binding stoichiometry is shown in blue. P: protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111168-g004: Spectrometric determination of hemin binding by Zn(II)Har and Zn(II)C97A.Hemin (Hm, 1–2 µM) in TBS was incubated with Zn(II)Har and Zn(II)C97A at protein to hemin molar ratios of 0∶1 to 4∶1 for 1 h and solution spectra collected on a Cary 50 UV-visible spectrometer (Varian). Lysozyme (Lys) was used as a negative control (green lines). (A) Absorption spectra of 2 µM free hemin, 8 µM Zn(II)Har or Zn(II)C97A, and hemin plus four equivalents of Zn(II)Har or Zn(II)C97A. Based on the hemin binding affinities of Zn(II)Har and Zn(II)C97A estimated in (B), at the starting protein:hemin molar ratio of 4∶1, free hemin in the equilibrium solution was 3.8% and 15.3% of the total hemin after reaction with Zn(II)Har and Zn(II)C97A, respectively. (B) Spectra of 1∶1 protein to hemin (2 µM) molar ratio are presented as a subtraction from the spectrum of hemin only (red line for Zn(II)Har, brown line for Zn(II)C97A, green line for lysozyme). The hemin binding affinity of the protein was estimated by fitting the absorbance changes at 419 nm for Zn(II)Har and Zn(II)C97A against protein concentrations (inset) using the biochemical analysis program Dynafit [31]. Inset: Fitted titration curves, apparent dissociation constants (Kd) and the titration data point sets of the normalised absorbance at 419 nm for Zn(II)Har and Zn(II)C97A. Estimation of binding stoichiometry is shown in blue. P: protein.
Mentions: Addition of Zn(II)Har or Zn(II)C97A to hemin induced a solution spectrum change that indicated the formation of a complex between hemin and each protein. UV-visible spectra of the Zn(II)Har-hemin and Zn(II)C97A-hemin complexes showed a blue shift of the typical hemin absorption peak at 388 nm in the near UV range by ∼16 nm (372 nm; Fig. 4A) consistent with hemin binding to HRMs [39] and indicating both proteins had affinity for hemin. Difference absorption spectra of the Zn(II)Har-hemin and Zn(II)C97A-hemin complexes showed a second absorption maxima at ∼420 nm (Fig. 4B). The intensities of the Soret maxima for Zn(II)C97A were reduced with respect to that of the wild-type spectrum. Addition of lysozyme to hemin resulted in no obvious change to the hemin spectrum. Titration of a hemin solution with Zn(II)Har or Zn(II)C97A showed that Zn(II)Har bound one hemin molecule per monomer with high affinity (Kd of 0.23±0.12 µM), and Zn(II)C97A had a four-fold lower affinity for hemin with a Kd of 1.00±0.37 µM (Fig. 4B, inset).

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