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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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EMSA of Zn(II)Har150 binding to DNA.(A). Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Lanes 2–7, 9 & 11 all contain 500 ng (0.2 µM) of a 240 bp PCR product encompassing the 33277 dnaA promoter sequence (PGN_0001). Additionally, lane 3 has 1 µg (2.8 µM) Zn(II)Har150; lane 4, 2 µg (5.6 µM) Zn(II)Har150; lane 5, 3 µg (8.4 µM) Zn(II)Har150; lane 6, 4 µg (11.2 µM) Zn(II)Har150 and lane 7, 5 µg (14 µM) Zn(II)Har150. Lane 2 contains DNA only whereas Lane 8 contains 5 µg (14 µM) Zn(II)Har150 only. Lanes 9 – 12 contain the negative control protein for DNA binding, BSA, where there is 5 µg BSA in lanes 9 & 10, and 3 µg BSA in lanes 11 & 12. (B). Agarose gel electrophoresis stained with SimplyBlue SafeStain (Life Technologies) for visualizing protein following DNA visualisation. Lanes are as described in (A). The position of the anode (+) and cathode (-) are noted. (C). EMSA competition experiment where an excess of unlabeled dnaA promoter DNA (1250 ng) competed with 250 ng (0.1 µM) FAM-labeled dnaA promoter DNA for binding to 3 µg (8.4 µM) Zn(II)Har150 (lane 3). Lane 1 contains 250 ng FAM-labeled DNA only, whereas lane 2 contains 250 ng FAM-labeled DNA bound to 3 µg Zn(II)Har150. Visualised is the fluorescence of the FAM-labeled DNA after agarose gel electrophoresis (D) EMSA experiment where the promoter-containing DNA of PGN_1308 (lanes 1–3) was shifted by its cognate transcriptional repressor (lane 2) but not by Zn(II)Har150 (lane 3). (E). Inhibition of Zn(II)Har150 DNA binding by hemin. The addition of increasing concentrations of hemin (lane 3, 0 µM; lane 4, 14 µM; lane 5, 70 µM; lane 6, 140 µM) to a constant amount of DNA (500 ng, lanes 2–6) and Zn(II)Har150 (14 µM, lanes 3–6) resulted in increasing inhibition of DNA binding by Zn(II)Har150. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA.
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pone-0111168-g006: EMSA of Zn(II)Har150 binding to DNA.(A). Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Lanes 2–7, 9 & 11 all contain 500 ng (0.2 µM) of a 240 bp PCR product encompassing the 33277 dnaA promoter sequence (PGN_0001). Additionally, lane 3 has 1 µg (2.8 µM) Zn(II)Har150; lane 4, 2 µg (5.6 µM) Zn(II)Har150; lane 5, 3 µg (8.4 µM) Zn(II)Har150; lane 6, 4 µg (11.2 µM) Zn(II)Har150 and lane 7, 5 µg (14 µM) Zn(II)Har150. Lane 2 contains DNA only whereas Lane 8 contains 5 µg (14 µM) Zn(II)Har150 only. Lanes 9 – 12 contain the negative control protein for DNA binding, BSA, where there is 5 µg BSA in lanes 9 & 10, and 3 µg BSA in lanes 11 & 12. (B). Agarose gel electrophoresis stained with SimplyBlue SafeStain (Life Technologies) for visualizing protein following DNA visualisation. Lanes are as described in (A). The position of the anode (+) and cathode (-) are noted. (C). EMSA competition experiment where an excess of unlabeled dnaA promoter DNA (1250 ng) competed with 250 ng (0.1 µM) FAM-labeled dnaA promoter DNA for binding to 3 µg (8.4 µM) Zn(II)Har150 (lane 3). Lane 1 contains 250 ng FAM-labeled DNA only, whereas lane 2 contains 250 ng FAM-labeled DNA bound to 3 µg Zn(II)Har150. Visualised is the fluorescence of the FAM-labeled DNA after agarose gel electrophoresis (D) EMSA experiment where the promoter-containing DNA of PGN_1308 (lanes 1–3) was shifted by its cognate transcriptional repressor (lane 2) but not by Zn(II)Har150 (lane 3). (E). Inhibition of Zn(II)Har150 DNA binding by hemin. The addition of increasing concentrations of hemin (lane 3, 0 µM; lane 4, 14 µM; lane 5, 70 µM; lane 6, 140 µM) to a constant amount of DNA (500 ng, lanes 2–6) and Zn(II)Har150 (14 µM, lanes 3–6) resulted in increasing inhibition of DNA binding by Zn(II)Har150. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA.

Mentions: Zn(II)Har150 without its lysine-rich tail was used for EMSA analyses to minimize non-specific DNA interactions as it has a lower predicted pI of 8.98 compared with Zn(II)Har (pI of 9.47). The promoter region of dnaA (PGN_0001) was used as the Zn(II)Har binding target because dnaA has been identified from the microarray results of the current study (vide infra) as being negatively regulated by Har. The EMSAs were run on agarose gels with the wells cast centrally to enable electrophoresis of both protein and DNA which are oppositely charged [40]. Zn(II)Har150 bound with the promoter region of dnaA forming a Zn(II)Har150-DNA complex preventing the DNA from entering the gel under the applied electric field. The negative control BSA which does not bind DNA did not affect the migration of the dnaA promoter region (Fig. 6A). The positively charged Zn(II)Har150 migrated towards the cathode (-) but this movement was retarded in the presence of the specific DNA due to the formation of the Zn(II)Har150-DNA complex (Fig. 6B). Unlabeled specific DNA competed for binding of Zn(II)Har150 to the FAM-labeled specific DNA (Fig. 6C), however non-specific DNA at a similar concentration to the specific DNA did not compete for binding indicating that the binding of Zn(II)Har150 was specific for the dnaA promoter. Zn(II)Har150 did not bind to the promoter region of PGN_1308 a DNA target for a different transcriptional regulator of P. gingivalis (PgMntR), indicating that Zn(II)Har150 does not bind DNA non-specifically (Fig. 6D). Increasing concentrations of hemin resulted in increasing dissociation of the Zn(II)Har150-DNA complex showing that the binding of Zn(II)Har150 to the dnaA promoter region was specifically inhibited by hemin at molar excess concentrations (Fig. 6E).


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)

EMSA of Zn(II)Har150 binding to DNA.(A). Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Lanes 2–7, 9 & 11 all contain 500 ng (0.2 µM) of a 240 bp PCR product encompassing the 33277 dnaA promoter sequence (PGN_0001). Additionally, lane 3 has 1 µg (2.8 µM) Zn(II)Har150; lane 4, 2 µg (5.6 µM) Zn(II)Har150; lane 5, 3 µg (8.4 µM) Zn(II)Har150; lane 6, 4 µg (11.2 µM) Zn(II)Har150 and lane 7, 5 µg (14 µM) Zn(II)Har150. Lane 2 contains DNA only whereas Lane 8 contains 5 µg (14 µM) Zn(II)Har150 only. Lanes 9 – 12 contain the negative control protein for DNA binding, BSA, where there is 5 µg BSA in lanes 9 & 10, and 3 µg BSA in lanes 11 & 12. (B). Agarose gel electrophoresis stained with SimplyBlue SafeStain (Life Technologies) for visualizing protein following DNA visualisation. Lanes are as described in (A). The position of the anode (+) and cathode (-) are noted. (C). EMSA competition experiment where an excess of unlabeled dnaA promoter DNA (1250 ng) competed with 250 ng (0.1 µM) FAM-labeled dnaA promoter DNA for binding to 3 µg (8.4 µM) Zn(II)Har150 (lane 3). Lane 1 contains 250 ng FAM-labeled DNA only, whereas lane 2 contains 250 ng FAM-labeled DNA bound to 3 µg Zn(II)Har150. Visualised is the fluorescence of the FAM-labeled DNA after agarose gel electrophoresis (D) EMSA experiment where the promoter-containing DNA of PGN_1308 (lanes 1–3) was shifted by its cognate transcriptional repressor (lane 2) but not by Zn(II)Har150 (lane 3). (E). Inhibition of Zn(II)Har150 DNA binding by hemin. The addition of increasing concentrations of hemin (lane 3, 0 µM; lane 4, 14 µM; lane 5, 70 µM; lane 6, 140 µM) to a constant amount of DNA (500 ng, lanes 2–6) and Zn(II)Har150 (14 µM, lanes 3–6) resulted in increasing inhibition of DNA binding by Zn(II)Har150. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA.
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Related In: Results  -  Collection

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pone-0111168-g006: EMSA of Zn(II)Har150 binding to DNA.(A). Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Lanes 2–7, 9 & 11 all contain 500 ng (0.2 µM) of a 240 bp PCR product encompassing the 33277 dnaA promoter sequence (PGN_0001). Additionally, lane 3 has 1 µg (2.8 µM) Zn(II)Har150; lane 4, 2 µg (5.6 µM) Zn(II)Har150; lane 5, 3 µg (8.4 µM) Zn(II)Har150; lane 6, 4 µg (11.2 µM) Zn(II)Har150 and lane 7, 5 µg (14 µM) Zn(II)Har150. Lane 2 contains DNA only whereas Lane 8 contains 5 µg (14 µM) Zn(II)Har150 only. Lanes 9 – 12 contain the negative control protein for DNA binding, BSA, where there is 5 µg BSA in lanes 9 & 10, and 3 µg BSA in lanes 11 & 12. (B). Agarose gel electrophoresis stained with SimplyBlue SafeStain (Life Technologies) for visualizing protein following DNA visualisation. Lanes are as described in (A). The position of the anode (+) and cathode (-) are noted. (C). EMSA competition experiment where an excess of unlabeled dnaA promoter DNA (1250 ng) competed with 250 ng (0.1 µM) FAM-labeled dnaA promoter DNA for binding to 3 µg (8.4 µM) Zn(II)Har150 (lane 3). Lane 1 contains 250 ng FAM-labeled DNA only, whereas lane 2 contains 250 ng FAM-labeled DNA bound to 3 µg Zn(II)Har150. Visualised is the fluorescence of the FAM-labeled DNA after agarose gel electrophoresis (D) EMSA experiment where the promoter-containing DNA of PGN_1308 (lanes 1–3) was shifted by its cognate transcriptional repressor (lane 2) but not by Zn(II)Har150 (lane 3). (E). Inhibition of Zn(II)Har150 DNA binding by hemin. The addition of increasing concentrations of hemin (lane 3, 0 µM; lane 4, 14 µM; lane 5, 70 µM; lane 6, 140 µM) to a constant amount of DNA (500 ng, lanes 2–6) and Zn(II)Har150 (14 µM, lanes 3–6) resulted in increasing inhibition of DNA binding by Zn(II)Har150. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA.
Mentions: Zn(II)Har150 without its lysine-rich tail was used for EMSA analyses to minimize non-specific DNA interactions as it has a lower predicted pI of 8.98 compared with Zn(II)Har (pI of 9.47). The promoter region of dnaA (PGN_0001) was used as the Zn(II)Har binding target because dnaA has been identified from the microarray results of the current study (vide infra) as being negatively regulated by Har. The EMSAs were run on agarose gels with the wells cast centrally to enable electrophoresis of both protein and DNA which are oppositely charged [40]. Zn(II)Har150 bound with the promoter region of dnaA forming a Zn(II)Har150-DNA complex preventing the DNA from entering the gel under the applied electric field. The negative control BSA which does not bind DNA did not affect the migration of the dnaA promoter region (Fig. 6A). The positively charged Zn(II)Har150 migrated towards the cathode (-) but this movement was retarded in the presence of the specific DNA due to the formation of the Zn(II)Har150-DNA complex (Fig. 6B). Unlabeled specific DNA competed for binding of Zn(II)Har150 to the FAM-labeled specific DNA (Fig. 6C), however non-specific DNA at a similar concentration to the specific DNA did not compete for binding indicating that the binding of Zn(II)Har150 was specific for the dnaA promoter. Zn(II)Har150 did not bind to the promoter region of PGN_1308 a DNA target for a different transcriptional regulator of P. gingivalis (PgMntR), indicating that Zn(II)Har150 does not bind DNA non-specifically (Fig. 6D). Increasing concentrations of hemin resulted in increasing dissociation of the Zn(II)Har150-DNA complex showing that the binding of Zn(II)Har150 to the dnaA promoter region was specifically inhibited by hemin at molar excess concentrations (Fig. 6E).

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