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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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P. gingivalis biofilm development.Orthogonal projections of CLSM images showing a representative region of the x-y plane over the depth of the biofilm in both xz and yz dimensions of the ATCC 33277 wild-type, har mutant ECR455 and har complement ECR 475 strains grown in excess hemin (A) or hemin-limitation (B). Comparison of the Biovolume (C), Average Thickness (D) and SA:Biovolume (E) calculated for each strain's biofilm growth in either excess hemin (dark bars) or limited hemin (light bars) over three independent experiments. All biometric parameters analysed for the biofilms formed by ATCC 33277 and ECR475 were significantly (p<0.005) altered when hemin was limited. * indicates a significant difference in excess hemin versus limited hemin (p<0.005); ** indicates a significant difference in ECR455 biovolume, average thickness and SA:biovolume when compared to the same biometric parameters of both the ATCC 33277 and ECR475 biofilms (p<0.001).
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pone-0111168-g008: P. gingivalis biofilm development.Orthogonal projections of CLSM images showing a representative region of the x-y plane over the depth of the biofilm in both xz and yz dimensions of the ATCC 33277 wild-type, har mutant ECR455 and har complement ECR 475 strains grown in excess hemin (A) or hemin-limitation (B). Comparison of the Biovolume (C), Average Thickness (D) and SA:Biovolume (E) calculated for each strain's biofilm growth in either excess hemin (dark bars) or limited hemin (light bars) over three independent experiments. All biometric parameters analysed for the biofilms formed by ATCC 33277 and ECR475 were significantly (p<0.005) altered when hemin was limited. * indicates a significant difference in excess hemin versus limited hemin (p<0.005); ** indicates a significant difference in ECR455 biovolume, average thickness and SA:biovolume when compared to the same biometric parameters of both the ATCC 33277 and ECR475 biofilms (p<0.001).

Mentions: Biometric analysis of the biofilms grown in either hemin-limited or non-limiting growth conditions showed that P. gingivalis ATCC 33277 wild-type and the har complemented ECR475 produced biofilms that were not statistically different (Fig. 8). The har mutant ECR455 produced biofilms that had significantly reduced biovolume and average thickness and an increased surface area (SA):biovolume ratio compared with the wild-type or ECR475 (Fig. 8). Comparison of the biofilm formed by each strain under hemin-limitation or non-limitation showed that hemin availability had no effect on the biovolume or average thickness of the biofilm formed by ECR455 whereas there was a significant reduction in the biovolume and average thickness of the wild-type and ECR475 biofilms grown under hemin limitation (Fig. 8). Planktonic growth of the three strains under the same growth conditions was similar (data not shown).


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)

P. gingivalis biofilm development.Orthogonal projections of CLSM images showing a representative region of the x-y plane over the depth of the biofilm in both xz and yz dimensions of the ATCC 33277 wild-type, har mutant ECR455 and har complement ECR 475 strains grown in excess hemin (A) or hemin-limitation (B). Comparison of the Biovolume (C), Average Thickness (D) and SA:Biovolume (E) calculated for each strain's biofilm growth in either excess hemin (dark bars) or limited hemin (light bars) over three independent experiments. All biometric parameters analysed for the biofilms formed by ATCC 33277 and ECR475 were significantly (p<0.005) altered when hemin was limited. * indicates a significant difference in excess hemin versus limited hemin (p<0.005); ** indicates a significant difference in ECR455 biovolume, average thickness and SA:biovolume when compared to the same biometric parameters of both the ATCC 33277 and ECR475 biofilms (p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111168-g008: P. gingivalis biofilm development.Orthogonal projections of CLSM images showing a representative region of the x-y plane over the depth of the biofilm in both xz and yz dimensions of the ATCC 33277 wild-type, har mutant ECR455 and har complement ECR 475 strains grown in excess hemin (A) or hemin-limitation (B). Comparison of the Biovolume (C), Average Thickness (D) and SA:Biovolume (E) calculated for each strain's biofilm growth in either excess hemin (dark bars) or limited hemin (light bars) over three independent experiments. All biometric parameters analysed for the biofilms formed by ATCC 33277 and ECR475 were significantly (p<0.005) altered when hemin was limited. * indicates a significant difference in excess hemin versus limited hemin (p<0.005); ** indicates a significant difference in ECR455 biovolume, average thickness and SA:biovolume when compared to the same biometric parameters of both the ATCC 33277 and ECR475 biofilms (p<0.001).
Mentions: Biometric analysis of the biofilms grown in either hemin-limited or non-limiting growth conditions showed that P. gingivalis ATCC 33277 wild-type and the har complemented ECR475 produced biofilms that were not statistically different (Fig. 8). The har mutant ECR455 produced biofilms that had significantly reduced biovolume and average thickness and an increased surface area (SA):biovolume ratio compared with the wild-type or ECR475 (Fig. 8). Comparison of the biofilm formed by each strain under hemin-limitation or non-limitation showed that hemin availability had no effect on the biovolume or average thickness of the biofilm formed by ECR455 whereas there was a significant reduction in the biovolume and average thickness of the wild-type and ECR475 biofilms grown under hemin limitation (Fig. 8). Planktonic growth of the three strains under the same growth conditions was similar (data not shown).

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