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.

A study was conducted to compare the efficiency of crude aqueous (CA) and solvent extracts (CM) of clove on the caries-inducing properties of Streptococcus mutans. The cariogenic properties investigated included the cell adhesion, cell-surface hydrophobicity and glucan synthesis activities of S. mutans. There was a significant difference between the effect of the CA and CM extracts on the adhesion of S. mutans (P < 0.05) within a concentration range of 5-15 mg/ml, the CM extract demonstrating a slightly higher inhibitory effect. However, the effect of the CM extract on the cell-surface hydrophobicity of S. mutans was weaker than that of the CA extract. The two extracts were found to reduce the synthesis of water-insoluble glucan (WIG) by almost 50% at a concentration as low as 0.5 mg/ml and the CM extract exhibited a significantly higher inhibitory effect than the CA extract (P < 0.05). The present findings indicate that both the CA and CM extracts exert inhibitory effects on the cariogenic properties of S. mutans and that the CA extract is as equally effective as the CM extract.

a b s t r a c tBackground: Transition metals including iron and manganese are necessary for life because of their ability to donate and accept electrons. Approximately one-third of all proteins require essential transition metal ions to perform catalytic, structural and regulatory functions. These essential metal ions react differently to the presence of oxygen radicals with iron directly involved in the formation of toxic reactive oxygen species, whilst manganese can protect against oxidative stress. Highlight: Anaerobic bacterial species have been poorly studied with regard to transition metal homoeostasis and behave differently in many respects when compared with aerobic or aerotolerant species. To optimise catabolism whilst protecting themselves from unwanted reactions bacterial cells must maintain intracellular metal levels in a very narrow range that varies, dependent on the environment. To maintain metal ion homoeostasis, bacteria have evolved complex regulatory mechanisms of metal uptake, secretion and storage. In this review we examine how iron, haem and manganese availability dictate the lifestyle and virulence of the anaerobic Gram-negative, periodontal pathogen Porphyromonas gingivalis. Conclusion: P. gingivalis has novel haem, iron and manganese transporters and metalloregulatory proteins that enable it to switch rapidly between an energy efficient iron-dependent virulent phase and a protective manganese-dependent survival phase.

PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10−11 M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10−10 M and Kd2 ≤ 6.0 x 10−8 M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner.