Characterisation of the novel Porphyromonas gingivalis transcriptional regulator PgMntR

Objectives: Bacterial transcriptional regulators control gene expression in respond to environmental challenges. Elucidation of the mechanism of action of these regulators in Porphyromonas gingivalis a major pathogen in chronic periodontitits, will provide us with a better understanding of how this organism survives in the healthy oral cavity and causes disease and may provide targets for small molecule inhibitors. We identified a novel member of the MntR family of transcriptional regulators in P. gingivalis (PgMntR) that uniquely contains two FeoA domains, which are usually associated with protein-protein interactions and ferrous iron transporters. MntRs usually function as a dimer regulating gene expression in response to the level of intracellular manganese. Aims: To characterise the metal binding site of PgMntR and the function of its novel two FeoA C-terminal domains.

Methods: Wild-type and mutant PgMntR proteins either lacking FeoA domains or with altered metal binding residues were expressed, purified and characterized using mass-spectrometry and circular-dichroism spectroscopy.

Results: Mass spectrometric analysis detected the monomeric form of the wild-type and mutated PgMntR but dimeric form of the proteins when both FeoA domains were removed. This indicated that the FeoA domains prevent dimerisation of the protein. The absence of metal ions in the purified proteins showed that metal ion is not an essential component to the monomeric form of PgMntR. Circular dichroism revealed that the mutations affected the secondary structure of PgMntR, with all of the amino acid substitutions making the protein more thermally stable. The addition of manganese increased the thermal stability of the wild-type, but decreased the stability of the mutants tested.

Conclusions: PgMntR appears to function differently to other members of the MntR family, given that the novel double FeoA domain prevents dimerisation of PgMntR. As MntR homologues normally function as a dimer, PgMntR must use a different mechanism to function, which is yet to be elucidated.