Characterisation of a Porphyromonas gingivalis Fur Orthologue

Objectives: The ferric uptake regulator (Fur) is a global bacterial transcriptional repressor using iron as a cofactor to maintain intracellular iron levels within a required range for bacterial growth. The Gram-negative obligate anaerobe, periodontal pathogen Porphyromonas gingivalis expresses a Fur orthologue (Pg-Fur, 166aa) with a novel lysine rich C-terminus. The present study is to characterize Pg-Fur by determining its metal and DNA binding activities. Methods: Recombinant Pg-Fur was expressed in E. coli and purified with chromatographic techniques. Fluorescence spectroscopic and mass spectrometric analyses were carried out to investigate the metal binding ability of Pg-Fur. Circular dichroism spectroscopy was used in determination of the secondary structure and thermostability of Pg-Fur in the absence and presence of metal ions. P. gingivalis genomic DNA fragments protected from DNAse I digestion in the presence of Pg-Fur were analysed with SDS-PAGE. Analytical size exclusion chromatography was used to analyse the polymerisation states of Pg-Fur. Results: Pg-Fur bound both zinc and ferrous ions with a higher binding ability for Zn²⁺. Addition of Zn²⁺, Fe²⁺ and Mn²⁺ to a solution of Pg-Fur-Zn(II) induced a significant change in secondary structure of the protein. In addition, the presence of Fe²⁺ enhanced the thermostability of the protein. Pg-Fur-Zn(II) existed as a dimer in solution in the range of pH 6.0-8.5 and has strong interactions with P. gingivalis genomic DNA so that it could protect a range of DNA fragments from digestion by DNAse I but the level of this protection was lower when the positively charged lysine rich C-terminus was removed. Conclusions: Pg-Fur has a typical divalent metal ion binding property as other Fur homologues. The lysine rich C-terminus of Pg-Fur lacking in most Fur homologues may play an additional role for DNA binding in gene regulation in P. gingivalis.