Porphyromonas gingivalis is a major pathogen associated with the microbial biofilm-mediated disease chronic periodontitis. Biofilm formation by P. gingivalis 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. Iron homeostasis is usually mediated in Gram-negative bacteria at the transcriptional level by the Ferric Uptake Regulator (Fur). There is a single predicted Fur superfamily orthologue in P. gingivalis which we have designated Har (heme associated regulator).
We expressed Har as a recombinant protein and showed that it formed dimers with the essential presence of zinc cations. Har bound heme with an apparent Kd of 0.23 µM and ferrous iron with an apparent Kd of 0.021 µM and the binding of either heme or ferrous iron resulted in changes in the secondary structure of the protein. Har lacks the conserved metal binding residues found in other Fur orthologues but we confirmed 97C was required for binding heme as part of the identified -97C-98P-99L- heme binding motif. Har bound the promoter region of PGN_0001 independently of heme or iron. Under heme excess conditions the expression of 35 genes was down-regulated in a Har mutant (ECR455) relative to the wild-type. Twenty six of these genes were previously found up-regulated in P. gingivalis during growth as a biofilm and 11 were up-regulated under heme limitation. There were no significant changes in cellular content of divalent metal ions in ECR455 compared with the wild-type, but Har was required for heme-responsive biofilm development.
P. gingivalis is unique as an iron-dependent Gram-negative bacterium with a single heme-binding Fur superfamily orthologue that does not regulate iron homeostasis, instead regulating heme-dependent biofilm formation.