BINDING AND ACCUMULATION OF HEMIN IN PORPHYROMONAS-GINGIVALIS ARE INDUCED BY HEMIN

Although hemin is an essential nutrient for the black-pigmented oral b acterium Porphyromonas gingivalis, the mechanisms involved in hemin bi nding and uptake are poorly defined. In this study, we have examined t he binding of hemin and Congo red (CR) to P. gingivalis whole cells an d have defined the conditions for maximal binding. Additionally, the a ccumulation of hemin by P. gingivalis under growing conditions has bee n characterized. P. gingivalis A7436 was grown under hemin- or iron-de plete conditions (basal medium [BM] or Schaedler broth with dipyridyl [SBD]) or under hemin- or iron-replete conditions (BM with hemin [BMH] or Schaedler broth [SB]), and hemin and CR binding were assessed spec trophotometrically. Binding of hemin by P.gingivalis whole cells was r apid and was observed in samples obtained from cells grown under hemin - and iron-replete and hemin-deplete conditions but was not observed i n cells grown under iron limitation. We also found that P. gingivalis whole cells bound more hemin when grown in BMH or SB than cells grown in BM or SBD. Binding of CR by P. gingivalis A7436 was also enhanced w hen cells were grown in the presence of hemin or when cells were incub ated with hemin prior to CR binding. Hemin binding and accumulation we re also assessed using [C-14]hemin and [Fe-59]hemin under growing cond itions. Both [C-14]hemin and [Fe-59]hemin were accumulated by P. gingi valis, indicating that iron and the porphyrin ring were taken into the cell. Binding and accumulation of hemin under growing conditions were also induced by growth of P. gingivalis in hemin-replete media. Hemin accumulation was inhibited by the addition of KCN to P. gingivalis cu ltures, indicating that active transport was required for hemin uptake . [C-14]hemin binding and accumulation were also inhibited by the addi tion of either cold hemin or protoporphyrin IX. Taken together, these results indicate that P. gingivalis transports the entire hemin moiety into the cell and that the binding and accumulation of hemin are indu ced by growth of cultures in the presence of hemin.