Characterization of a novel outer membrane hemin-binding protein of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative, anaerobic coccobacillus that h as been implicated as a major etiological agent in the development of chron ic periodontitis. In this paper, we report the characterization of a protei n, IhtB (iron heme transport; formerly designated Pga30), that is an outer membrane hemin-binding protein potentially involved in iron assimilation by P. gingivalis. IhtB was localized to the cell surface of P. gingivalis by Western blot analysis of a Sarkosyl-insoluble outer membrane preparation an d by immunocytochemical staining of whole cells using IhtB peptide-specific antisera. The protein, released from the cell surface, was shown to bind t o hemin using hemin-agarose, The growth of heme-limited, but not heme-reple te, P,gingivalis cells was inhibited by preincubation with IhtB peptide-spe cific antisera. The IhtB gene was located between an open reading frame enc oding a putative TonB-linked outer membrane receptor and three open reading frames that have sequence similarity to ATP binding cassette transport sys tem operons in of her bacteria. Analysis of the deduced amino acid sequence of IhtB showed significant similarity to the Salmonella typhimurium protei n CbiK, a cobalt chelatase that is structurally related to the ATP-independ ent family of ferrochelatases, Molecular modeling indicated that the IhtB a mino acid sequence could be threaded onto the CbiK fold with the IHtB struc tural model containing the active-site residues critical for chelatase acti vity. These results suggest that IhtB is a peripheral outer membrane chelat ase that may remove iron from heme prior to uptake by P. gingivalis.