Porphyromonas gingivalis is a gram-negative, anaerobic coccobacillus that has been implicated as a major etiological agent in the development of chronic periodontitis. In this paper, we report the characterization of a protein, 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 and by immunocytochemical staining of whole cells using IhtB peptide-specific antisera. The protein, released from the cell surface, was shown to bind to hemin using hemin-agarose. The growth of heme-limited, but not heme-replete, P. gingivalis cells was inhibited by preincubation with IhtB peptide-specific antisera. The ihtB gene was located between an open reading frame encoding a putative TonB-linked outer membrane receptor and three open reading frames that have sequence similarity to ATP binding cassette transport system operons in other bacteria. Analysis of the deduced amino acid sequence of IhtB showed significant similarity to the Salmonella typhimurium protein CbiK, a cobalt chelatase that is structurally related to the ATP-independent family of ferrochelatases. Molecular modeling indicated that the IhtB amino acid sequence could be threaded onto the CbiK fold with the IhtB structural model containing the active-site residues critical for chelatase activity. These results suggest that IhtB is a peripheral outer membrane chelatase that may remove iron from heme prior to uptake by P. gingivalis.

Porphyromonas gingivalis is a black-pigmented, gram-negative bacterium that has been implicated as a major pathogen in the development of adult periodontitis. In an approach to identify a P. gingivalis antigen uniquely seroreactive with healthy subjects, we produced a surface and periplasmic extract of P. gingivalis, separated that extract into 36 fractions using anion-exchange chromatography and screened each fraction for reactivity in an enzyme-linked immunosorbent assay (ELISA) using sera from eight periodontitis patients and eight age- and sex-matched healthy controls. All of the diseased subjects harboured subgingival P. gingivalis by DNA probe analysis and exhibited similar seroreactivity profiles to the anion exchange fractions. However, only two of the healthy subjects (C10 and C15) were seroreactive with the fractions. The highest reactivity for all the seropositive subjects was with the same anion-exchange fractions 13-15. The anion exchange fraction (14) with the highest seroreactivity was subjected to gel filtration chromatography, and fraction 22 from this chromatography exhibited the highest reactivity with all the seropositive subjects. However, fraction 27 was found to be uniquely seroreactive with healthy subject C10, as it was not recognized by sera from any of the diseased or the other healthy subjects. This fraction was further purified by reversed-phase high-pressure liquid chromatography and shown to contain a 30-kDa protein as determined by SDS-PAGE. Control subject C10 had no pocket depths greater than 3 mm and no sites that bled on gentle probing; however, P. gingivalis was detected in subgingival plaque samples at a level of 10(5)-10(6) cells per site in two of the ten sites sampled. This subject was also unusual in that he exhibited a seroreactivity profile similar to that of diseased subjects, which was not characteristic of the healthy control subjects. The unique reactivity of the 30-kDa antigen, designated Pga30, with subject C10 serum was confirmed in a Western blot with the purified antigen. N-Terminal sequence analysis of Pga30 produced a single, unambiguous protein sequence confirming the purity of the protein. A search of the database using the N-terminal sequence obtained did not reveal any significant sequence similarity. In conclusion, we have identified a P. gingivalis antigen that was uniquely reactive in an ELISA and Western blot with serum from a subject with no clinical signs of periodontitis who harbored P. gingivalis in subgingival plaque.

We have determined the transcription start points (tsp) for recently identified Porphyromonas gingivalis W50 genes, kgp, rgpA, rgpB (formerly designated prtK, prtR, and prtRII respectively), fetB and the mcmAB operon. Alignment of the DNA upstream of these tsp and those from the literature has enabled us to identify consensus sequences that may represent a P. gingivalis promoter. There is a potential -10 hexamer sequence, 5'-TATATT-3' centred on average at -10/11 nt which is repeated at -19/20 nt and an upstream consensus, 5'-CAGAT(A/G)-3' which is centred at -39/40 nt.