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Comparative Study
. 2000 Nov;182(22):6456-62.
doi: 10.1128/JB.182.22.6456-6462.2000.

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

S G Dashper  1 , A Hendtlass, N Slakeski, C Jackson, K J Cross, L Brownfield, R Hamilton, I Barr, E C Reynolds
Affiliations
Comparative Study

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

S G Dashper et al. J Bacteriol. 2000 Nov.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Northern blot analysis of P. gingivalis RNA. Hybridization with an oligonucleotide probe specific for ihtB showed a single transcript of 1.3 kb. The relative positions of RNA molecular size markers are indicated.
FIG. 2
FIG. 2
The ihtABCDE genetic locus of P. gingivalis that encodes a proposed iron transport system. The putative function and size of the product of each gene are shown. The arrows indicate the relative positions of each of the predicted coding regions, and the shaded areas indicate the presence of leader sequences. aa, amino acids.
FIG. 3
FIG. 3
Western blot analysis of cell fractions produced by French pressure treatment of P. gingivalis and probed with IhtB peptide-specific antisera. Mw, molecular size markers; lane 1, cytoplasmic fraction; lane 2, Sarkosyl-insoluble outer membrane fraction.
FIG. 4
FIG. 4
Western blot analysis of P. gingivalis outer membrane and periplasmic proteins eluted from hemin-agarose with 2 M guanidine-HCl. This Western blot analysis used IhtB peptide-specific antisera. Mw, molecular size markers; lane 1, control omitting the hemin-agarose; lane 2, hemin-agarose eluant.
FIG. 5
FIG. 5
Growth of P. gingivalis in batch culture in basal medium with hemin (1 μg/ml). Heme-depleted cells of P. gingivalis were preincubated for 1 h at 37°C in IhtB peptide-specific antiserum which had been heat inactivated at 56°C for 30 min (⧫), heat-inactivated NRS (▴), or basal medium with hemin (■). Points represent the mean of three independent determinations. OD, optical density.
FIG. 6
FIG. 6
(A) Alignment of CbiK and IhtB deduced amino acid sequences generated by GeneFold. Shading indicates identical residues, and bold indicates conservative substitutions. Residues that are conserved across the entire anaerobic, ATP-independent cobalt and ferrochelatase class of proteins (40) are indicated by asterisks. Numbering indicates the position in the IhtB sequence of the His residues (His174 and His236) proposed to be critical for chelatase activity. The N-terminal sequence obtained for the purified N-terminally truncated protein (17) is underlined. (B) Ribbon diagram of the active site of the IhtB conformational model showing the putative heme-binding site constructed using the Sybyl program. Residues believed to be important for substrate binding and active-site conformation are labeled and have side chains displayed.
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