Characterization of a novel outer membrane hemin-binding protein of Porphyromonas gingivalis.
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.
Dashper
S G
SG
School of Dental Science, The University of Melbourne, Melbourne, Victoria, Australia.
Hendtlass
A
A
Slakeski
N
N
Jackson
C
C
Cross
K J
KJ
Brownfield
L
L
Hamilton
R
R
Barr
I
I
Reynolds
E C
EC
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D003470:Culture Media; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007106:Immune Sera / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003470:Culture Media; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; C416080:IhtB protein, Porphyromonas gingivalis; D007106:Immune Sera; D007501:Iron