Cloning and Expression of Two Novel Hemin Binding Protein Genes fromTreponema denticola

Abstract

Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed, T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of the hbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in several T. denticola ATCC strains and clinical isolates, but not in Treponema pectinovorum, Treponema socranskii, or Escherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, called hbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. The hbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

Expression and Characterization of an Iron-Regulated Hemin-Binding Protein, HbpA, fromLeptospira interrogansSerovar Lai

“…Some bacterial outer membrane proteins are toxic when overexpressed in E. coli (39), with the toxicity being attributed to the 5Ј region of the gene that encodes the signal peptide. We found this to be true for the full-length HbpA, with the formation of small colonies and a low level of expression of the 81-kDa rHbpA protein.…”

Redox properties of extracellular polymeric substances (EPS) from electroactive bacteria

“…Heme-binding protein electrophoresis was conducted to identify the presence of this redox component in the EPS, which was confirmed by lithium dodecyl sulfate (LDS)-PAGE and staining with tetramethylbenzidine (TMBZ, a chromogen that specifically binds to proteins with heme-associated peroxidase activity)3738. Before analysis, the EPS samples were dissolved in deionized water, and the final concentration was adjusted to 6 g/L.…”

Upregulation of Intercellular Adhesion Molecule 1 and Proinflammatory Cytokines by the Major Surface Proteins of Treponema maltophilum and Treponema lecithinolyticum , the Phylogenetic Group IV Oral Spirochetes Associated with Periodontitis and Endodontic Infections

“…A protein homologous to this protein was also identified in Treponema vincentii. The 44-and 43-kDa outer sheath polypeptides of T. denticola have strong hemin-binding activity, which might be involved in iron acquisition (9,55). A major surface protein, MompA, was identified in Treponema pectinovorum, but its function is unclear (51).…”