Cloning and expression of two novel hemin binding protein genes from Treponema denticola

Treponema denticola does not appear to produce siderophores, so it must acq uire iron by other pathways. Indeed, T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding abi lity. To characterize the HbpA protein, its gene was cloned from genomic DN A libraries of T, denticola. Sequence analysis of the hbpA open reading fra me 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. de nticola ATCC strains and clinical isolates, but not in Treponema pectinovor um, Treponema socranskii, or Escherichia call, HbpA, expressed as a recombi nant protein in E, call and purified by antibody affinity chromatography, h as hemin binding activity as determined by lithium dodecyl sulfate-polyacry lamide gel electrophoresis with tetramethylbenzidine staining. Northern blo t analysis showed that there were two hbpA-containing transcripts, of appro ximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Int erestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA, This downstream gene, called hbpB, was cloned and sequenc ed and its product was expressed as a fusion protein in E. coli, The hbpB g ene product is 49% identical to HbpA and binds hemin, Thus, T. denticola ha s two novel hemin binding proteins which may be part of a previously unreco gnized iron acquisition pathway.