Cloning and expression of two novel hemin binding protein genes from Treponema denticola.
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.
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Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Holt
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Kolodrubetz
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001547:Benzaldehydes; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006427:Hemin / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling / Q000379:methods; D014208:Treponema / Q000235:genetics*
D001426:Bacterial Proteins; D001547:Benzaldehydes; D002352:Carrier Proteins; D004269:DNA, Bacterial; C097952:HbpA protein, Haemophilus influenzae; D008074:Lipoproteins; D012967:Sodium Dodecyl Sulfate; C034031:2,4,5-trimethoxybenzaldehyde; D006427:Hemin; C028913:dodecyl sulfate; D007501:Iron