Characterization and expression of HmuR, a TonB-dependent hemoglobin receptor of Porphyromonas gingivalis

The gram-negative pathogen Porphyromonas gingivalis requires hemin for grow th. Hemoglobin bound to haptoglobin and hemin complexed to hemopexin can be used as heme sources, indicating that P. gingivalis must have a means to r emove the hemin from these host iron-binding proteins. However, the specifi c mechanisms utilized by P. gingivalis for the extraction of heme from heme -binding proteins and for iron transport are poorly understood. In this stu dy we have determined that a newly identified TonB-dependent hemoglobin-hem in receptor (HmuR) is involved in hemoglobin binding and utilization in P. gingivalis A7436. HmuR shares amino acid homolog with TonB-dependent outer membrane receptors of gram-negative bacteria involved in the acquisition of iron from hemin and hemoglobin, including HemR of Yersinia enterocolitica, ShuA of Shigella dysenteriae, HpuB of Neisseria gonorrhoeae and N. meningi tidis, HmbR of N. meningitidis, HgbA of Haemophilus ducreyi, and HgpB of H. influenzae. Southern blot analysis confirmed the presence of the hmuR gene and revealed genetic variability in the carboxy terminus of hmuR in P. gin givalis strains 33277, 381, W50, and 53977. We also identified directly ups tream of the hmuR gene a gene which we designated hmuY. Upstream of the hmu Y start codon, a region with homology to the Fur binding consensus sequence was identified. Reverse transcription-PCR analysis revealed that hmuR and hmuY were cotranscribed and that transcription was negatively regulated by iron. Inactivation of hmuR resulted in a decreased ability of P. gingivalis to bind hemoglobin and to grow with hemoglobin or hemin as sole iron sourc es. Escherichia coli cells expressing recombinant HmuR were shown to bind h emoglobin and hemin. Furthermore, purified recombinant HmuR was demonstrate d to bind hemoglobin. Taken together, these results indicate that HmuR sen es as the major TonB-dependent outer membrane receptor involved in the util ization of both hemin and hemoglobin in P. gingivalis.