Characterization of a novel 24 kDa hemin-binding protein, HmuY', in Porphyromonas gingivalis W50

Abstract

Porphyromonas gingivalis is a black-pigmented, anaerobic Gram-negative bacterium that is important in the progression of chronic and severe periodontitis. This organism has an essential requirement for iron, which is usually obtained from heme. In this study, we report the characterization of a novel 24 kDa hemin-binding protein, HmuYa??, in P. gingivalis W50. The hmuYa?? gene is 651 bp long and encodes for a protein of 217 amino acids, which was found to differ from the previously identified HmuY protein by an additional 74 amino acids at the N-terminus. Recombinant HmuYa?? demonstrated hemin-binding ability by LDS-PAGE and TMBZ staining. Northern analyses showed hmuYa?? to be transcribed as a 750 bp transcript and that hmuYa?? mRNA was regulated mainly by growth phase changes. Transcription start site and putative promoter regions of hmuYa?? were also identified. A P. gingivalis isogenic mutant deficient in hmuYa?? gene was successfully created and it exhibited significant growth retardation under hemin-limited conditions. Further, HmuYa?? was found to be localized to the outer cell surface by transmission electron microscopy. Taken together, these results suggest HmuYa?? to be an outer-membrane hemin-binding protein, important for the growth of P. gingivalis.