Archives of Microbiology

, Volume 189, Issue 3, pp 197–210 | Cite as

Porphyromonas gingivalis HmuY and HmuR: further characterization of a novel mechanism of heme utilization

  • Teresa OlczakEmail author
  • Aneta Sroka
  • Jan Potempa
  • Mariusz Olczak
Original Paper


Porphyromonas gingivalis HmuY is a putative heme-binding lipoprotein associated with the outer membrane. It is part of an operon together with a gene encoding an outer-membrane hemin utilization receptor (HmuR) and four uncharacterized genes. A similar operon organization was found in Bacteroides fragilis and B. thetaiotaomicron, with the former containing an additional HmuY homologue encoded upstream of the hmuR-like gene. In P. gingivalis cultured under heme-limited conditions, a ∼1-kb hmuY transcript was produced at high levels along with some ∼3.5 and ∼9-kb transcripts. Compared with the parental strain, mutants deficient in hmuY or hmuR or hmuYhmuR gene function grew more slowly and bound lower amounts of hemin and hemoglobin. Significantly, they grew more slowly or were unable to grow when human serum was used as the sole iron/heme source. Analysis of the hmu promoter showed that it is regulated by iron. The HmuY protein normally occurs as a homodimer, but in the presence of hemin it may form tetramers. These results show that HmuY may be the first reported member of a new class of proteins in Porphyromonas and Bacteroides species involved in heme utilization, a function being exerted in conjunction with HmuR, an outer-membrane heme transporter.


Porphyromonas gingivalis HmuR Heme outer-membrane receptor HmuY Heme-binding protein Heme uptake Lipoprotein His-tag 



Dr. C.A. Genco (Section of infectious diseases, Boston University School of Medicine, USA) is gratefully acknowledged for giving T.O. the opportunity to continue studies on P. gingivalis heme utilization. The authors thank Dr. Klaus Hantke (University of Tübingen, Germany) for generously supplying us with the E. coli K-12 EB53 strain. This work was supported in part by grants nos. 3 P05A 113 24 and N401 029 32/0742 from the Department of Scientific Research, Ministry of Science and Higher Education, Poland (T.O.) and NIH grant (DE 09761), USA (J.P.). J.P. is a Subsydium Profesorskie award recipient from the Foundation for Polish Science (Warsaw, Poland). Part of the data was presented at the FEBS Congress and the 9th IUBMB Conference (Budapest, Olczak et al. 2005, FEBS J 272S1:15).

Supplementary material

203_2007_309_MOESM1_ESM.doc (77 kb)
ESM1 (DOC 77 kb)
203_2007_309_MOESM2_ESM.doc (64 kb)
ESM2 (DOC 65 kb)


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Bairoch A, Boeekmann B (1991) The SWISS-PROT protein sequence data bank. Nucleic Acids Res 19:2247–2249PubMedGoogle Scholar
  3. Burgess NA, Kirke DF, Williams P, Winzer K, Hardie KR, Meyers NL, Aduse-Opoku J, Curtis MA, Camara M (2002) LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence. Microbiology 148:763–772PubMedGoogle Scholar
  4. Cerdeno-Tarraga AM, Patrick S, Crossman LC, Blakely G, Abratt V, Lennard N, Poxton I, Duerden B, Harris B, Quail MA, Barron A, Clark L, Corton C, Doggett J, Holden MT, Larke N, Line A, Lord A, Norbertczak H, Ormond D, Price C, Rabbinowitsch E, Woodward J, Barrell B, Parkhill J (2005) Extensive DNA inversions in the B. fragilis genome control variable gene expression. Science 307:1463–1465PubMedCrossRefGoogle Scholar
  5. Chan AC, Lelj-Garolla B, Rosell F, Pedersen KA, Mauk AG, Murphy ME (2006) Cofacial heme binding is linked to dimerization by a bacterial heme transport protein. J Mol Biol 362:1108–1119PubMedCrossRefGoogle Scholar
  6. Chung WO, Park Y, Lamont RJ, McNab R, Barbieri B, Demuth DR (2001) Signaling system in Porphyromonas gingivalis based on a LuxS protein. J Bacteriol 183:3903–3909PubMedCrossRefGoogle Scholar
  7. Comstock LE, Coyne MJ, Tzianabos AO, Kasper DL (1999) Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R. J Bacteriol 181:6192–6196PubMedGoogle Scholar
  8. Eberspacher B, Braun V (1980) The involvement of cytochromes in the uptake of ferrichrome by Escherichia coli K-12. FEMS Microbiol Lett 7:61–64CrossRefGoogle Scholar
  9. Fong KP, Chung WO, Lamont RJ, Demuth DR (2001) Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS. Infect Immun 69:7625–7634PubMedCrossRefGoogle Scholar
  10. Frandsen EV, Poulson K, Curtis MA, Kilian M (2001) Evidence of recombination in Porphyromonas gingivalis and random distribution of putative virulence markers. Infect Immun 69:4479–4485PubMedCrossRefGoogle Scholar
  11. Garrido ME, Bosch M, Medina R, Bigas A, Llagostera M, Perez de Rozas AM, Badiola I, Barbe J (2003) fur-independent regulation of the Pasteurella multocida hbpA gene encoding a haemin-binding protein. Microbiology 149:2273–2281PubMedCrossRefGoogle Scholar
  12. Genco CA, Dixon DW (2001) Emerging strategies in microbial heme capture. Mol Microbiol 391:1–11CrossRefGoogle Scholar
  13. Geourjon C, Deleage G (1995) Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11:681–684PubMedGoogle Scholar
  14. Gray-Owen SD, Schryvers AS (1996) Bacterial transferrin and lactoferrin receptors. Trends Microbiol 4:185–191PubMedCrossRefGoogle Scholar
  15. James CE, Hasegawa Y, Park Y, Yeung V, Tribble GD, Kuboniwa M, Demuth DR, Lamont RJ (2006) LuxS involvement in the regulation of genes coding for hemin and iron acquisition systems in Porphyromonas gingivalis. Infect Immun 74:3834–3844PubMedCrossRefGoogle Scholar
  16. Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403–405PubMedCrossRefGoogle Scholar
  17. Juncker AS, Willenbrock H, von Heijne G, Nielsen H, Brunak S, Krogh A (2003) Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci 12:1652–1662PubMedCrossRefGoogle Scholar
  18. Heck C, Balzer A, Fuhrmann O, Klug G (2000) Initial events in the degradation of the polycistronic puf mRNA in Rhodobacter capsulatus and consequences for further processing steps. Mol Microbiol 35:90–100PubMedCrossRefGoogle Scholar
  19. Karunakaran T, Madden T, Kuramitsu H (1997) Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis. J Bacteriol 179:1898–1908PubMedGoogle Scholar
  20. Kim SJ, Chu L, Holt SC (1996) Isolation and characterization of a hemin-binding cell envelope protein from Porphyromonas gingivalis. Microb Pathog 21:65–70PubMedCrossRefGoogle Scholar
  21. Koehler A, Karch H, Beikler T, Flemmig TF, Suerbaum S, Schmidt H (2003) Multilocus sequence analysis of Porphyromonas gingivalis indicates frequent recombination. Microbiology 149:2407–2415PubMedCrossRefGoogle Scholar
  22. Kuwahara T, Yamashita A, Hirakawa H, Nakayama H, Toh H, Okada N, Kuhara S, Hattori M, Hayashi T, Ohnishi Y (2004) Genomic analysis of Bacteroides fragilis reveals extensive DNA inversions regulating cell surface adaptation. Proc Natl Acad Sci USA 101:14919–14924PubMedCrossRefGoogle Scholar
  23. Letoffe S, Ghigo JM, Wandersman C (1994) Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein. Proc Natl Acad Sci USA 91:9876–9880PubMedCrossRefGoogle Scholar
  24. Lewis LA, Gray E, Wang YP, Roe BA, Dyer DW (1997) Molecular characterization of hpuAB, the haemoglobin–haptoglobin-utilization operon of Neisseria meningitidis. Mol Microbiol 23:737–749PubMedCrossRefGoogle Scholar
  25. Lewis JP, Plata K, Fan Y, Rosato A, Anaya C (2006) Transcriptional organization, regulation and role of the Porphyromonas gingivalis W83 hmu haemin-uptake locus. Microbiology 152:3367–3382PubMedCrossRefGoogle Scholar
  26. Limberger RJ, Slivienski LL, Izard J, Samsonoff WA (1999) Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks. J Bacteriol 181:3743–3750PubMedGoogle Scholar
  27. Liu X, Sroka A, Potempa J, Genco CA (2004) Coordinate expression of the Porphyromonas gingivalis lysine-specific gingipain proteinase, Kgp, arginine-specific gingipain proteinase, RgpA, and the heme/hemoglobin receptor, HmuR. Biol Chem 385:1049–1057PubMedCrossRefGoogle Scholar
  28. Liu X, Olczak T, Guo HC, Dixon DW, Genco CA (2006) Identification of essential amino acid residues required for hemoprotein utilization in the Porphyromonas gingivalis heme receptor HmuR. Infect Immun 74:1222–1232PubMedCrossRefGoogle Scholar
  29. Mihara J, Holt SC (1993a) Purification and characterization of fibroblast-activating factor isolated from Porphyromonas gingivalis W50. Infect Immun 61:588–595PubMedGoogle Scholar
  30. Mihara J, Holt SC (1993b) Modulation of growth and function of human gingival fibroblast-activating factor derived from Porphyromonas gingivalis W50. Infect Immun 61:596–601PubMedGoogle Scholar
  31. Mihara J, Yoneda T, Holt SC (1993) Role of Porphyromonas gingivalis-derived fibroblast-activating factor in bone resorption. Infect Immun 61:3562–3564PubMedGoogle Scholar
  32. Miller JH (1972) Experiments in molecular genetics. Cold Springer Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  33. Mills M, Payne SM (1995) Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7. J Bacteriol 177:3004–3009PubMedGoogle Scholar
  34. Nelson KE, Fleischmann RD, DeBoy RT, Paulsen IT, Fouts DE, Eisen JA, Daugherty SC, Dodson RJ, Durkin AS, Gwinn M, Haft DH, Kolonay JF, Nelson WC, Mason T, Tallon L, Gray J, Granger D, Tettelin H, Dong H, Galvin JL, Duncan MJ, Dewhirst FE, Fraser CM (2003) Complete genome sequence of the oral pathogenic bacterium Porphyromonas gingivalis strain W83. J Bacteriol 185:5591–5601PubMedCrossRefGoogle Scholar
  35. Nienaber A, Hennecke H, Fischer HM (2001) Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum. Mol Microbiol 41:787–800PubMedCrossRefGoogle Scholar
  36. Ochsner UA, Johnson Z, Vasil ML (2000) Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa. Microbiology 146:185–198PubMedGoogle Scholar
  37. Olczak T (2006) Heme receptor HmuR from Porphyromonas gingivalis: towards a further understanding of heme uptake. Arch Microbiol 186:393–402PubMedCrossRefGoogle Scholar
  38. Olczak T, Dixon DW, Genco CA (2001) Binding specificity of the Porphyromonas gingivalis heme and hemoglobin receptor HmuR, gingipain K, and gingipain R1 for heme, porphyrins, and metalloporphyrins. J Bacteriol 183:5599–5608PubMedCrossRefGoogle Scholar
  39. Olczak T, Simpson W, Liu X, Genco CA (2005) Iron and heme utilization in Porphyromonas gingivalis. FEMS Microbiol Rev 29:119–144PubMedCrossRefGoogle Scholar
  40. Olczak T, Siudeja K, Olczak M (2006) Purification and initial characterization of a novel Porphyromonas gingivalis HmuY protein expressed in Escherichia coli and insect cells. Protein Expr Purif 49:299–306PubMedCrossRefGoogle Scholar
  41. Potempa J, Sroka A, Imamura T, Travis J (2003) Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes. Curr Protein Pept Sci 4:397–407PubMedCrossRefGoogle Scholar
  42. Rasmussen JL, Odelson DA, Macrina FL (1986) Complete nucleotide sequence and transcription of ermF, a macrolide-lincosamide-streptogramin B resistance determinant from Bacteroides fragilis. J Bacteriol 168:523–533PubMedGoogle Scholar
  43. Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS (2003) Comparative genomics of the vitamin B12 metabolism and regulation in Prokaryotes. J Biol Chem 278:41148–41159PubMedCrossRefGoogle Scholar
  44. Sawers RG (2005) Evidence for novel processing of the anaerobically inducible dicistronic focA-pfl mRNA transcript in Escherichia coli. Mol Microbiol 58:1441–1453PubMedCrossRefGoogle Scholar
  45. Simpson W, Wang CY, Bond V, Potempa J, Mikolajczyk-Pawlinska J, Travis J, Genco CA (1999). Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis. Infect Immun 67:5012–5020PubMedGoogle Scholar
  46. Simpson W, Olczak T, Genco CA (2000) Characterization and expression of HmuR, a TonB-dependent hemoglobin receptor of Porphyromonas gingivalis. J Bacteriol 182:5737–5748PubMedCrossRefGoogle Scholar
  47. Simpson W, Olczak T, Genco CA (2004) Lysine-specific gingipain K and heme/hemoglobin receptor HmuR are involved in heme utilization in Porphyromonas gingivalis. Acta Biochim Pol 51:253–262Google Scholar
  48. Stojiljkovic I, Hantke K (1992) Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria. EMBO J 11:4359–4376PubMedGoogle Scholar
  49. Thompson JM, Jones HA, Perry RD (1999) Molecular characterization of the hemin uptake locus (hmu) from Yersinia pestis and analysis of hmu mutants for hemin and hemoprotein utilization. Infect Immun 67:3879–3892PubMedGoogle Scholar
  50. Wang BY, Chi B, Kuramitsu HK (2002) Genetic exchange between Treponema denticola and Streptomyces gordonii in biofilms. Oral Microbiol Immunol 17:108–112PubMedCrossRefGoogle Scholar
  51. Xu J, Bjursell MK, Himrod J, Deng S, Carmichael LK, Chiang HC, Hooper LV (2003) A genomic view of the human-Bacteroides thetaiotaomicron symbiosis. Science 299:2074–2076PubMedCrossRefGoogle Scholar
  52. Zor T, Selinger Z (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem 236:302–308PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Teresa Olczak
    • 1
    Email author
  • Aneta Sroka
    • 2
  • Jan Potempa
    • 2
  • Mariusz Olczak
    • 1
  1. 1.Laboratory of Biochemistry, Faculty of BiotechnologyUniversity of WroclawWroclawPoland
  2. 2.Department of Microbiology, Faculty of Biochemistry, Biophysics and BiotechnologyJagiellonian UniversityKrakowPoland

Personalised recommendations