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

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Abstract

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 hmuY–hmuR 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.

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References

  • 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 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Bairoch A, Boeekmann B (1991) The SWISS-PROT protein sequence data bank. Nucleic Acids Res 19:2247–2249

    PubMed  CAS  Google Scholar 

  • 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 148:763–772

    PubMed  CAS  Google Scholar 

  • 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 307:1463–1465

    Article  PubMed  CAS  Google Scholar 

  • 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 362:1108–1119

    Article  PubMed  CAS  Google Scholar 

  • 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 183:3903–3909

    Article  PubMed  CAS  Google Scholar 

  • 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 181:6192–6196

    PubMed  CAS  Google Scholar 

  • Eberspacher B, Braun V (1980) The involvement of cytochromes in the uptake of ferrichrome by Escherichia coli K-12. FEMS Microbiol Lett 7:61–64

    Article  Google Scholar 

  • Fong KP, Chung WO, Lamont RJ, Demuth DR (2001) Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS. Infect Immun 69:7625–7634

    Article  PubMed  CAS  Google Scholar 

  • Frandsen EV, Poulson K, Curtis MA, Kilian M (2001) Evidence of recombination in Porphyromonas gingivalis and random distribution of putative virulence markers. Infect Immun 69:4479–4485

    Article  PubMed  CAS  Google Scholar 

  • 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 149:2273–2281

    Article  PubMed  CAS  Google Scholar 

  • Genco CA, Dixon DW (2001) Emerging strategies in microbial heme capture. Mol Microbiol 391:1–11

    Article  Google Scholar 

  • Geourjon C, Deleage G (1995) Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11:681–684

    PubMed  CAS  Google Scholar 

  • Gray-Owen SD, Schryvers AS (1996) Bacterial transferrin and lactoferrin receptors. Trends Microbiol 4:185–191

    Article  PubMed  CAS  Google Scholar 

  • 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 74:3834–3844

    Article  PubMed  CAS  Google Scholar 

  • Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403–405

    Article  PubMed  CAS  Google Scholar 

  • 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 12:1652–1662

    Article  PubMed  CAS  Google Scholar 

  • 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 35:90–100

    Article  PubMed  CAS  Google Scholar 

  • Karunakaran T, Madden T, Kuramitsu H (1997) Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis. J Bacteriol 179:1898–1908

    PubMed  CAS  Google Scholar 

  • Kim SJ, Chu L, Holt SC (1996) Isolation and characterization of a hemin-binding cell envelope protein from Porphyromonas gingivalis. Microb Pathog 21:65–70

    Article  PubMed  CAS  Google Scholar 

  • Koehler A, Karch H, Beikler T, Flemmig TF, Suerbaum S, Schmidt H (2003) Multilocus sequence analysis of Porphyromonas gingivalis indicates frequent recombination. Microbiology 149:2407–2415

    Article  PubMed  CAS  Google Scholar 

  • 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 101:14919–14924

    Article  PubMed  CAS  Google Scholar 

  • Letoffe S, Ghigo JM, Wandersman C (1994) Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein. Proc Natl Acad Sci USA 91:9876–9880

    Article  PubMed  CAS  Google Scholar 

  • 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 23:737–749

    Article  PubMed  CAS  Google Scholar 

  • 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 152:3367–3382

    Article  PubMed  CAS  Google Scholar 

  • 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 181:3743–3750

    PubMed  CAS  Google Scholar 

  • 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 385:1049–1057

    Article  PubMed  CAS  Google Scholar 

  • 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 74:1222–1232

    Article  PubMed  CAS  Google Scholar 

  • Mihara J, Holt SC (1993a) Purification and characterization of fibroblast-activating factor isolated from Porphyromonas gingivalis W50. Infect Immun 61:588–595

    PubMed  CAS  Google Scholar 

  • Mihara J, Holt SC (1993b) Modulation of growth and function of human gingival fibroblast-activating factor derived from Porphyromonas gingivalis W50. Infect Immun 61:596–601

    PubMed  CAS  Google Scholar 

  • Mihara J, Yoneda T, Holt SC (1993) Role of Porphyromonas gingivalis-derived fibroblast-activating factor in bone resorption. Infect Immun 61:3562–3564

    PubMed  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Springer Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • 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 177:3004–3009

    PubMed  CAS  Google Scholar 

  • 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 185:5591–5601

    Article  PubMed  CAS  Google Scholar 

  • Nienaber A, Hennecke H, Fischer HM (2001) Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum. Mol Microbiol 41:787–800

    Article  PubMed  CAS  Google Scholar 

  • Ochsner UA, Johnson Z, Vasil ML (2000) Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa. Microbiology 146:185–198

    PubMed  CAS  Google Scholar 

  • Olczak T (2006) Heme receptor HmuR from Porphyromonas gingivalis: towards a further understanding of heme uptake. Arch Microbiol 186:393–402

    Article  PubMed  CAS  Google Scholar 

  • 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 183:5599–5608

    Article  PubMed  CAS  Google Scholar 

  • Olczak T, Simpson W, Liu X, Genco CA (2005) Iron and heme utilization in Porphyromonas gingivalis. FEMS Microbiol Rev 29:119–144

    Article  PubMed  CAS  Google Scholar 

  • 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 49:299–306

    Article  PubMed  CAS  Google Scholar 

  • 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 4:397–407

    Article  PubMed  CAS  Google Scholar 

  • 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 168:523–533

    PubMed  CAS  Google Scholar 

  • Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS (2003) Comparative genomics of the vitamin B12 metabolism and regulation in Prokaryotes. J Biol Chem 278:41148–41159

    Article  PubMed  CAS  Google Scholar 

  • Sawers RG (2005) Evidence for novel processing of the anaerobically inducible dicistronic focA-pfl mRNA transcript in Escherichia coli. Mol Microbiol 58:1441–1453

    Article  PubMed  CAS  Google Scholar 

  • 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 67:5012–5020

    PubMed  CAS  Google Scholar 

  • Simpson W, Olczak T, Genco CA (2000) Characterization and expression of HmuR, a TonB-dependent hemoglobin receptor of Porphyromonas gingivalis. J Bacteriol 182:5737–5748

    Article  PubMed  CAS  Google Scholar 

  • 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 51:253–262

    CAS  Google Scholar 

  • Stojiljkovic I, Hantke K (1992) Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria. EMBO J 11:4359–4376

    PubMed  CAS  Google Scholar 

  • 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 67:3879–3892

    PubMed  CAS  Google Scholar 

  • Wang BY, Chi B, Kuramitsu HK (2002) Genetic exchange between Treponema denticola and Streptomyces gordonii in biofilms. Oral Microbiol Immunol 17:108–112

    Article  PubMed  CAS  Google Scholar 

  • 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 299:2074–2076

    Article  PubMed  CAS  Google Scholar 

  • Zor T, Selinger Z (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem 236:302–308

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

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).

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Authors and Affiliations

  1. Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137, Wroclaw, Poland

    Teresa Olczak & Mariusz Olczak

  2. Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland

    Aneta Sroka & Jan Potempa

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  1. Teresa Olczak
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Correspondence to Teresa Olczak.

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Communicated by Wolfgang Buckel.

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Olczak, T., Sroka, A., Potempa, J. et al. Porphyromonas gingivalis HmuY and HmuR: further characterization of a novel mechanism of heme utilization. Arch Microbiol 189, 197–210 (2008). https://doi.org/10.1007/s00203-007-0309-7

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  • DOI: https://doi.org/10.1007/s00203-007-0309-7

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