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. 1997 Feb;179(3):838–845. doi: 10.1128/jb.179.3.838-845.1997

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

M P Schmitt 1
PMCID: PMC178768  PMID: 9006041

Abstract

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.

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Selected References

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  1. Boyd J., Oza M. N., Murphy J. R. Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5968–5972. doi: 10.1073/pnas.87.15.5968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Braun V., Gross R., Köster W., Zimmermann L. Plasmid and chromosomal mutants in the iron(III)-aerobactin transport system of Escherichia coli. Use of streptonigrin for selection. Mol Gen Genet. 1983;192(1-2):131–139. doi: 10.1007/BF00327658. [DOI] [PubMed] [Google Scholar]
  3. Cone R., Hasan S. K., Lown J. W., Morgan A. R. The mechanism of the degradation of DNA by streptonigrin. Can J Biochem. 1976 Mar;54(3):219–223. doi: 10.1139/o76-034. [DOI] [PubMed] [Google Scholar]
  4. Cryz S. J., Jr, Russell L. M., Holmes R. K. Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production. J Bacteriol. 1983 Apr;154(1):245–252. doi: 10.1128/jb.154.1.245-252.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Doukhan L., Predich M., Nair G., Dussurget O., Mandic-Mulec I., Cole S. T., Smith D. R., Smith I. Genomic organization of the mycobacterial sigma gene cluster. Gene. 1995 Nov 7;165(1):67–70. doi: 10.1016/0378-1119(95)00427-8. [DOI] [PubMed] [Google Scholar]
  6. Dyer D. W., McKenna W., Woods J. P., Sparling P. F. Isolation by streptonigrin enrichment and characterization of a transferrin-specific iron uptake mutant of Neisseria meningitidis. Microb Pathog. 1987 Nov;3(5):351–363. doi: 10.1016/0882-4010(87)90005-2. [DOI] [PubMed] [Google Scholar]
  7. Efstratiou A., George R. C., Begg N. T. Non-toxigenic Corynebacterium diphtheriae var gravis in England. Lancet. 1993 Jun 19;341(8860):1592–1593. doi: 10.1016/0140-6736(93)90727-x. [DOI] [PubMed] [Google Scholar]
  8. Elkins C. Identification and purification of a conserved heme-regulated hemoglobin-binding outer membrane protein from Haemophilus ducreyi. Infect Immun. 1995 Apr;63(4):1241–1245. doi: 10.1128/iai.63.4.1241-1245.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Francis R. T., Jr, Booth J. W., Becker R. R. Uptake of iron from hemoglobin and the haptoglobin-hemoglobin complex by hemolytic bacteria. Int J Biochem. 1985;17(7):767–773. doi: 10.1016/0020-711x(85)90262-9. [DOI] [PubMed] [Google Scholar]
  10. Furman M., Fica A., Saxena M., Di Fabio J. L., Cabello F. C. Salmonella typhi iron uptake mutants are attenuated in mice. Infect Immun. 1994 Sep;62(9):4091–4094. doi: 10.1128/iai.62.9.4091-4094.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Genco C. A., Chen C. Y., Arko R. J., Kapczynski D. R., Morse S. A. Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers. J Gen Microbiol. 1991 Jun;137(6):1313–1321. doi: 10.1099/00221287-137-6-1313. [DOI] [PubMed] [Google Scholar]
  12. Greenfield L., Bjorn M. J., Horn G., Fong D., Buck G. A., Collier R. J., Kaplan D. A. Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6853–6857. doi: 10.1073/pnas.80.22.6853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Günter-Seeboth K., Schupp T. Cloning and sequence analysis of the Corynebacterium diphtheriae dtxR homologue from Streptomyces lividans and S. pilosus encoding a putative iron repressor protein. Gene. 1995 Dec 1;166(1):117–119. doi: 10.1016/0378-1119(95)00628-7. [DOI] [PubMed] [Google Scholar]
  14. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  15. Hantke K. Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12. Mol Gen Genet. 1984;197(2):337–341. doi: 10.1007/BF00330982. [DOI] [PubMed] [Google Scholar]
  16. Henderson D. P., Payne S. M. Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins. J Bacteriol. 1994 Jun;176(11):3269–3277. doi: 10.1128/jb.176.11.3269-3277.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holmes R. K., Barksdale L. Genetic analysis of tox+ and tox- bacteriophages of Corynebacterium diphtheriae. J Virol. 1969 Jun;3(6):586–598. doi: 10.1128/jvi.3.6.586-598.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jin H., Ren Z., Pozsgay J. M., Elkins C., Whitby P. W., Morton D. J., Stull T. L. Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae. Infect Immun. 1996 Aug;64(8):3134–3141. doi: 10.1128/iai.64.8.3134-3141.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee B. C. Quelling the red menace: haem capture by bacteria. Mol Microbiol. 1995 Nov;18(3):383–390. doi: 10.1111/j.1365-2958.1995.mmi_18030383.x. [DOI] [PubMed] [Google Scholar]
  20. Litwin C. M., Calderwood S. B. Role of iron in regulation of virulence genes. Clin Microbiol Rev. 1993 Apr;6(2):137–149. doi: 10.1128/cmr.6.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Meyer J. M., Neely A., Stintzi A., Georges C., Holder I. A. Pyoverdin is essential for virulence of Pseudomonas aeruginosa. Infect Immun. 1996 Feb;64(2):518–523. doi: 10.1128/iai.64.2.518-523.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mietzner T. A., Morse S. A. The role of iron-binding proteins in the survival of pathogenic bacteria. Annu Rev Nutr. 1994;14:471–493. doi: 10.1146/annurev.nu.14.070194.002351. [DOI] [PubMed] [Google Scholar]
  23. Mills M., Payne S. M. Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7. J Bacteriol. 1995 Jun;177(11):3004–3009. doi: 10.1128/jb.177.11.3004-3009.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Oguiza J. A., Tao X., Marcos A. T., Martín J. F., Murphy J. R. Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR homolog from Brevibacterium lactofermentum. J Bacteriol. 1995 Jan;177(2):465–467. doi: 10.1128/jb.177.2.465-467.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Otto B. R., Verweij-van Vught A. M., MacLaren D. M. Transferrins and heme-compounds as iron sources for pathogenic bacteria. Crit Rev Microbiol. 1992;18(3):217–233. doi: 10.3109/10408419209114559. [DOI] [PubMed] [Google Scholar]
  26. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  27. Rogers H. J. Iron-Binding Catechols and Virulence in Escherichia coli. Infect Immun. 1973 Mar;7(3):445–456. doi: 10.1128/iai.7.3.445-456.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Russell L. M., Cryz S. J., Jr, Holmes R. K. Genetic and biochemical evidence for a siderophore-dependent iron transport system in Corynebacterium diphtheriae. Infect Immun. 1984 Jul;45(1):143–149. doi: 10.1128/iai.45.1.143-149.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Russell L. M., Holmes R. K. Initial characterization of the ferric iron transport system of Corynebacterium diphtheriae. J Bacteriol. 1983 Sep;155(3):1439–1442. doi: 10.1128/jb.155.3.1439-1442.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schiller J., Groman N., Coyle M. Plasmids in Corynebacterium diphtheriae and diphtheroids mediating erythromycin resistance. Antimicrob Agents Chemother. 1980 Nov;18(5):814–821. doi: 10.1128/aac.18.5.814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schmitt M. P., Holmes R. K. Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron. J Bacteriol. 1994 Feb;176(4):1141–1149. doi: 10.1128/jb.176.4.1141-1149.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmitt M. P., Holmes R. K. Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains. Infect Immun. 1991 Jun;59(6):1899–1904. doi: 10.1128/iai.59.6.1899-1904.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schmitt M. P., Predich M., Doukhan L., Smith I., Holmes R. K. Characterization of an iron-dependent regulatory protein (IdeR) of Mycobacterium tuberculosis as a functional homolog of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae. Infect Immun. 1995 Nov;63(11):4284–4289. doi: 10.1128/iai.63.11.4284-4289.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmitt M. P., Twiddy E. M., Holmes R. K. Purification and characterization of the diphtheria toxin repressor. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7576–7580. doi: 10.1073/pnas.89.16.7576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schneider R., Hantke K. Iron-hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein-dependent transport system. Mol Microbiol. 1993 Apr;8(1):111–121. doi: 10.1111/j.1365-2958.1993.tb01208.x. [DOI] [PubMed] [Google Scholar]
  37. Serwold-Davis T. M., Groman N., Rabin M. Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae plasmid pNG2. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4964–4968. doi: 10.1073/pnas.84.14.4964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stoebner J. A., Payne S. M. Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae. Infect Immun. 1988 Nov;56(11):2891–2895. doi: 10.1128/iai.56.11.2891-2895.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stojiljkovic I., Hantke K. Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria. EMBO J. 1992 Dec;11(12):4359–4367. doi: 10.1002/j.1460-2075.1992.tb05535.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stojiljkovic I., Hantke K. Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica. Mol Microbiol. 1994 Aug;13(4):719–732. doi: 10.1111/j.1365-2958.1994.tb00465.x. [DOI] [PubMed] [Google Scholar]
  41. Tai S. P., Krafft A. E., Nootheti P., Holmes R. K. Coordinate regulation of siderophore and diphtheria toxin production by iron in Corynebacterium diphtheriae. Microb Pathog. 1990 Oct;9(4):267–273. doi: 10.1016/0882-4010(90)90015-i. [DOI] [PubMed] [Google Scholar]
  42. Tai S. S., Lee C. J., Winter R. E. Hemin utilization is related to virulence of Streptococcus pneumoniae. Infect Immun. 1993 Dec;61(12):5401–5405. doi: 10.1128/iai.61.12.5401-5405.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tao X., Murphy J. R. Binding of the metalloregulatory protein DtxR to the diphtheria tox operator requires a divalent heavy metal ion and protects the palindromic sequence from DNase I digestion. J Biol Chem. 1992 Oct 25;267(30):21761–21764. [PubMed] [Google Scholar]
  44. Tao X., Murphy J. R. Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9646–9650. doi: 10.1073/pnas.91.20.9646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tao X., Schiering N., Zeng H. Y., Ringe D., Murphy J. R. Iron, DtxR, and the regulation of diphtheria toxin expression. Mol Microbiol. 1994 Oct;14(2):191–197. doi: 10.1111/j.1365-2958.1994.tb01280.x. [DOI] [PubMed] [Google Scholar]
  46. Weinberg E. D. Iron and infection. Microbiol Rev. 1978 Mar;42(1):45–66. doi: 10.1128/mr.42.1.45-66.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. White J. R. Streptonigrin-transition metal complexes: binding to DNA and biological activity. Biochem Biophys Res Commun. 1977 Jul 11;77(1):387–391. doi: 10.1016/s0006-291x(77)80209-x. [DOI] [PubMed] [Google Scholar]
  48. White J. R., Yeowell H. N. Iron enhances the bactericidal action of streptonigrin. Biochem Biophys Res Commun. 1982 May 31;106(2):407–411. doi: 10.1016/0006-291x(82)91125-1. [DOI] [PubMed] [Google Scholar]
  49. Wilks A., Ortiz de Montellano P. R., Sun J., Loehr T. M. Heme oxygenase (HO-1): His-132 stabilizes a distal water ligand and assists catalysis. Biochemistry. 1996 Jan 23;35(3):930–936. doi: 10.1021/bi952405f. [DOI] [PubMed] [Google Scholar]
  50. Williams P. H., Carbonetti N. H. Iron, siderophores, and the pursuit of virulence: independence of the aerobactin and enterochelin iron uptake systems in Escherichia coli. Infect Immun. 1986 Mar;51(3):942–947. doi: 10.1128/iai.51.3.942-947.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Williams P. H. Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of Escherichia coli. Infect Immun. 1979 Dec;26(3):925–932. doi: 10.1128/iai.26.3.925-932.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yoshida T., Biro P., Cohen T., Müller R. M., Shibahara S. Human heme oxygenase cDNA and induction of its mRNA by hemin. Eur J Biochem. 1988 Feb 1;171(3):457–461. doi: 10.1111/j.1432-1033.1988.tb13811.x. [DOI] [PubMed] [Google Scholar]

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