Skip to main content
Infection and Immunity logoLink to Infection and Immunity
. 1994 Oct;62(10):4515–4525. doi: 10.1128/iai.62.10.4515-4525.1994

Identification of a locus involved in the utilization of iron by Haemophilus influenzae.

J D Sanders 1, L D Cope 1, E J Hansen 1
PMCID: PMC303138  PMID: 7927717

Abstract

Haemophilus influenzae has an absolute requirement for heme for aerobic growth. This organism can satisfy this requirement by synthesizing heme from iron and protoporphyrin IX (PPIX). H. influenzae type b (Hib) strain DL42 was found to be unable to form single colonies when grown on a medium containing free iron and PPIX in place of heme. In contrast, the nontypeable H. influenzae (NTHI) strain TN106 grew readily on the same medium. A genomic library from NTHI strain TN106 was used to transform Hib strain DL42, and recombinants were selected on a medium containing iron and PPIX in place of heme. A recombinant plasmid with an 11.5-kb NTHI DNA insert was shown to confer on Hib strain DL42 the ability to grow on iron and PPIX. Nucleotide sequence analysis revealed that this NTHI DNA insert contained three genes, designated hitA, hitB, and hitC, which encoded products similar to the SfuABC proteins of Serratia marcescens, which have been shown to constitute a periplasmic binding protein-dependent iron transport system in this enteric organism. The NTHI HitA protein also was 69% identical to the ferric-binding protein of Neisseria gonorrhoeae. Inactivation of the cloned NTHI hitC gene by insertion of an antibiotic resistance cartridge eliminated the ability of the recombinant plasmid to complement the growth deficiency of Hib DL42. Construction of an isogenic NTHI TN106 mutant lacking a functional hitC gene revealed that this mutation prevented this strain from growing on a medium containing iron and PPIX in place of heme. This NTHI hitC mutant was also unable to utilize either iron bound to transferrin or iron chelates. These results suggest that the products encoded by the hitABC genes are essential for the utilization of iron by NTHI.

Full text

PDF
4523

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Angerer A., Gaisser S., Braun V. Nucleotide sequences of the sfuA, sfuB, and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism. J Bacteriol. 1990 Feb;172(2):572–578. doi: 10.1128/jb.172.2.572-578.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angerer A., Klupp B., Braun V. Iron transport systems of Serratia marcescens. J Bacteriol. 1992 Feb;174(4):1378–1387. doi: 10.1128/jb.174.4.1378-1387.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bagg A., Neilands J. B. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol Rev. 1987 Dec;51(4):509–518. doi: 10.1128/mr.51.4.509-518.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barcak G. J., Chandler M. S., Redfield R. J., Tomb J. F. Genetic systems in Haemophilus influenzae. Methods Enzymol. 1991;204:321–342. doi: 10.1016/0076-6879(91)04016-h. [DOI] [PubMed] [Google Scholar]
  5. Berish S. A., Mietzner T. A., Mayer L. W., Genco C. A., Holloway B. P., Morse S. A. Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae. J Exp Med. 1990 May 1;171(5):1535–1546. doi: 10.1084/jem.171.5.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanton K. J., Biswas G. D., Tsai J., Adams J., Dyer D. W., Davis S. M., Koch G. G., Sen P. K., Sparling P. F. Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin. J Bacteriol. 1990 Sep;172(9):5225–5235. doi: 10.1128/jb.172.9.5225-5235.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bouvier J., Richaud C., Higgins W., Bögler O., Stragier P. Cloning, characterization, and expression of the dapE gene of Escherichia coli. J Bacteriol. 1992 Aug;174(16):5265–5271. doi: 10.1128/jb.174.16.5265-5271.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bullen J. J., Rogers H. J., Griffiths E. Role of iron in bacterial infection. Curr Top Microbiol Immunol. 1978;80:1–35. doi: 10.1007/978-3-642-66956-9_1. [DOI] [PubMed] [Google Scholar]
  9. Chen C. Y., Berish S. A., Morse S. A., Mietzner T. A. The ferric iron-binding protein of pathogenic Neisseria spp. functions as a periplasmic transport protein in iron acquisition from human transferrin. Mol Microbiol. 1993 Oct;10(2):311–318. doi: 10.1111/j.1365-2958.1993.tb01957.x. [DOI] [PubMed] [Google Scholar]
  10. Cope L. D., Yogev R., Mertsola J., Latimer J. L., Hanson M. S., McCracken G. H., Jr, Hansen E. J. Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type B. Mol Microbiol. 1991 May;5(5):1113–1124. doi: 10.1111/j.1365-2958.1991.tb01884.x. [DOI] [PubMed] [Google Scholar]
  11. Cornelissen C. N., Biswas G. D., Sparling P. F. Expression of gonococcal transferrin-binding protein 1 causes Escherichia coli to bind human transferrin. J Bacteriol. 1993 Apr;175(8):2448–2450. doi: 10.1128/jb.175.8.2448-2450.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crosa J. H. Genetics and molecular biology of siderophore-mediated iron transport in bacteria. Microbiol Rev. 1989 Dec;53(4):517–530. doi: 10.1128/mr.53.4.517-530.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Evans N. M., Smith D. D., Wicken A. J. Haemin and nicotinamide adenine dinucleotide requirements of Haemophilus influenzae and Haemophilus parainfluenzae. J Med Microbiol. 1974 Aug;7(3):359–365. doi: 10.1099/00222615-7-3-359. [DOI] [PubMed] [Google Scholar]
  15. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Griffiths E. Iron and bacterial virulence--a brief overview. Biol Met. 1991;4(1):7–13. doi: 10.1007/BF01135551. [DOI] [PubMed] [Google Scholar]
  17. Hansen E. J., Gonzales F. R., Chamberlain N. R., Norgard M. V., Miller E. E., Cope L. D., Pelzel S. E., Gaddy B., Clausell A. Cloning of the gene encoding the major outer membrane protein of Haemophilus influenzae type b. Infect Immun. 1988 Oct;56(10):2709–2716. doi: 10.1128/iai.56.10.2709-2716.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hansen E. J., Latimer J. L., Thomas S. E., Helminen M., Albritton W. L., Radolf J. D. Use of electroporation to construct isogenic mutants of Haemophilus ducreyi. J Bacteriol. 1992 Aug;174(16):5442–5449. doi: 10.1128/jb.174.16.5442-5449.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hansen E. J., Pelzel S. E., Orth K., Moomaw C. R., Radolf J. D., Slaughter C. A. Structural and antigenic conservation of the P2 porin protein among strains of Haemophilus influenzae type b. Infect Immun. 1989 Nov;57(11):3270–3275. doi: 10.1128/iai.57.11.3270-3275.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hanson M. S., Pelzel S. E., Latimer J., Muller-Eberhard U., Hansen E. J. Identification of a genetic locus of Haemophilus influenzae type b necessary for the binding and utilization of heme bound to human hemopexin. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1973–1977. doi: 10.1073/pnas.89.5.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Harkness R. E., Chong P., Klein M. H. Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae. J Bacteriol. 1992 Apr;174(8):2425–2430. doi: 10.1128/jb.174.8.2425-2430.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Herrington D. A., Sparling P. F. Haemophilus influenzae can use human transferrin as a sole source for required iron. Infect Immun. 1985 Apr;48(1):248–251. doi: 10.1128/iai.48.1.248-251.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Herriott R. M., Meyer E. M., Vogt M. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J Bacteriol. 1970 Feb;101(2):517–524. doi: 10.1128/jb.101.2.517-524.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Higgins C. F., Gallagher M. P., Mimmack M. L., Pearce S. R. A family of closely related ATP-binding subunits from prokaryotic and eukaryotic cells. Bioessays. 1988 Apr;8(4):111–116. doi: 10.1002/bies.950080406. [DOI] [PubMed] [Google Scholar]
  25. Holland J., Langford P. R., Towner K. J., Williams P. Evidence for in vivo expression of transferrin-binding proteins in Haemophilus influenzae type b. Infect Immun. 1992 Jul;60(7):2986–2991. doi: 10.1128/iai.60.7.2986-2991.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  27. Kimura A., Gulig P. A., McCracken G. H., Jr, Loftus T. A., Hansen E. J. A minor high-molecular-weight outer membrane protein of Haemophilus influenzae type b is a protective antigen. Infect Immun. 1985 Jan;47(1):253–259. doi: 10.1128/iai.47.1.253-259.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee B. C. Isolation of an outer membrane hemin-binding protein of Haemophilus influenzae type b. Infect Immun. 1992 Mar;60(3):810–816. doi: 10.1128/iai.60.3.810-816.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Legrain M., Mazarin V., Irwin S. W., Bouchon B., Quentin-Millet M. J., Jacobs E., Schryvers A. B. Cloning and characterization of Neisseria meningitidis genes encoding the transferrin-binding proteins Tbp1 and Tbp2. Gene. 1993 Aug 16;130(1):73–80. doi: 10.1016/0378-1119(93)90348-7. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  32. McGehee J. L., Radolf J. D., Toews G. B., Hansen E. J. Effect of primary immunization on pulmonary clearance of nontypable Haemophilus influenzae. Am J Respir Cell Mol Biol. 1989 Sep;1(3):201–210. doi: 10.1165/ajrcmb/1.3.201. [DOI] [PubMed] [Google Scholar]
  33. Mietzner T. A., Bolan G., Schoolnik G. K., Morse S. A. Purification and characterization of the major iron-regulated protein expressed by pathogenic Neisseriae. J Exp Med. 1987 Apr 1;165(4):1041–1057. doi: 10.1084/jem.165.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Morse S. A., Chen C. Y., LeFaou A., Mietzner T. A. A potential role for the major iron-regulated protein expressed by pathogenic Neisseria species. Rev Infect Dis. 1988 Jul-Aug;10 (Suppl 2):S306–S310. doi: 10.1093/cid/10.supplement_2.s306. [DOI] [PubMed] [Google Scholar]
  35. Morton D. J., Williams P. Characterization of the outer-membrane proteins of Haemophilus parainfluenzae expressed under iron-sufficient and iron-restricted conditions. J Gen Microbiol. 1989 Feb;135(Pt 2):445–451. doi: 10.1099/00221287-135-2-445. [DOI] [PubMed] [Google Scholar]
  36. Payne S. M. Iron and virulence in the family Enterobacteriaceae. Crit Rev Microbiol. 1988;16(2):81–111. doi: 10.3109/10408418809104468. [DOI] [PubMed] [Google Scholar]
  37. Pidcock K. A., Wooten J. A., Daley B. A., Stull T. L. Iron acquisition by Haemophilus influenzae. Infect Immun. 1988 Apr;56(4):721–725. doi: 10.1128/iai.56.4.721-725.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Robertson S. M., Frisch C. F., Gulig P. A., Kettman J. R., Johnston K. H., Hansen E. J. Monoclonal antibodies directed against a cell surface-exposed outer membrane protein of Haemophilus influenzae type b. Infect Immun. 1982 Apr;36(1):80–88. doi: 10.1128/iai.36.1.80-88.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Robinson A. C., Kenan D. J., Sweeney J., Donachie W. D. Further evidence for overlapping transcriptional units in an Escherichia coli cell envelope-cell division gene cluster: DNA sequence and transcriptional organization of the ddl ftsQ region. J Bacteriol. 1986 Sep;167(3):809–817. doi: 10.1128/jb.167.3.809-817.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sanders J. D., Cope L. D., Jarosik G. P., Maciver I., Latimer J. L., Toews G. B., Hansen E. J. Reconstitution of a porin-deficient mutant of Haemophilus influenzae type b with a porin gene from nontypeable H. influenzae. Infect Immun. 1993 Sep;61(9):3966–3975. doi: 10.1128/iai.61.9.3966-3975.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schryvers A. B. Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae. Mol Microbiol. 1988 Jul;2(4):467–472. doi: 10.1111/j.1365-2958.1988.tb00052.x. [DOI] [PubMed] [Google Scholar]
  42. Schryvers A. B. Identification of the transferrin- and lactoferrin-binding proteins in Haemophilus influenzae. J Med Microbiol. 1989 Jun;29(2):121–130. doi: 10.1099/00222615-29-2-121. [DOI] [PubMed] [Google Scholar]
  43. Schryvers A. B., Lee B. C. Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae. Can J Microbiol. 1989 Mar;35(3):409–415. doi: 10.1139/m89-063. [DOI] [PubMed] [Google Scholar]
  44. Setlow J. K., Brown D. C., Boling M. E., Mattingly A., Gordon M. P. Repair of deoxyribonucleic acid in Haemophilus influenzae. I. X-ray sensitivity of ultraviolet-sensitive mutants and their behavior as hosts to ultraviolet-irradiated bacteriophage and transforming deoxyribonucleic acid. J Bacteriol. 1968 Feb;95(2):546–558. doi: 10.1128/jb.95.2.546-558.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stuy J. H., Walter R. B. Effect of glycerol on plasmid transfer in genetically competent Haemophilus influenzae. Mol Gen Genet. 1986 May;203(2):296–299. doi: 10.1007/BF00333969. [DOI] [PubMed] [Google Scholar]
  46. Tomb J. F., Barcak G. J., Chandler M. S., Redfield R. J., Smith H. O. Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd. J Bacteriol. 1989 Jul;171(7):3796–3802. doi: 10.1128/jb.171.7.3796-3802.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. WHITE D. C., GRANICK S. HEMIN BIOSYNTHESIS IN HAEMOPHILUS. J Bacteriol. 1963 Apr;85:842–850. doi: 10.1128/jb.85.4.842-850.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weinberg E. D. Iron withholding: a defense against infection and neoplasia. Physiol Rev. 1984 Jan;64(1):65–102. doi: 10.1152/physrev.1984.64.1.65. [DOI] [PubMed] [Google Scholar]
  49. Whitchurch C. B., Hobbs M., Livingston S. P., Krishnapillai V., Mattick J. S. Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene. 1991 May 15;101(1):33–44. doi: 10.1016/0378-1119(91)90221-v. [DOI] [PubMed] [Google Scholar]
  50. Williams P., Morton D. J., Towner K. J., Stevenson P., Griffiths E. Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus. J Gen Microbiol. 1990 Dec;136(12):2343–2350. doi: 10.1099/00221287-136-12-2343. [DOI] [PubMed] [Google Scholar]
  51. Willson P. J., Albritton W. L., Slaney L., Setlow J. K. Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi. Antimicrob Agents Chemother. 1989 Sep;33(9):1627–1630. doi: 10.1128/aac.33.9.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wu B., Georgopoulos C., Ang D. The essential Escherichia coli msgB gene, a multicopy suppressor of a temperature-sensitive allele of the heat shock gene grpE, is identical to dapE. J Bacteriol. 1992 Aug;174(16):5258–5264. doi: 10.1128/jb.174.16.5258-5264.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zimmermann L., Angerer A., Braun V. Mechanistically novel iron(III) transport system in Serratia marcescens. J Bacteriol. 1989 Jan;171(1):238–243. doi: 10.1128/jb.171.1.238-243.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES