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. 1995 Dec;177(24):7194–7201. doi: 10.1128/jb.177.24.7194-7201.1995

Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters.

U A Ochsner 1, A I Vasil 1, M L Vasil 1
PMCID: PMC177600  PMID: 8522528

Abstract

The cloned Pseudomonas aeruginosa fur (ferric uptake regulator) gene was overexpressed in P. aeruginosa by using a T7 expression system, and the Fur protein (PA-Fur) was purified by using a combination of ion-exchange chromatography and metal affinity chromatography. The DNA binding activity of the PA-Fur protein was confirmed by gel mobility shift assays and DNase I footprints of the synthetic DNA fragment GATAAT GATAATCATTATC, representing a perfect "Fur box". In addition, it was shown that PA-Fur is capable of binding to promoter and operator determinants of the tightly iron-regulated Escherichia coli fepA-fes enterobactin gene system. The activity of PA-Fur on the promoters of iron-regulated genes involved in the production of two siderophores, pyochelin and pyoverdin, and in the expression of exotoxin A was investigated. Data indicating that the promoters of the pchR gene, encoding a transcriptional activator for pyochelin synthesis, and of the pvdS gene, encoding a positive regulator for pyoverdin production, are specifically recognized by Fur-Fe(II) are presented, suggesting that PA-Fur represses expression of pchR and pvdS during growth in an iron-replete environment. However, neither the promoter region of the gene encoding exotoxin A (toxA) nor the promoters of the regAB operon, required for toxA expression, interacted with high concentrations of purified PA-Fur. These data indicate that iron regulation of exotoxin A production involves additional factors which may ultimately be under the control of PA-Fur.

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Selected References

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  1. Ankenbauer R. G., Quan H. N. FptA, the Fe(III)-pyochelin receptor of Pseudomonas aeruginosa: a phenolate siderophore receptor homologous to hydroxamate siderophore receptors. J Bacteriol. 1994 Jan;176(2):307–319. doi: 10.1128/jb.176.2.307-319.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bagg A., Neilands J. B. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry. 1987 Aug 25;26(17):5471–5477. doi: 10.1021/bi00391a039. [DOI] [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. Berish S. A., Subbarao S., Chen C. Y., Trees D. L., Morse S. A. Identification and cloning of a fur homolog from Neisseria gonorrhoeae. Infect Immun. 1993 Nov;61(11):4599–4606. doi: 10.1128/iai.61.11.4599-4606.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bjorn M. J., Iglewski B. H., Ives S. K., Sadoff J. C., Vasil M. L. Effect of iron on yields of exotoxin A in cultures of Pseudomonas aeruginosa PA-103. Infect Immun. 1978 Mar;19(3):785–791. doi: 10.1128/iai.19.3.785-791.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Briat J. F. Iron assimilation and storage in prokaryotes. J Gen Microbiol. 1992 Dec;138(12):2475–2483. doi: 10.1099/00221287-138-12-2475. [DOI] [PubMed] [Google Scholar]
  7. Brickman T. J., Armstrong S. K. Bordetella pertussis fur gene restores iron repressibility of siderophore and protein expression to deregulated Bordetella bronchiseptica mutants. J Bacteriol. 1995 Jan;177(1):268–270. doi: 10.1128/jb.177.1.268-270.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brickman T. J., Ozenberger B. A., McIntosh M. A. Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli. J Mol Biol. 1990 Apr 20;212(4):669–682. doi: 10.1016/0022-2836(90)90229-F. [DOI] [PubMed] [Google Scholar]
  9. Brumlik M. J., Storey D. G. Zinc and iron regulate translation of the gene encoding Pseudomonas aeruginosa elastase. Mol Microbiol. 1992 Feb;6(3):337–344. doi: 10.1111/j.1365-2958.1992.tb01476.x. [DOI] [PubMed] [Google Scholar]
  10. Brunschwig E., Darzins A. A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa. Gene. 1992 Feb 1;111(1):35–41. doi: 10.1016/0378-1119(92)90600-t. [DOI] [PubMed] [Google Scholar]
  11. Butterton J. R., Stoebner J. A., Payne S. M., Calderwood S. B. Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J Bacteriol. 1992 Jun;174(11):3729–3738. doi: 10.1128/jb.174.11.3729-3738.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Calderwood S. B., Mekalanos J. J. Confirmation of the Fur operator site by insertion of a synthetic oligonucleotide into an operon fusion plasmid. J Bacteriol. 1988 Feb;170(2):1015–1017. doi: 10.1128/jb.170.2.1015-1017.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Calderwood S. B., Mekalanos J. J. Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus. J Bacteriol. 1987 Oct;169(10):4759–4764. doi: 10.1128/jb.169.10.4759-4764.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chen S. T., Jordan E. M., Wilson R. B., Draper R. K., Clowes R. C. Transcription and expression of the exotoxin A gene of Pseudomonas aeruginosa. J Gen Microbiol. 1987 Nov;133(11):3081–3091. doi: 10.1099/00221287-133-11-3081. [DOI] [PubMed] [Google Scholar]
  15. Cox C. D., Adams P. Siderophore activity of pyoverdin for Pseudomonas aeruginosa. Infect Immun. 1985 Apr;48(1):130–138. doi: 10.1128/iai.48.1.130-138.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cox C. D. Iron uptake with ferripyochelin and ferric citrate by Pseudomonas aeruginosa. J Bacteriol. 1980 May;142(2):581–587. doi: 10.1128/jb.142.2.581-587.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cunliffe H. E., Merriman T. R., Lamont I. L. Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor. J Bacteriol. 1995 May;177(10):2744–2750. doi: 10.1128/jb.177.10.2744-2750.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. De Lorenzo V., Herrero M., Giovannini F., Neilands J. B. Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli. Eur J Biochem. 1988 May 2;173(3):537–546. doi: 10.1111/j.1432-1033.1988.tb14032.x. [DOI] [PubMed] [Google Scholar]
  19. Ernst J. F., Bennett R. L., Rothfield L. I. Constitutive expression of the iron-enterochelin and ferrichrome uptake systems in a mutant strain of Salmonella typhimurium. J Bacteriol. 1978 Sep;135(3):928–934. doi: 10.1128/jb.135.3.928-934.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Foster J. W., Hall H. K. Effect of Salmonella typhimurium ferric uptake regulator (fur) mutations on iron- and pH-regulated protein synthesis. J Bacteriol. 1992 Jul;174(13):4317–4323. doi: 10.1128/jb.174.13.4317-4323.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Frank D. W., Iglewski B. H. Kinetics of toxA and regA mRNA accumulation in Pseudomonas aeruginosa. J Bacteriol. 1988 Oct;170(10):4477–4483. doi: 10.1128/jb.170.10.4477-4483.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Frank D. W., Storey D. G., Hindahl M. S., Iglewski B. H. Differential regulation by iron of regA and toxA transcript accumulation in Pseudomonas aeruginosa. J Bacteriol. 1989 Oct;171(10):5304–5313. doi: 10.1128/jb.171.10.5304-5313.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Goldberg M. B., Boyko S. A., Calderwood S. B. Transcriptional regulation by iron of a Vibrio cholerae virulence gene and homology of the gene to the Escherichia coli fur system. J Bacteriol. 1990 Dec;172(12):6863–6870. doi: 10.1128/jb.172.12.6863-6870.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Grant C. C., Vasil M. L. Analysis of transcription of the exotoxin A gene of Pseudomonas aeruginosa. J Bacteriol. 1986 Dec;168(3):1112–1119. doi: 10.1128/jb.168.3.1112-1119.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Griggs D. W., Konisky J. Mechanism for iron-regulated transcription of the Escherichia coli cir gene: metal-dependent binding of fur protein to the promoters. J Bacteriol. 1989 Feb;171(2):1048–1054. doi: 10.1128/jb.171.2.1048-1054.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hantke K. Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol Gen Genet. 1981;182(2):288–292. doi: 10.1007/BF00269672. [DOI] [PubMed] [Google Scholar]
  27. Hedstrom R. C., Funk C. R., Kaper J. B., Pavlovskis O. R., Galloway D. R. Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa. Infect Immun. 1986 Jan;51(1):37–42. doi: 10.1128/iai.51.1.37-42.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Heinrichs D. E., Poole K. Cloning and sequence analysis of a gene (pchR) encoding an AraC family activator of pyochelin and ferripyochelin receptor synthesis in Pseudomonas aeruginosa. J Bacteriol. 1993 Sep;175(18):5882–5889. doi: 10.1128/jb.175.18.5882-5889.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Heinrichs D. E., Young L., Poole K. Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein. Infect Immun. 1991 Oct;59(10):3680–3684. doi: 10.1128/iai.59.10.3680-3684.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hennecke H. Regulation of bacterial gene expression by metal-protein complexes. Mol Microbiol. 1990 Oct;4(10):1621–1628. doi: 10.1111/j.1365-2958.1990.tb00538.x. [DOI] [PubMed] [Google Scholar]
  31. Hickey E. K., Cianciotto N. P. Cloning and sequencing of the Legionella pneumophila fur gene. Gene. 1994 May 27;143(1):117–121. doi: 10.1016/0378-1119(94)90615-7. [DOI] [PubMed] [Google Scholar]
  32. Hindahl M. S., Frank D. W., Hamood A., Iglewski B. H. Characterization of a gene that regulates toxin A synthesis in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Jun 24;16(12):5699–5699. doi: 10.1093/nar/16.12.5699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hunt M. D., Pettis G. S., McIntosh M. A. Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system. J Bacteriol. 1994 Jul;176(13):3944–3955. doi: 10.1128/jb.176.13.3944-3955.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Höfte M., Buysens S., Koedam N., Cornelis P. Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2. Biometals. 1993 Summer;6(2):85–91. doi: 10.1007/BF00140108. [DOI] [PubMed] [Google Scholar]
  35. Jaeger K. E., Ransac S., Dijkstra B. W., Colson C., van Heuvel M., Misset O. Bacterial lipases. FEMS Microbiol Rev. 1994 Sep;15(1):29–63. doi: 10.1111/j.1574-6976.1994.tb00121.x. [DOI] [PubMed] [Google Scholar]
  36. Karkhoff-Schweizer R. R., Schryvers A. B., Schweizer H. P. Cloning and sequence analysis of the fur gene encoding an iron-regulatory protein of Neisseria meningitidis. Gene. 1994 Apr 8;141(1):139–140. doi: 10.1016/0378-1119(94)90143-0. [DOI] [PubMed] [Google Scholar]
  37. Le Cam E., Frechon D., Barray M., Fourcade A., Delain E. Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11816–11820. doi: 10.1073/pnas.91.25.11816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Litwin C. M., Boyko S. A., Calderwood S. B. Cloning, sequencing, and transcriptional regulation of the Vibrio cholerae fur gene. J Bacteriol. 1992 Mar;174(6):1897–1903. doi: 10.1128/jb.174.6.1897-1903.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Litwin C. M., Calderwood S. B. Analysis of the complexity of gene regulation by fur in Vibrio cholerae. J Bacteriol. 1994 Jan;176(1):240–248. doi: 10.1128/jb.176.1.240-248.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Liu P. V. Extracellular toxins of Pseudomonas aeruginosa. J Infect Dis. 1974 Nov;130 (Suppl)(0):S94–S99. doi: 10.1093/infdis/130.supplement.s94. [DOI] [PubMed] [Google Scholar]
  41. Lory S. Effect of iron on accumulation of exotoxin A-specific mRNA in Pseudomonas aeruginosa. J Bacteriol. 1986 Dec;168(3):1451–1456. doi: 10.1128/jb.168.3.1451-1456.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Meyer J. M., Hohnadel D., Khan A., Cornelis P. Pyoverdin-facilitated iron uptake in Pseudomonas aeruginosa: immunological characterization of the ferripyoverdin receptor. Mol Microbiol. 1990 Aug;4(8):1401–1405. doi: 10.1111/j.1365-2958.1990.tb00719.x. [DOI] [PubMed] [Google Scholar]
  43. Niederhoffer E. C., Naranjo C. M., Bradley K. L., Fee J. A. Control of Escherichia coli superoxide dismutase (sodA and sodB) genes by the ferric uptake regulation (fur) locus. J Bacteriol. 1990 Apr;172(4):1930–1938. doi: 10.1128/jb.172.4.1930-1938.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. O'Sullivan D. J., Dowling D. N., deLorenzo V., O'Gara F. Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter. FEMS Microbiol Lett. 1994 Apr 15;117(3):327–332. doi: 10.1016/0378-1097(94)90579-7. [DOI] [PubMed] [Google Scholar]
  45. Payne S. M. Iron acquisition in microbial pathogenesis. Trends Microbiol. 1993 May;1(2):66–69. doi: 10.1016/0966-842x(93)90036-q. [DOI] [PubMed] [Google Scholar]
  46. Payne S. M. Iron and virulence in Shigella. Mol Microbiol. 1989 Sep;3(9):1301–1306. doi: 10.1111/j.1365-2958.1989.tb00281.x. [DOI] [PubMed] [Google Scholar]
  47. Prince R. W., Cox C. D., Vasil M. L. Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J Bacteriol. 1993 May;175(9):2589–2598. doi: 10.1128/jb.175.9.2589-2598.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Prince R. W., Storey D. G., Vasil A. I., Vasil M. L. Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01. Mol Microbiol. 1991 Nov;5(11):2823–2831. doi: 10.1111/j.1365-2958.1991.tb01991.x. [DOI] [PubMed] [Google Scholar]
  49. Schrum L. W., Hassan H. M. The effects of fur on the transcriptional and post-transcriptional regulation of MnSOD gene (sodA) in Escherichia coli. Arch Biochem Biophys. 1994 Mar;309(2):288–292. doi: 10.1006/abbi.1994.1115. [DOI] [PubMed] [Google Scholar]
  50. Schäffer S., Hantke K., Braun V. Nucleotide sequence of the iron regulatory gene fur. Mol Gen Genet. 1985;200(1):110–113. doi: 10.1007/BF00383321. [DOI] [PubMed] [Google Scholar]
  51. Sokol P. A., Woods D. E. Demonstration of an iron-siderophore-binding protein in the outer membrane of Pseudomonas aeruginosa. Infect Immun. 1983 May;40(2):665–669. doi: 10.1128/iai.40.2.665-669.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Staggs T. M., Fetherston J. D., Perry R. D. Pleiotropic effects of a Yersinia pestis fur mutation. J Bacteriol. 1994 Dec;176(24):7614–7624. doi: 10.1128/jb.176.24.7614-7624.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Staggs T. M., Perry R. D. Fur regulation in Yersinia species. Mol Microbiol. 1992 Sep;6(17):2507–2516. doi: 10.1111/j.1365-2958.1992.tb01427.x. [DOI] [PubMed] [Google Scholar]
  54. Staggs T. M., Perry R. D. Identification and cloning of a fur regulatory gene in Yersinia pestis. J Bacteriol. 1991 Jan;173(2):417–425. doi: 10.1128/jb.173.2.417-425.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Storey D. G., Frank D. W., Farinha M. A., Kropinski A. M., Iglewski B. H. Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene. Mol Microbiol. 1990 Mar;4(3):499–503. doi: 10.1111/j.1365-2958.1990.tb00616.x. [DOI] [PubMed] [Google Scholar]
  56. Vasil M. L., Grant C. C., Prince R. W. Regulation of exotoxin A synthesis in Pseudomonas aeruginosa: characterization of toxA-lacZ fusions in wild-type and mutant strains. Mol Microbiol. 1989 Mar;3(3):371–381. doi: 10.1111/j.1365-2958.1989.tb00182.x. [DOI] [PubMed] [Google Scholar]
  57. Venturi V., Ottevanger C., Bracke M., Weisbeek P. Iron regulation of siderophore biosynthesis and transport in Pseudomonas putida WCS358: involvement of a transcriptional activator and of the Fur protein. Mol Microbiol. 1995 Mar;15(6):1081–1093. doi: 10.1111/j.1365-2958.1995.tb02283.x. [DOI] [PubMed] [Google Scholar]
  58. Walker S. L., Hiremath L. S., Wozniak D. J., Galloway D. R. ToxR (RegA)-mediated in vitro transcription of Pseudomonas aeruginosa toxA. Gene. 1994 Dec 2;150(1):87–92. doi: 10.1016/0378-1119(94)90863-x. [DOI] [PubMed] [Google Scholar]
  59. Wee S., Neilands J. B., Bittner M. L., Hemming B. C., Haymore B. L., Seetharam R. Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12. Biol Met. 1988;1(1):62–68. doi: 10.1007/BF01128019. [DOI] [PubMed] [Google Scholar]
  60. Wertheimer A. M., Tolmasky M. E., Actis L. A., Crosa J. H. Structural and functional analyses of mutant Fur proteins with impaired regulatory function. J Bacteriol. 1994 Aug;176(16):5116–5122. doi: 10.1128/jb.176.16.5116-5122.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wick M. J., Frank D. W., Storey D. G., Iglewski B. H. Identification of regB, a gene required for optimal exotoxin A yields in Pseudomonas aeruginosa. Mol Microbiol. 1990 Mar;4(3):489–497. doi: 10.1111/j.1365-2958.1990.tb00615.x. [DOI] [PubMed] [Google Scholar]
  62. Wooldridge K. G., Williams P. H., Ketley J. M. Iron-responsive genetic regulation in Campylobacter jejuni: cloning and characterization of a fur homolog. J Bacteriol. 1994 Sep;176(18):5852–5856. doi: 10.1128/jb.176.18.5852-5856.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. de Lorenzo V., Giovannini F., Herrero M., Neilands J. B. Metal ion regulation of gene expression. Fur repressor-operator interaction at the promoter region of the aerobactin system of pColV-K30. J Mol Biol. 1988 Oct 20;203(4):875–884. doi: 10.1016/0022-2836(88)90113-1. [DOI] [PubMed] [Google Scholar]
  64. de Lorenzo V., Wee S., Herrero M., Neilands J. B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987 Jun;169(6):2624–2630. doi: 10.1128/jb.169.6.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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