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. 1994 Aug;176(16):5108–5115. doi: 10.1128/jb.176.16.5108-5115.1994

Vibrio cholerae fur mutations associated with loss of repressor activity: implications for the structural-functional relationships of fur.

M S Lam 1, C M Litwin 1, P A Carroll 1, S B Calderwood 1
PMCID: PMC196351  PMID: 8051024

Abstract

We used the Vibrio cholerae Fur protein as a model of iron-sensitive repressor proteins in gram-negative bacteria. Utilizing manganese mutagenesis, we isolated twelve independent mutations in V. cholerae fur that resulted in partial or complete loss of Fur repressor function. The mutant fur genes were recovered by PCR and sequenced; 11 of the 12 contained point mutations (two of which were identical), and one contained a 7-bp insertion that resulted in premature truncation of Fur. All of the mutants, except that containing the prematurely truncated Fur, produced protein by Western blot (immunoblot) analysis, although several had substantially smaller amounts of Fur and two made an immunoreactive protein that migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nine of the 11 point mutations altered amino acids that are identical in all of the fur genes sequenced so far, suggesting that these amino acids may play important structural or functional roles in Fur activity. Eight of the point mutations occurred in the amino-terminal half of Fur, which is thought to mediate DNA binding; most of these mutations occurred in conserved amino acids that have been previously suggested to play a role in the interaction between adjacent alpha-helices of the protein. Three of the point mutations occurred in the carboxy-terminal half of Fur, which is thought to bind iron. One mutation at histidine-90 was associated with complete loss of Fur function; this amino acid is within a motif previously suggested as being involved in iron binding by Fur. The fur allele mutant at histidine-90 interfered with iron regulation by wild-type fur in the same cell when the mutant allele was present at higher copy number; wild-type fur was dominant over all other fur mutant alleles studied. These results are analyzed with respect to previous models of the structure and function of Fur as an iron-sensitive repressor.

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Selected References

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  1. Arnold F. H., Haymore B. L. Engineered metal-binding proteins: purification to protein folding. Science. 1991 Jun 28;252(5014):1796–1797. doi: 10.1126/science.1648261. [DOI] [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. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Coy M., Neilands J. B. Structural dynamics and functional domains of the fur protein. Biochemistry. 1991 Aug 20;30(33):8201–8210. doi: 10.1021/bi00247a016. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. De Grandis S., Ginsberg J., Toone M., Climie S., Friesen J., Brunton J. Nucleotide sequence and promoter mapping of the Escherichia coli Shiga-like toxin operon of bacteriophage H-19B. J Bacteriol. 1987 Sep;169(9):4313–4319. doi: 10.1128/jb.169.9.4313-4319.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Goldberg M. B., Boyko S. A., Butterton J. R., Stoebner J. A., Payne S. M., Calderwood S. B. Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins. Mol Microbiol. 1992 Aug;6(16):2407–2418. doi: 10.1111/j.1365-2958.1992.tb01415.x. [DOI] [PubMed] [Google Scholar]
  15. Goldberg M. B., Boyko S. A., Calderwood S. B. Positive transcriptional regulation of an iron-regulated virulence gene in Vibrio cholerae. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1125–1129. doi: 10.1073/pnas.88.4.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Goldberg M. B., DiRita V. J., Calderwood S. B. Identification of an iron-regulated virulence determinant in Vibrio cholerae, using TnphoA mutagenesis. Infect Immun. 1990 Jan;58(1):55–60. doi: 10.1128/iai.58.1.55-60.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hamed M. Y. Binding of the ferric uptake regulation repressor protein (Fur) to Mn(II), Fe(II), Co(II), and Cu(II) ions as co-repressors: electronic absorption, equilibrium, and 57Fe Mössbauer studies. J Inorg Biochem. 1993 May 15;50(3):193–210. doi: 10.1016/0162-0134(93)80025-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Hantke K. Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism. Mol Gen Genet. 1987 Nov;210(1):135–139. doi: 10.1007/BF00337769. [DOI] [PubMed] [Google Scholar]
  22. Hovde C. J., Calderwood S. B., Mekalanos J. J., Collier R. J. Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2568–2572. doi: 10.1073/pnas.85.8.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Litwin C. M., Calderwood S. B. Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. J Bacteriol. 1993 Feb;175(3):706–715. doi: 10.1128/jb.175.3.706-715.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  28. Michaelis S., Inouye H., Oliver D., Beckwith J. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol. 1983 Apr;154(1):366–374. doi: 10.1128/jb.154.1.366-374.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Neilands J. B. A brief history of iron metabolism. Biol Met. 1991;4(1):1–6. doi: 10.1007/BF01135550. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Saito T., Duly D., Williams R. J. The histidines of the iron-uptake regulation protein, Fur. Eur J Biochem. 1991 Apr 10;197(1):39–42. doi: 10.1111/j.1432-1033.1991.tb15879.x. [DOI] [PubMed] [Google Scholar]
  32. Saito T., Williams R. J. The binding of the ferric uptake regulation protein to a DNA fragment. Eur J Biochem. 1991 Apr 10;197(1):43–47. doi: 10.1111/j.1432-1033.1991.tb15880.x. [DOI] [PubMed] [Google Scholar]
  33. Saito T., Wormald M. R., Williams R. J. Some structural features of the iron-uptake regulation protein. Eur J Biochem. 1991 Apr 10;197(1):29–38. doi: 10.1111/j.1432-1033.1991.tb15878.x. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Sigel S. P., Payne S. M. Effect of iron limitation on growth, siderophore production, and expression of outer membrane proteins of Vibrio cholerae. J Bacteriol. 1982 Apr;150(1):148–155. doi: 10.1128/jb.150.1.148-155.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stoebner J. A., Butterton J. R., Calderwood S. B., Payne S. M. Identification of the vibriobactin receptor of Vibrio cholerae. J Bacteriol. 1992 May;174(10):3270–3274. doi: 10.1128/jb.174.10.3270-3274.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tautz D., Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983 Jul 1;132(1):14–19. doi: 10.1016/0003-2697(83)90419-0. [DOI] [PubMed] [Google Scholar]
  43. Waldbeser L. S., Tolmasky M. E., Actis L. A., Crosa J. H. Mechanisms for negative regulation by iron of the fatA outer membrane protein gene expression in Vibrio anguillarum 775. J Biol Chem. 1993 May 15;268(14):10433–10439. [PubMed] [Google Scholar]
  44. 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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